Research ArticleLoop-Mediated Isothermal Amplification Assay for Detectionof Generic and Verocytotoxin-Producing Escherichia coli amongIndigenous Individuals in Malaysia

Cindy Shuan Ju Teh1 Kek Heng Chua2 Yvonne Ai Lian Lim3

Soo Ching Lee3 and Kwai Lin Thong4

1 Department of Medical Microbiology Faculty of Medicine University of Malaya 50603 Kuala Lumpur Malaysia2 Department of Biomedical Science Faculty of Medicine University of Malaya 50603 Kuala Lumpur Malaysia3 Department of Parasitology Faculty of Medicine University of Malaya 50603 Kuala Lumpur Malaysia4 Institute of Biological Sciences Faculty of Science University of Malaya 50603 Kuala Lumpur Malaysia

Correspondence should be addressed to Kwai LinThong thongklumedumy

Received 4 April 2014 Accepted 6 May 2014 Published 19 May 2014

Academic Editor Amparo Latorre

Copyright copy 2014 Cindy Shuan Ju Teh et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

We have successfully developed a Loop-mediated isothermal amplification (LAMP) assay that could specifically detect genericEscherichia coli (E coli) This assay was tested on 85 bacterial strains and successfully identified 54 E coli strains (average thresholdtime Tt = 2126) The sensitivity of this assay was evaluated on serial dilutions of bacterial cultures and spiked faeces The assaycould detect 102 CFUmL for bacterial culture with Tt = 3330 while the detection limit for spiked faeces was 103 CFUmL (Tt =3112)We have also detected 46 generic E coli from 50 faecal samples obtained from indigenous individuals with 16 of the positivesamples being verocytotoxin-producing E coli (VTEC) positive VT1VT2 allele was present in one faecal sample while the ratio ofVT1 to VT2was 6 1 Overall our study had demonstrated high risk of VTEC infection among the indigenous community andmostof the asymptomatic infection occurred among those aged below 15 years The role of asymptomatic human carriers as a source ofdissemination should not be underestimated Large scale screening of the VTEC infection among indigenous populations and thepotential contamination sources will be possible and easy with the aid of this newly developed rapid and simple LAMP assay

1 Introduction

The human body is inhabited by a vast number of microor-ganisms It has been reported that there are 1014 bacterial cellspresent in our body [1] Besides fulfilling their physiologicalfunctions the microbiota has a direct impact on humanhealth [2] Basically the microbiota plays important rolesin combating aggression from other microorganisms [3] aswell as maintaining the balance of the intestinal mucosa andproviding an innate defence [4]

Escherichia coli (E coli) is an inhabitant of the intestinesand faeces of humans and animals It is among the firstbacterial species to colonize the intestine during infancyCommensal E coli strains found mainly in the caecum andthe colon provide some benefits to their host by inducing

colonization resistance in the host through the productionof bacteriocins and through other mechanisms [5] Mostof the E coli strains are harmless but they can also causeextraintestinal E coli (ExPEC) infections in urinary tractmeninges and bloodstream

Nevertheless there are certain serotypes that can causediseases in humans and animals [6 7] Verocytotoxin-producing Escherichia coli (VTEC) is a serious public healthconcern as it has been responsible for outbreaks in Europeancountries [8 9] and has been associated with haemolytic-uraemic syndrome (HUS) the leading cause of acute renalfailure in children [7 10] On the other hand VTEC couldalso colonize the human intestine without causing any symp-toms [6 11] This infection might not be harmful to the hostbut remains a threat to the public as asymptomatic carriers are

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 457839 6 pageshttpdxdoiorg1011552014457839

2 The Scientific World Journal

the potential source for transmission ofVTEC hence promptdetection of carrier is important

Given that instantaneous detection of the infectiousagents is important hence molecular approaches have beenutilized as the detection is more rapid than conventionalbiochemical tests Compared to PCR loop-mediated isother-mal amplification (LAMP) produces more specific resultsas there are 4 primers targeting 6 regions within a geneBesides LAMP is alsomore rapid (reaction occurs at constanttemperature) and eliminates the need for electrophoresis asthe results can be monitored through real-time turbidimeteror visually based on turbidity or with the aid of SYBR I dye[12]

In Malaysia studies on sanitation-related diseases suchas parasitic infections are commonly carried out among theindigenous communities due to their poor living conditionsinappropriate hygiene practices and the lack of functioningtoilet facilities in their houses [13] Asymptomatic parasiticinfections among indigenous individuals have been reported[14 15] however the study on bacterial infections is relativelyscarce Previously Rahim et al [16] had conducted a study onthe prevalence of Helicobacter pylori (HP) infection amongthe indigenous individuals A total of 19 asymptomaticindigenous individuals were tested positive for the HP infec-tion The limited amount of information regarding bacterialinfections may have been contributed to the lack of rapidtechnique which has the potential to be applied in the fieldThis is crucial as many of the indigenous communities aresituated in remote areas which is logistically challenging ifsamples were to be processed within a few hours Develop-ment of cost-effective and rapidmodern tools which have thepotential to overcome these limitations will be crucial

In the present study we aim to develop a LAMP assaythat can specifically detect genericE coliThedeveloped assaywill be applied in combination with another VTEC-specificLAMP assay previously developed by Hara-Kudo et al [17] todetect the presence of E coli and VTEC among Orang Aslicommunity in Malaysia

2 Materials and Methods

21 Bacteria Strains Fifty E coli strains (5 verocytotoxin-producing and 45 nonverocytotoxin-producing E coli) and35 bacterial species other than E coli (including 10 Shigellaspp) were revived from the culture collections in the Lab-oratory of Biomedical Science and Molecular MicrobiologyInstitute of Graduate Studies University of Malaya A loopfulof colonies of overnight culture was suspended in 100 120583LddH2O and was subjected to boiling at 99∘C for 5min

The suspension was snapped cool and centrifuged Thesupernatant was quantified and 15 120583L (equivalent to sim50 ng)was used as the DNA template for LAMP assay and PCR

22 LAMP Assay mGenomeSubtractor [18] and web-basedArtemis Comparison Tool (WebACT) (httpwwwwebactorgWebACT) were used to compare the publishedgenomes of E coli and other Enterobacteriaceae such asShigella spp Salmonella Yersinia spp and Klebsiella spp

A list of conserved regions for E coli was generated and wasagain blasted against the database by using NCBI BLAST(httpblastncbinlmnihgovBlastcgi) A CDS encodingglycerate kinase (EcolC 3109 Accession number CP000946)was selected and used for primers design Two pairs ofprimers including F3 (51015840-GGTAGATCGAACGGTCATCG-31015840) B3 (51015840-GGCCAGCAACGGATTACG-31015840) forward innerprimer (FIP) (51015840-CGCAGACTTCAAGCGTCACGA-TCGAAGGAACGGTGGATGC-31015840) and backward innerprimer (BIP) (51015840-CCTTACCGGCGACGGGAAAA-CTTTTCAGGCGCGACCAG-31015840) as well as a loop primer(LB) (51015840-TGAGATGGCGGCAGCAAGTG-31015840) weredesigned by using online PrimerExplorer V4 program(PrimerExplorer Eiken Chemical Co Ltd)

The LAMP assay was optimized on E coli strains usingLoopamp DNA amplification kit (Eiken Chemical Co LtdTokyo Japan) Briefly the reaction was carried out in a totalof 25 120583L containing 40 pmol of each FIP and BIP primers5 pmol of each F3 and B3 primers 10 pmol of LB primer125 120583L of 2X reaction mixture 1 120583L of Bst DNA polymeraseand 15 120583L of DNA (approximately 100 ng) The reactionmixtures were incubated at 62∘C for 80min followed byenzyme inactivation at 80∘C for 2min in the Loopamp real-time turbidimeter (LA-320 TeramecsCo Ltd Kyoto Japan)The positive result was indicated when the turbidity reached01 within 60min at 65 nM and Tt was recorded

23 PCR Validation PCR was performed in parallel tovalidate the results produced by LAMP assay Briefly PCRtargeting phoA gene forE coli detection andVT1 orVT2 genefor verocytotoxin producer was carried out as described byHo et al [19]

24 Detection Limit of LAMP Assay on Bacterial Cultureand Spiked Faeces Overnight cultures of E coli were seriallydiluted 10-fold An aliquot of 100 120583L of each dilutionwas usedfor the DNA template preparation and plated on lysogenyagar for CFU count On the other hand the overnight culturewas also spiked in 900 120583L of stools (diluted with brain-heartinfusion broth to avoid inhibition during PCR) followed by a10-fold serial dilution An aliquot of 100120583L of the dilutionwasplated on the agar for viable count while another 100 120583L wasused for the extraction of DNA by using crude lysate methodThe DNA templates were subjected to LAMP assay

25 Field Evaluation of LAMPAssay for the Detection of VTECamong Indigenous Individuals A total of 50 indigenousindividualswith positive parasitic infectionswere recruited inthe study (Ethics committeeIRB reference number 82411)Sample collection procedure has also been approved by theDepartment of Orang Asli Development (JAKOA) Consentwas given by the participating indigenous individuals eithervia signature or via thumbprint prior to sample collectionFaecal samples were subjected to DNA extraction usingQIAamp DNA Stool Mini Kit (QIAGEN The Netherlands)The extracted total DNA was quantified and stored at minus20∘Cuntil further use

The Scientific World Journal 3

minus01

0

01

02

03

04

05

06

07

0 1000 2000 3000 4000 5000

BC 106

SF 106

BC 105

SF 105

BC 104

SF 104

BC 103

BC 102

SF 103

SF 102

Figure 1 Sensitivity of LAMP assay of bacterial culture (BC) andspiked faecal (SF) specimens on different CFU (from 106 to 102)

The LAMP assay developed in this study was used todetect generic strains of E coli that are present in the faecalsample while VT1 and VT2 producer were determined byusing LAMP assay that was previously described by Hara-Kudo et al [17] The reaction consisted of approximately200ndash400 ng total DNA incubated at 62∘C for 45min andthe positive reactions were detected based on the colourchange after the addition of a SYBR green 1 10 dilution to thereaction tube

3 Results

All the 50 E coli strains showed positive reaction within25min with an average Tt value of 2126 None of the non-Ecoli strains showed positive amplification This indicates thehigh specificity of the LAMP assay for E coli detection Fur-thermore the detection limit of the LAMP assay was as lowas 102 CFUmL and 103 CFUmL on bacterial culture (Tt =3330) and the spiked faeces (Tt = 3112) respectively Forbacterial culture the reaction with more than 105 CFUmLof cells yielded positive results within 18min while forfaecal samples only those reactions which have more than106 CFUmL of cells showed positive amplification before18min (Figure 1)

The usefulness of the newly developed E coli LAMP assaywas evaluated using the faecal samples collected from OrangAsli Overall the results obtained by using LAMP assay werein concordance with PCR except for 1 sample (Table 1) Toverify the result of LAMP assay the amplicon of F3 and B3 ofthis particular sample was sent for sequencingThe ampliconwas 100 matched with the E coli glycerate kinase genesegment

On the other hand all E coli positive samples were alsosubjected to VT1 and VT2 detection as described in Ho et al[19] Among the 46 E coli positive samples VT1 was detected

in 6 volunteers (ge15 years old) while VT2 was detected in oneadultrsquos sample Only one sample obtained from a 5-year-oldgirl was detected with VT1 and VT2 producing E coli

4 Discussion

In this study a LAMP assay targeting glycerate kinase of Ecoli was developedThe assay demonstrated a high specificityand was fairly sensitive when applied on bacterial culture(102) and spiked faecal sample (103) None of the Shigellaspp showed positive reaction and this indicated that thedeveloped LAMP assay could specifically differentiate Ecoli from Shigella Therefore this assay could be potentiallyapplied on food and animal samples

LAMP assays targeting different strainspathotypes of Ecoli have been described previously [17 20ndash22] Among thepublished studies Hill et al [21] had developed a LAMPassay targeting common strains of E coli and this assay wasevaluated using urine samples malB gene was used for theprimer design however this gene was also found in ShigellaThe developed assay worked well in the identification ofurinary E coli (as Shigella is rare in extraintestinal infection)but will be restricted to other applications such as detectionof common E coli strains from faecal food soil and watersamples

E coli and Shigella are closely related The validity ofShigella genus has become a contentious issue over the pastdecades Based on the conventional view Shigella and E colishared greater than 90 homology by DNA-DNA reassoci-ation analysis and Shigella are E coli strains that undergothe convergent evolution of Shigella phenotypic propertiesthat contribute to the Shigella pathotype [23ndash25] Howeverin terms of clinical manifestation Shigella spp are frankpathogens that readily cause disease in humans while E colinormally lives in the intestines of humans and animals (withthe exception of pathogenic clones) Prompt and reliableidentification of these infectious agents is important for theright choice of treatment and to avoid any complications

Differentiation between E coli and Shigella is difficultbut achievable based on physiological biochemical andserological typing [26] For a more rapid method alkalinephosphatase (phoA) gene has been used in PCRs for commonE coli strains detection demonstrating high specificity [2728]Therefore this PCRwas selected as a tool for validation ofthe results obtained by using LAMP in this study In fact theLAMP assay showed higher specificity in detecting genericE coli in the indigenous samples compared to PCR targetingphoA gene where the PCR could not detect E coli in one ofthe samples (PG31)

The rate of verocytotoxin-producing E coli in asymp-tomatic human carrier in this study was considered high(16) compared to previous studies that reported the rateof 76 and 4 conducted by Hong et al [6] and Stephanand Hoelzle [11] respectively In the studies of Hong et al [6]and Stephan andHoelzle [11] workers in slaughterhouse weretargeted as they were considered at higher risk for carriageor excretion of VTEC Thus the finding in this study hashighlighted the attention on hygiene practices and living

4 The Scientific World Journal

Table 1 PCR and LAMP results for the samples obtained from indigenous individuals which were previously detected with different parasiticinfections

ID AGE Parasitic infection PCR LAMP (E coli)Generic E coli VT producer

PG 122 8 Giardia + +PG 201 12 Giardia Entamoeba coli minus minus

PG 31 10 Giardia minus +PB 111 73 Giardia + +PB 145 5 Giardia + + VT1VT2PM 192 7 Giardia + +PM 252 7 Giardia Entamoeba coli + +PM 312 8 Giardia + +PM 531 10 Giardia minus minus

PM 532 9 Giardia + +PM 541 10 Giardia + +SP 111 6 Giardia + +SP 73 1 Giardia + +DB 117 15 Giardia + + VT1DB 21 30 Giardia Trichuris Ascaris Entamoeba coli Iodamoeba + +BP 15 4 Giardia Trichuris + +BP 142 10 Giardia Trichuris Ascaris + +SB 33 11 Giardia + + VT1DK 104 7 Giardia Trichuris + +DK 173 6 Giardia TrichurisHookworm Entamoeba coli + +DK 176 13 Giardia Trichuris Entamoeba coli + +DK 1910 8 Giardia Trichuris Ascaris + +DK 271 10 Giardia Trichuris + +DK 291 7 Giardia Trichuris + +UK 152 32 Giardia + +UK 155 4 Giardia Trichuris Ascaris + +UK 173 7 Giardia Ascaris + +UK 62 45 Giardia Ascaris + +UK 65 13 Giardia Ascaris + + VT1UL 132 5 Giardia Trichuris Ascaris + + VT1UL 33 12 Giardia Trichuris AscarisHookworm minus minus

UL 44 1 Giardia + +UK 153 9 Giardia Ascaris + +UK 171 32 Giardia Ascaris minus minus

UK 63 18 Giardia + +PP 11 58 Giardia Trichuris Ascaris + +PS 73 11 Giardia + +NT 44 13 Giardia Trichuris Ascaris + +NT 62 30 Giardia Ascaris + + VT2NT 63 8 Giardia Ascaris Entamoeba coli + +PG 27 17 GiardiaHookworm + +PG 262 43 Giardia + +JJ 72 36 Giardia + +JJ 114 7 Giardia + + VT1JJ 115 5 Giardia + + VT1SW 53 10 Giardia + +SW 101 31 Giardia + +SW 112 1 Giardia + +SW 143 4 Giardia E coli + +SW 163 32 GiardiaHookworm + +

The Scientific World Journal 5

conditions of the indigenous populations in Malaysia giventhe fact that these communities are afflicted with parasiticinfections not only as reported previously [14 15] but alsoas a reservoir for VTEC

In this study all the volunteers were infected by differentparasites such as Giardia Trichuris Ascaris HookwormEntamoeba coli and Iodamoeba There is no statistical evi-dence that showed correlation between parasitic infectionand VTEC colonization Our results also did not suggest anygroups (single or multiple parasitic infections) that are proneto VTEC colonization Interestingly we found that E coliwas hardly detected in the stool samples containing multipleparasites as in the first run of LAMP assay (sample PM 252DB 21 BP 15 BP 142 DK 173 DK 176 DK 1910 DK 271UK 155 UL 132 NT 62 NT 63 and SW 163) Reasonsthat influenced the sensitivity of this LAMP assay will befurther investigated Meanwhile the 4 samples which werenot detected with E coli might be due to recent antibiotictreatments

5 Conclusion

We have successfully developed a LAMP assay that canspecifically detect generic E coli and further performed incombination with the LAMP assay that detect verocytotoxin-related gene Our study suggests the high risk of indigenouscommunity for carriage and excretion of VTEC Due to thehigh specificity and sensitivity of the generic E coli LAMPassay developed in this study we proposed the application ofthis assay in food and animals as an alternative approach forrapid and more specific detection of E coli

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank University of Malaya for support andfacilities This study was funded by Higher Impact ResearchGrant (UMC6251HIRMOHE02) andGrant GA 013-2013(Molecular Diagnostics of Bacterial Pathogens)

References

[1] R E Ley D A Peterson and J I Gordon ldquoEcological andevolutionary forces shaping microbial diversity in the humanintestinerdquo Cell vol 124 no 4 pp 837ndash848 2006

[2] J C Clemente L K Ursell L W Parfrey and R Knight ldquoTheimpact of the gut microbiota on human health an integrativeviewrdquo Cell vol 148 no 6 pp 1258ndash1270 2012

[3] R Burcelin E Luche M Serino and J Amar ldquoThe gutmicrobiota ecology a new opportunity for the treatment ofmetabolic diseasesrdquo Frontiers in Bioscience vol 14 no 13 pp5107ndash5117 2009

[4] H Tlaskalova-Hogenova R Stpankova H Kozakova et alldquoThe role of gut microbiota (commensal bacteria) and themucosal barrier in the pathogenesis of inflammatory and

autoimmune diseases and cancer contribution of germ-freeand gnotobiotic animalmodels of human diseasesrdquoCellular andMolecular Immunology vol 8 no 2 pp 110ndash120 2011

[5] O Tenaillon D Skurnik B Picard and E Denamur ldquoThepopulation genetics of commensal Escherichia colirdquo NatureReviews Microbiology vol 8 no 3 pp 207ndash217 2010

[6] S Hong K H Oh S H Cho et al ldquoAsymptomatic healthyslaughterhouse workers in South Korea carrying Shiga toxin-producing Escherichia colirdquo FEMS Immunology and MedicalMicrobiology vol 56 no 1 pp 41ndash47 2009

[7] M A Karmali V Gannon and J M Sargeant ldquoVerocytotoxin-producing Escherichia coli (VTEC)rdquo Veterinary Microbiologyvol 140 no 3-4 pp 360ndash370 2010

[8] B Soborg S G Lassen L Muller et al ldquoA verocytotoxin-producing E coli outbreak with a surprisingly high risk ofhaemolytic uraemic syndrome Denmark September-October2012rdquo Euro Surveillance vol 18 no 2 2013

[9] T Dallman G P Smith B OrsquoBrien et al ldquoCharacterizationof a verocytotoxin-producing enteroaggregative Escherichia coliserogroupO111H21 strain associatedwith a household outbreakin Northern Irelandrdquo Journal of Clinical Microbiology vol 50no 12 pp 4116ndash4119 2012

[10] F Scheutz E M Nielsen J Frimodt-Moslashller et al ldquoCharacteris-tics of the enteroaggregative Shiga toxinverotoxin-producingEscherichia coli O104 H4 strain causing the outbreak ofhaemolytic uraemic syndrome in Germany May to June 2011rdquoEurosurveillance vol 16 no 24 2011

[11] R Stephan and L E Hoelzle ldquoCharacterization of shigatoxin type 2 variant B-subunit in Escherichia coli strains fromasymptomatic human carriers by PCR-RFLPrdquo Letters in AppliedMicrobiology vol 31 no 2 pp 139ndash142 2000

[12] K T Lim C S Teh and K L Thong ldquoLoop-mediated isother-mal amplification assay for the rapid detection of Staphylococcusaureusrdquo BioMed Research International vol 2013 Article ID895816 5 pages 2013

[13] N A Nasr H M Al-Mekhlafi A Ahmed M A Roslan andA Bulgiba ldquoTowards an effective control programme of soil-transmitted helminth infections among Orang Asli in ruralMalaysiamdashpart 2 knowledge attitude and practicesrdquo ParasitVectors vol 6 article 28 2013

[14] R Ngui Y A Lim S C Chow J A de Bruyne and C KLiam ldquoPrevalence of bronchial asthma among Orang Asli inPeninsular Malaysiardquo Medical Journal of Malaysia vol 66 no1 pp 27ndash31 2011

[15] A K Mahdy J Surin A Mohd-Adnan K L Wan andY A Lim ldquoMolecular characterization of Giardia duodenalisisolated from Semai Pahang Orang Asli (Peninsular Malaysiaaborigines)rdquo Parasitology vol 136 no 11 pp 1237ndash1241 2009

[16] A A Rahim Y Y Lee N A Majid et al ldquoHelicobacterpylori infection among Aborigines (the Orang Asli) in thenortheastern region of Peninsular Malaysiardquo The AmericanJournal of TropicalMedicine andHygiene vol 83 no 5 pp 1119ndash1122 2010

[17] Y Hara-Kudo J Nemoto K Ohtsuka et al ldquoSensitive andrapid detection of Vero toxin-producing Escherichia coli usingloop-mediated isothermal amplificationrdquo Journal of MedicalMicrobiology vol 56 no 3 pp 398ndash406 2007

[18] Y Shao X He E M Harrison et al ldquomGenomeSubtractor aweb-based tool for parallel in silico subtractive hybridizationanalysis of multiple bacterial genomesrdquo Nucleic Acids Researchvol 38 no 2 pp W194ndashW200 2010

6 The Scientific World Journal

[19] W S Ho L K Tan P T Ooi C C Yeo and K L ThongldquoPrevalence and characterization of verotoxigenic-Escherichiacoli isolates from pigs in Malaysiardquo BMC Veterinary Researchvol 9 article 109 2013

[20] A Yano R Ishimaru and R Hujikata ldquoRapid and sensitivedetection of heat-labile I and heat-stable I enterotoxin genes ofenterotoxigenic Escherichia coli by Loop-Mediated IsothermalAmplificationrdquo Journal of Microbiological Methods vol 68 no2 pp 414ndash420 2007

[21] J Hill S Beriwal I Chandra et al ldquoLoop-mediated isothermalamplification assay for rapid detection of common strains ofEscherichia colirdquo Journal of Clinical Microbiology vol 46 no 8pp 2800ndash2804 2008

[22] X Zhao Y Li L Wang et al ldquoDevelopment and applicationof a loop-mediated isothermal amplification method on rapiddetection Escherichia coli O157 strains from food samplesrdquoMolecular Biology Reports vol 37 no 5 pp 2183ndash2188 2010

[23] A T Maurelli R E Fernandez C A Bloch C K Rode andA Fasano ldquolsquoBlack holesrsquo and bacterial pathogenicity a largegenomic deletion that enhances the virulence of Shigella sppand enteroinvasive Escherichia colirdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 95 no7 pp 3943ndash3948 1998

[24] G M Pupo R Lan and P R Reeves ldquoMultiple independentorigins of Shigella clones of Escherichia coli and convergentevolution of many of their characteristicsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 97 no 19 pp 10567ndash10572 2000

[25] G E Sims and S H Kim ldquoWhole-genome phylogeny ofEscherichia coliShigella group by feature frequency profiles(FFPs)rdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 108 no 20 pp 8329ndash8334 2011

[26] M J van den Beld and F A Reubsaet ldquoDifferentiation betweenShigella enteroinvasive Escherichia coli (EIEC) and noninvasiveEscherichia colirdquo European Journal of Clinical Microbiology ampInfectious Diseases vol 31 no 6 pp 899ndash904 2012

[27] QHu J Tu XHan Y ZhuCDing and S Yu ldquoDevelopment ofmultiplex PCR assay for rapid detection of Riemerella anatipes-tifer Escherichia coli and Salmonella enterica simultaneouslyfrom ducksrdquo Journal of Microbiological Methods vol 87 no 1pp 64ndash69 2011

[28] K L Thong M Y Lai C S J Teh and K H ChualdquoSimultaneous detection of methicillin-resistant Staphylococcusaureus Acinetobacter baumannii Escherichia coli Klebsiellapneumoniae and Pseudomonas aeruginosa by multiplex PCRrdquoTropical Biomedicine vol 28 no 1 pp 21ndash31 2011

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International Journal of

Microbiology

2 The Scientific World Journal

the potential source for transmission ofVTEC hence promptdetection of carrier is important

Given that instantaneous detection of the infectiousagents is important hence molecular approaches have beenutilized as the detection is more rapid than conventionalbiochemical tests Compared to PCR loop-mediated isother-mal amplification (LAMP) produces more specific resultsas there are 4 primers targeting 6 regions within a geneBesides LAMP is alsomore rapid (reaction occurs at constanttemperature) and eliminates the need for electrophoresis asthe results can be monitored through real-time turbidimeteror visually based on turbidity or with the aid of SYBR I dye[12]

In Malaysia studies on sanitation-related diseases suchas parasitic infections are commonly carried out among theindigenous communities due to their poor living conditionsinappropriate hygiene practices and the lack of functioningtoilet facilities in their houses [13] Asymptomatic parasiticinfections among indigenous individuals have been reported[14 15] however the study on bacterial infections is relativelyscarce Previously Rahim et al [16] had conducted a study onthe prevalence of Helicobacter pylori (HP) infection amongthe indigenous individuals A total of 19 asymptomaticindigenous individuals were tested positive for the HP infec-tion The limited amount of information regarding bacterialinfections may have been contributed to the lack of rapidtechnique which has the potential to be applied in the fieldThis is crucial as many of the indigenous communities aresituated in remote areas which is logistically challenging ifsamples were to be processed within a few hours Develop-ment of cost-effective and rapidmodern tools which have thepotential to overcome these limitations will be crucial

In the present study we aim to develop a LAMP assaythat can specifically detect genericE coliThedeveloped assaywill be applied in combination with another VTEC-specificLAMP assay previously developed by Hara-Kudo et al [17] todetect the presence of E coli and VTEC among Orang Aslicommunity in Malaysia

2 Materials and Methods

21 Bacteria Strains Fifty E coli strains (5 verocytotoxin-producing and 45 nonverocytotoxin-producing E coli) and35 bacterial species other than E coli (including 10 Shigellaspp) were revived from the culture collections in the Lab-oratory of Biomedical Science and Molecular MicrobiologyInstitute of Graduate Studies University of Malaya A loopfulof colonies of overnight culture was suspended in 100 120583LddH2O and was subjected to boiling at 99∘C for 5min

The suspension was snapped cool and centrifuged Thesupernatant was quantified and 15 120583L (equivalent to sim50 ng)was used as the DNA template for LAMP assay and PCR

22 LAMP Assay mGenomeSubtractor [18] and web-basedArtemis Comparison Tool (WebACT) (httpwwwwebactorgWebACT) were used to compare the publishedgenomes of E coli and other Enterobacteriaceae such asShigella spp Salmonella Yersinia spp and Klebsiella spp

A list of conserved regions for E coli was generated and wasagain blasted against the database by using NCBI BLAST(httpblastncbinlmnihgovBlastcgi) A CDS encodingglycerate kinase (EcolC 3109 Accession number CP000946)was selected and used for primers design Two pairs ofprimers including F3 (51015840-GGTAGATCGAACGGTCATCG-31015840) B3 (51015840-GGCCAGCAACGGATTACG-31015840) forward innerprimer (FIP) (51015840-CGCAGACTTCAAGCGTCACGA-TCGAAGGAACGGTGGATGC-31015840) and backward innerprimer (BIP) (51015840-CCTTACCGGCGACGGGAAAA-CTTTTCAGGCGCGACCAG-31015840) as well as a loop primer(LB) (51015840-TGAGATGGCGGCAGCAAGTG-31015840) weredesigned by using online PrimerExplorer V4 program(PrimerExplorer Eiken Chemical Co Ltd)

The LAMP assay was optimized on E coli strains usingLoopamp DNA amplification kit (Eiken Chemical Co LtdTokyo Japan) Briefly the reaction was carried out in a totalof 25 120583L containing 40 pmol of each FIP and BIP primers5 pmol of each F3 and B3 primers 10 pmol of LB primer125 120583L of 2X reaction mixture 1 120583L of Bst DNA polymeraseand 15 120583L of DNA (approximately 100 ng) The reactionmixtures were incubated at 62∘C for 80min followed byenzyme inactivation at 80∘C for 2min in the Loopamp real-time turbidimeter (LA-320 TeramecsCo Ltd Kyoto Japan)The positive result was indicated when the turbidity reached01 within 60min at 65 nM and Tt was recorded

23 PCR Validation PCR was performed in parallel tovalidate the results produced by LAMP assay Briefly PCRtargeting phoA gene forE coli detection andVT1 orVT2 genefor verocytotoxin producer was carried out as described byHo et al [19]

24 Detection Limit of LAMP Assay on Bacterial Cultureand Spiked Faeces Overnight cultures of E coli were seriallydiluted 10-fold An aliquot of 100 120583L of each dilutionwas usedfor the DNA template preparation and plated on lysogenyagar for CFU count On the other hand the overnight culturewas also spiked in 900 120583L of stools (diluted with brain-heartinfusion broth to avoid inhibition during PCR) followed by a10-fold serial dilution An aliquot of 100120583L of the dilutionwasplated on the agar for viable count while another 100 120583L wasused for the extraction of DNA by using crude lysate methodThe DNA templates were subjected to LAMP assay

25 Field Evaluation of LAMPAssay for the Detection of VTECamong Indigenous Individuals A total of 50 indigenousindividualswith positive parasitic infectionswere recruited inthe study (Ethics committeeIRB reference number 82411)Sample collection procedure has also been approved by theDepartment of Orang Asli Development (JAKOA) Consentwas given by the participating indigenous individuals eithervia signature or via thumbprint prior to sample collectionFaecal samples were subjected to DNA extraction usingQIAamp DNA Stool Mini Kit (QIAGEN The Netherlands)The extracted total DNA was quantified and stored at minus20∘Cuntil further use

The Scientific World Journal 3

minus01

0

01

02

03

04

05

06

07

0 1000 2000 3000 4000 5000

BC 106

SF 106

BC 105

SF 105

BC 104

SF 104

BC 103

BC 102

SF 103

SF 102

Figure 1 Sensitivity of LAMP assay of bacterial culture (BC) andspiked faecal (SF) specimens on different CFU (from 106 to 102)

The LAMP assay developed in this study was used todetect generic strains of E coli that are present in the faecalsample while VT1 and VT2 producer were determined byusing LAMP assay that was previously described by Hara-Kudo et al [17] The reaction consisted of approximately200ndash400 ng total DNA incubated at 62∘C for 45min andthe positive reactions were detected based on the colourchange after the addition of a SYBR green 1 10 dilution to thereaction tube

3 Results

All the 50 E coli strains showed positive reaction within25min with an average Tt value of 2126 None of the non-Ecoli strains showed positive amplification This indicates thehigh specificity of the LAMP assay for E coli detection Fur-thermore the detection limit of the LAMP assay was as lowas 102 CFUmL and 103 CFUmL on bacterial culture (Tt =3330) and the spiked faeces (Tt = 3112) respectively Forbacterial culture the reaction with more than 105 CFUmLof cells yielded positive results within 18min while forfaecal samples only those reactions which have more than106 CFUmL of cells showed positive amplification before18min (Figure 1)

The usefulness of the newly developed E coli LAMP assaywas evaluated using the faecal samples collected from OrangAsli Overall the results obtained by using LAMP assay werein concordance with PCR except for 1 sample (Table 1) Toverify the result of LAMP assay the amplicon of F3 and B3 ofthis particular sample was sent for sequencingThe ampliconwas 100 matched with the E coli glycerate kinase genesegment

On the other hand all E coli positive samples were alsosubjected to VT1 and VT2 detection as described in Ho et al[19] Among the 46 E coli positive samples VT1 was detected

in 6 volunteers (ge15 years old) while VT2 was detected in oneadultrsquos sample Only one sample obtained from a 5-year-oldgirl was detected with VT1 and VT2 producing E coli

4 Discussion

In this study a LAMP assay targeting glycerate kinase of Ecoli was developedThe assay demonstrated a high specificityand was fairly sensitive when applied on bacterial culture(102) and spiked faecal sample (103) None of the Shigellaspp showed positive reaction and this indicated that thedeveloped LAMP assay could specifically differentiate Ecoli from Shigella Therefore this assay could be potentiallyapplied on food and animal samples

LAMP assays targeting different strainspathotypes of Ecoli have been described previously [17 20ndash22] Among thepublished studies Hill et al [21] had developed a LAMPassay targeting common strains of E coli and this assay wasevaluated using urine samples malB gene was used for theprimer design however this gene was also found in ShigellaThe developed assay worked well in the identification ofurinary E coli (as Shigella is rare in extraintestinal infection)but will be restricted to other applications such as detectionof common E coli strains from faecal food soil and watersamples

E coli and Shigella are closely related The validity ofShigella genus has become a contentious issue over the pastdecades Based on the conventional view Shigella and E colishared greater than 90 homology by DNA-DNA reassoci-ation analysis and Shigella are E coli strains that undergothe convergent evolution of Shigella phenotypic propertiesthat contribute to the Shigella pathotype [23ndash25] Howeverin terms of clinical manifestation Shigella spp are frankpathogens that readily cause disease in humans while E colinormally lives in the intestines of humans and animals (withthe exception of pathogenic clones) Prompt and reliableidentification of these infectious agents is important for theright choice of treatment and to avoid any complications

Differentiation between E coli and Shigella is difficultbut achievable based on physiological biochemical andserological typing [26] For a more rapid method alkalinephosphatase (phoA) gene has been used in PCRs for commonE coli strains detection demonstrating high specificity [2728]Therefore this PCRwas selected as a tool for validation ofthe results obtained by using LAMP in this study In fact theLAMP assay showed higher specificity in detecting genericE coli in the indigenous samples compared to PCR targetingphoA gene where the PCR could not detect E coli in one ofthe samples (PG31)

The rate of verocytotoxin-producing E coli in asymp-tomatic human carrier in this study was considered high(16) compared to previous studies that reported the rateof 76 and 4 conducted by Hong et al [6] and Stephanand Hoelzle [11] respectively In the studies of Hong et al [6]and Stephan andHoelzle [11] workers in slaughterhouse weretargeted as they were considered at higher risk for carriageor excretion of VTEC Thus the finding in this study hashighlighted the attention on hygiene practices and living

4 The Scientific World Journal

Table 1 PCR and LAMP results for the samples obtained from indigenous individuals which were previously detected with different parasiticinfections

ID AGE Parasitic infection PCR LAMP (E coli)Generic E coli VT producer

PG 122 8 Giardia + +PG 201 12 Giardia Entamoeba coli minus minus

PG 31 10 Giardia minus +PB 111 73 Giardia + +PB 145 5 Giardia + + VT1VT2PM 192 7 Giardia + +PM 252 7 Giardia Entamoeba coli + +PM 312 8 Giardia + +PM 531 10 Giardia minus minus

PM 532 9 Giardia + +PM 541 10 Giardia + +SP 111 6 Giardia + +SP 73 1 Giardia + +DB 117 15 Giardia + + VT1DB 21 30 Giardia Trichuris Ascaris Entamoeba coli Iodamoeba + +BP 15 4 Giardia Trichuris + +BP 142 10 Giardia Trichuris Ascaris + +SB 33 11 Giardia + + VT1DK 104 7 Giardia Trichuris + +DK 173 6 Giardia TrichurisHookworm Entamoeba coli + +DK 176 13 Giardia Trichuris Entamoeba coli + +DK 1910 8 Giardia Trichuris Ascaris + +DK 271 10 Giardia Trichuris + +DK 291 7 Giardia Trichuris + +UK 152 32 Giardia + +UK 155 4 Giardia Trichuris Ascaris + +UK 173 7 Giardia Ascaris + +UK 62 45 Giardia Ascaris + +UK 65 13 Giardia Ascaris + + VT1UL 132 5 Giardia Trichuris Ascaris + + VT1UL 33 12 Giardia Trichuris AscarisHookworm minus minus

UL 44 1 Giardia + +UK 153 9 Giardia Ascaris + +UK 171 32 Giardia Ascaris minus minus

UK 63 18 Giardia + +PP 11 58 Giardia Trichuris Ascaris + +PS 73 11 Giardia + +NT 44 13 Giardia Trichuris Ascaris + +NT 62 30 Giardia Ascaris + + VT2NT 63 8 Giardia Ascaris Entamoeba coli + +PG 27 17 GiardiaHookworm + +PG 262 43 Giardia + +JJ 72 36 Giardia + +JJ 114 7 Giardia + + VT1JJ 115 5 Giardia + + VT1SW 53 10 Giardia + +SW 101 31 Giardia + +SW 112 1 Giardia + +SW 143 4 Giardia E coli + +SW 163 32 GiardiaHookworm + +

The Scientific World Journal 5

conditions of the indigenous populations in Malaysia giventhe fact that these communities are afflicted with parasiticinfections not only as reported previously [14 15] but alsoas a reservoir for VTEC

In this study all the volunteers were infected by differentparasites such as Giardia Trichuris Ascaris HookwormEntamoeba coli and Iodamoeba There is no statistical evi-dence that showed correlation between parasitic infectionand VTEC colonization Our results also did not suggest anygroups (single or multiple parasitic infections) that are proneto VTEC colonization Interestingly we found that E coliwas hardly detected in the stool samples containing multipleparasites as in the first run of LAMP assay (sample PM 252DB 21 BP 15 BP 142 DK 173 DK 176 DK 1910 DK 271UK 155 UL 132 NT 62 NT 63 and SW 163) Reasonsthat influenced the sensitivity of this LAMP assay will befurther investigated Meanwhile the 4 samples which werenot detected with E coli might be due to recent antibiotictreatments

5 Conclusion

We have successfully developed a LAMP assay that canspecifically detect generic E coli and further performed incombination with the LAMP assay that detect verocytotoxin-related gene Our study suggests the high risk of indigenouscommunity for carriage and excretion of VTEC Due to thehigh specificity and sensitivity of the generic E coli LAMPassay developed in this study we proposed the application ofthis assay in food and animals as an alternative approach forrapid and more specific detection of E coli

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank University of Malaya for support andfacilities This study was funded by Higher Impact ResearchGrant (UMC6251HIRMOHE02) andGrant GA 013-2013(Molecular Diagnostics of Bacterial Pathogens)

References

[1] R E Ley D A Peterson and J I Gordon ldquoEcological andevolutionary forces shaping microbial diversity in the humanintestinerdquo Cell vol 124 no 4 pp 837ndash848 2006

[2] J C Clemente L K Ursell L W Parfrey and R Knight ldquoTheimpact of the gut microbiota on human health an integrativeviewrdquo Cell vol 148 no 6 pp 1258ndash1270 2012

[3] R Burcelin E Luche M Serino and J Amar ldquoThe gutmicrobiota ecology a new opportunity for the treatment ofmetabolic diseasesrdquo Frontiers in Bioscience vol 14 no 13 pp5107ndash5117 2009

[4] H Tlaskalova-Hogenova R Stpankova H Kozakova et alldquoThe role of gut microbiota (commensal bacteria) and themucosal barrier in the pathogenesis of inflammatory and

autoimmune diseases and cancer contribution of germ-freeand gnotobiotic animalmodels of human diseasesrdquoCellular andMolecular Immunology vol 8 no 2 pp 110ndash120 2011

[5] O Tenaillon D Skurnik B Picard and E Denamur ldquoThepopulation genetics of commensal Escherichia colirdquo NatureReviews Microbiology vol 8 no 3 pp 207ndash217 2010

[6] S Hong K H Oh S H Cho et al ldquoAsymptomatic healthyslaughterhouse workers in South Korea carrying Shiga toxin-producing Escherichia colirdquo FEMS Immunology and MedicalMicrobiology vol 56 no 1 pp 41ndash47 2009

[7] M A Karmali V Gannon and J M Sargeant ldquoVerocytotoxin-producing Escherichia coli (VTEC)rdquo Veterinary Microbiologyvol 140 no 3-4 pp 360ndash370 2010

[8] B Soborg S G Lassen L Muller et al ldquoA verocytotoxin-producing E coli outbreak with a surprisingly high risk ofhaemolytic uraemic syndrome Denmark September-October2012rdquo Euro Surveillance vol 18 no 2 2013

[9] T Dallman G P Smith B OrsquoBrien et al ldquoCharacterizationof a verocytotoxin-producing enteroaggregative Escherichia coliserogroupO111H21 strain associatedwith a household outbreakin Northern Irelandrdquo Journal of Clinical Microbiology vol 50no 12 pp 4116ndash4119 2012

[10] F Scheutz E M Nielsen J Frimodt-Moslashller et al ldquoCharacteris-tics of the enteroaggregative Shiga toxinverotoxin-producingEscherichia coli O104 H4 strain causing the outbreak ofhaemolytic uraemic syndrome in Germany May to June 2011rdquoEurosurveillance vol 16 no 24 2011

[11] R Stephan and L E Hoelzle ldquoCharacterization of shigatoxin type 2 variant B-subunit in Escherichia coli strains fromasymptomatic human carriers by PCR-RFLPrdquo Letters in AppliedMicrobiology vol 31 no 2 pp 139ndash142 2000

[12] K T Lim C S Teh and K L Thong ldquoLoop-mediated isother-mal amplification assay for the rapid detection of Staphylococcusaureusrdquo BioMed Research International vol 2013 Article ID895816 5 pages 2013

[13] N A Nasr H M Al-Mekhlafi A Ahmed M A Roslan andA Bulgiba ldquoTowards an effective control programme of soil-transmitted helminth infections among Orang Asli in ruralMalaysiamdashpart 2 knowledge attitude and practicesrdquo ParasitVectors vol 6 article 28 2013

[14] R Ngui Y A Lim S C Chow J A de Bruyne and C KLiam ldquoPrevalence of bronchial asthma among Orang Asli inPeninsular Malaysiardquo Medical Journal of Malaysia vol 66 no1 pp 27ndash31 2011

[15] A K Mahdy J Surin A Mohd-Adnan K L Wan andY A Lim ldquoMolecular characterization of Giardia duodenalisisolated from Semai Pahang Orang Asli (Peninsular Malaysiaaborigines)rdquo Parasitology vol 136 no 11 pp 1237ndash1241 2009

[16] A A Rahim Y Y Lee N A Majid et al ldquoHelicobacterpylori infection among Aborigines (the Orang Asli) in thenortheastern region of Peninsular Malaysiardquo The AmericanJournal of TropicalMedicine andHygiene vol 83 no 5 pp 1119ndash1122 2010

[17] Y Hara-Kudo J Nemoto K Ohtsuka et al ldquoSensitive andrapid detection of Vero toxin-producing Escherichia coli usingloop-mediated isothermal amplificationrdquo Journal of MedicalMicrobiology vol 56 no 3 pp 398ndash406 2007

[18] Y Shao X He E M Harrison et al ldquomGenomeSubtractor aweb-based tool for parallel in silico subtractive hybridizationanalysis of multiple bacterial genomesrdquo Nucleic Acids Researchvol 38 no 2 pp W194ndashW200 2010

6 The Scientific World Journal

[19] W S Ho L K Tan P T Ooi C C Yeo and K L ThongldquoPrevalence and characterization of verotoxigenic-Escherichiacoli isolates from pigs in Malaysiardquo BMC Veterinary Researchvol 9 article 109 2013

[20] A Yano R Ishimaru and R Hujikata ldquoRapid and sensitivedetection of heat-labile I and heat-stable I enterotoxin genes ofenterotoxigenic Escherichia coli by Loop-Mediated IsothermalAmplificationrdquo Journal of Microbiological Methods vol 68 no2 pp 414ndash420 2007

[21] J Hill S Beriwal I Chandra et al ldquoLoop-mediated isothermalamplification assay for rapid detection of common strains ofEscherichia colirdquo Journal of Clinical Microbiology vol 46 no 8pp 2800ndash2804 2008

[22] X Zhao Y Li L Wang et al ldquoDevelopment and applicationof a loop-mediated isothermal amplification method on rapiddetection Escherichia coli O157 strains from food samplesrdquoMolecular Biology Reports vol 37 no 5 pp 2183ndash2188 2010

[23] A T Maurelli R E Fernandez C A Bloch C K Rode andA Fasano ldquolsquoBlack holesrsquo and bacterial pathogenicity a largegenomic deletion that enhances the virulence of Shigella sppand enteroinvasive Escherichia colirdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 95 no7 pp 3943ndash3948 1998

[24] G M Pupo R Lan and P R Reeves ldquoMultiple independentorigins of Shigella clones of Escherichia coli and convergentevolution of many of their characteristicsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 97 no 19 pp 10567ndash10572 2000

[25] G E Sims and S H Kim ldquoWhole-genome phylogeny ofEscherichia coliShigella group by feature frequency profiles(FFPs)rdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 108 no 20 pp 8329ndash8334 2011

[26] M J van den Beld and F A Reubsaet ldquoDifferentiation betweenShigella enteroinvasive Escherichia coli (EIEC) and noninvasiveEscherichia colirdquo European Journal of Clinical Microbiology ampInfectious Diseases vol 31 no 6 pp 899ndash904 2012

[27] QHu J Tu XHan Y ZhuCDing and S Yu ldquoDevelopment ofmultiplex PCR assay for rapid detection of Riemerella anatipes-tifer Escherichia coli and Salmonella enterica simultaneouslyfrom ducksrdquo Journal of Microbiological Methods vol 87 no 1pp 64ndash69 2011

[28] K L Thong M Y Lai C S J Teh and K H ChualdquoSimultaneous detection of methicillin-resistant Staphylococcusaureus Acinetobacter baumannii Escherichia coli Klebsiellapneumoniae and Pseudomonas aeruginosa by multiplex PCRrdquoTropical Biomedicine vol 28 no 1 pp 21ndash31 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World Journal 3

minus01

0

01

02

03

04

05

06

07

0 1000 2000 3000 4000 5000

BC 106

SF 106

BC 105

SF 105

BC 104

SF 104

BC 103

BC 102

SF 103

SF 102

Figure 1 Sensitivity of LAMP assay of bacterial culture (BC) andspiked faecal (SF) specimens on different CFU (from 106 to 102)

The LAMP assay developed in this study was used todetect generic strains of E coli that are present in the faecalsample while VT1 and VT2 producer were determined byusing LAMP assay that was previously described by Hara-Kudo et al [17] The reaction consisted of approximately200ndash400 ng total DNA incubated at 62∘C for 45min andthe positive reactions were detected based on the colourchange after the addition of a SYBR green 1 10 dilution to thereaction tube

3 Results

All the 50 E coli strains showed positive reaction within25min with an average Tt value of 2126 None of the non-Ecoli strains showed positive amplification This indicates thehigh specificity of the LAMP assay for E coli detection Fur-thermore the detection limit of the LAMP assay was as lowas 102 CFUmL and 103 CFUmL on bacterial culture (Tt =3330) and the spiked faeces (Tt = 3112) respectively Forbacterial culture the reaction with more than 105 CFUmLof cells yielded positive results within 18min while forfaecal samples only those reactions which have more than106 CFUmL of cells showed positive amplification before18min (Figure 1)

The usefulness of the newly developed E coli LAMP assaywas evaluated using the faecal samples collected from OrangAsli Overall the results obtained by using LAMP assay werein concordance with PCR except for 1 sample (Table 1) Toverify the result of LAMP assay the amplicon of F3 and B3 ofthis particular sample was sent for sequencingThe ampliconwas 100 matched with the E coli glycerate kinase genesegment

On the other hand all E coli positive samples were alsosubjected to VT1 and VT2 detection as described in Ho et al[19] Among the 46 E coli positive samples VT1 was detected

in 6 volunteers (ge15 years old) while VT2 was detected in oneadultrsquos sample Only one sample obtained from a 5-year-oldgirl was detected with VT1 and VT2 producing E coli

4 Discussion

In this study a LAMP assay targeting glycerate kinase of Ecoli was developedThe assay demonstrated a high specificityand was fairly sensitive when applied on bacterial culture(102) and spiked faecal sample (103) None of the Shigellaspp showed positive reaction and this indicated that thedeveloped LAMP assay could specifically differentiate Ecoli from Shigella Therefore this assay could be potentiallyapplied on food and animal samples

LAMP assays targeting different strainspathotypes of Ecoli have been described previously [17 20ndash22] Among thepublished studies Hill et al [21] had developed a LAMPassay targeting common strains of E coli and this assay wasevaluated using urine samples malB gene was used for theprimer design however this gene was also found in ShigellaThe developed assay worked well in the identification ofurinary E coli (as Shigella is rare in extraintestinal infection)but will be restricted to other applications such as detectionof common E coli strains from faecal food soil and watersamples

E coli and Shigella are closely related The validity ofShigella genus has become a contentious issue over the pastdecades Based on the conventional view Shigella and E colishared greater than 90 homology by DNA-DNA reassoci-ation analysis and Shigella are E coli strains that undergothe convergent evolution of Shigella phenotypic propertiesthat contribute to the Shigella pathotype [23ndash25] Howeverin terms of clinical manifestation Shigella spp are frankpathogens that readily cause disease in humans while E colinormally lives in the intestines of humans and animals (withthe exception of pathogenic clones) Prompt and reliableidentification of these infectious agents is important for theright choice of treatment and to avoid any complications

Differentiation between E coli and Shigella is difficultbut achievable based on physiological biochemical andserological typing [26] For a more rapid method alkalinephosphatase (phoA) gene has been used in PCRs for commonE coli strains detection demonstrating high specificity [2728]Therefore this PCRwas selected as a tool for validation ofthe results obtained by using LAMP in this study In fact theLAMP assay showed higher specificity in detecting genericE coli in the indigenous samples compared to PCR targetingphoA gene where the PCR could not detect E coli in one ofthe samples (PG31)

The rate of verocytotoxin-producing E coli in asymp-tomatic human carrier in this study was considered high(16) compared to previous studies that reported the rateof 76 and 4 conducted by Hong et al [6] and Stephanand Hoelzle [11] respectively In the studies of Hong et al [6]and Stephan andHoelzle [11] workers in slaughterhouse weretargeted as they were considered at higher risk for carriageor excretion of VTEC Thus the finding in this study hashighlighted the attention on hygiene practices and living

4 The Scientific World Journal

Table 1 PCR and LAMP results for the samples obtained from indigenous individuals which were previously detected with different parasiticinfections

ID AGE Parasitic infection PCR LAMP (E coli)Generic E coli VT producer

PG 122 8 Giardia + +PG 201 12 Giardia Entamoeba coli minus minus

PG 31 10 Giardia minus +PB 111 73 Giardia + +PB 145 5 Giardia + + VT1VT2PM 192 7 Giardia + +PM 252 7 Giardia Entamoeba coli + +PM 312 8 Giardia + +PM 531 10 Giardia minus minus

PM 532 9 Giardia + +PM 541 10 Giardia + +SP 111 6 Giardia + +SP 73 1 Giardia + +DB 117 15 Giardia + + VT1DB 21 30 Giardia Trichuris Ascaris Entamoeba coli Iodamoeba + +BP 15 4 Giardia Trichuris + +BP 142 10 Giardia Trichuris Ascaris + +SB 33 11 Giardia + + VT1DK 104 7 Giardia Trichuris + +DK 173 6 Giardia TrichurisHookworm Entamoeba coli + +DK 176 13 Giardia Trichuris Entamoeba coli + +DK 1910 8 Giardia Trichuris Ascaris + +DK 271 10 Giardia Trichuris + +DK 291 7 Giardia Trichuris + +UK 152 32 Giardia + +UK 155 4 Giardia Trichuris Ascaris + +UK 173 7 Giardia Ascaris + +UK 62 45 Giardia Ascaris + +UK 65 13 Giardia Ascaris + + VT1UL 132 5 Giardia Trichuris Ascaris + + VT1UL 33 12 Giardia Trichuris AscarisHookworm minus minus

UL 44 1 Giardia + +UK 153 9 Giardia Ascaris + +UK 171 32 Giardia Ascaris minus minus

UK 63 18 Giardia + +PP 11 58 Giardia Trichuris Ascaris + +PS 73 11 Giardia + +NT 44 13 Giardia Trichuris Ascaris + +NT 62 30 Giardia Ascaris + + VT2NT 63 8 Giardia Ascaris Entamoeba coli + +PG 27 17 GiardiaHookworm + +PG 262 43 Giardia + +JJ 72 36 Giardia + +JJ 114 7 Giardia + + VT1JJ 115 5 Giardia + + VT1SW 53 10 Giardia + +SW 101 31 Giardia + +SW 112 1 Giardia + +SW 143 4 Giardia E coli + +SW 163 32 GiardiaHookworm + +

The Scientific World Journal 5

conditions of the indigenous populations in Malaysia giventhe fact that these communities are afflicted with parasiticinfections not only as reported previously [14 15] but alsoas a reservoir for VTEC

In this study all the volunteers were infected by differentparasites such as Giardia Trichuris Ascaris HookwormEntamoeba coli and Iodamoeba There is no statistical evi-dence that showed correlation between parasitic infectionand VTEC colonization Our results also did not suggest anygroups (single or multiple parasitic infections) that are proneto VTEC colonization Interestingly we found that E coliwas hardly detected in the stool samples containing multipleparasites as in the first run of LAMP assay (sample PM 252DB 21 BP 15 BP 142 DK 173 DK 176 DK 1910 DK 271UK 155 UL 132 NT 62 NT 63 and SW 163) Reasonsthat influenced the sensitivity of this LAMP assay will befurther investigated Meanwhile the 4 samples which werenot detected with E coli might be due to recent antibiotictreatments

5 Conclusion

We have successfully developed a LAMP assay that canspecifically detect generic E coli and further performed incombination with the LAMP assay that detect verocytotoxin-related gene Our study suggests the high risk of indigenouscommunity for carriage and excretion of VTEC Due to thehigh specificity and sensitivity of the generic E coli LAMPassay developed in this study we proposed the application ofthis assay in food and animals as an alternative approach forrapid and more specific detection of E coli

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank University of Malaya for support andfacilities This study was funded by Higher Impact ResearchGrant (UMC6251HIRMOHE02) andGrant GA 013-2013(Molecular Diagnostics of Bacterial Pathogens)

References

[1] R E Ley D A Peterson and J I Gordon ldquoEcological andevolutionary forces shaping microbial diversity in the humanintestinerdquo Cell vol 124 no 4 pp 837ndash848 2006

[2] J C Clemente L K Ursell L W Parfrey and R Knight ldquoTheimpact of the gut microbiota on human health an integrativeviewrdquo Cell vol 148 no 6 pp 1258ndash1270 2012

[3] R Burcelin E Luche M Serino and J Amar ldquoThe gutmicrobiota ecology a new opportunity for the treatment ofmetabolic diseasesrdquo Frontiers in Bioscience vol 14 no 13 pp5107ndash5117 2009

[4] H Tlaskalova-Hogenova R Stpankova H Kozakova et alldquoThe role of gut microbiota (commensal bacteria) and themucosal barrier in the pathogenesis of inflammatory and

autoimmune diseases and cancer contribution of germ-freeand gnotobiotic animalmodels of human diseasesrdquoCellular andMolecular Immunology vol 8 no 2 pp 110ndash120 2011

[5] O Tenaillon D Skurnik B Picard and E Denamur ldquoThepopulation genetics of commensal Escherichia colirdquo NatureReviews Microbiology vol 8 no 3 pp 207ndash217 2010

[6] S Hong K H Oh S H Cho et al ldquoAsymptomatic healthyslaughterhouse workers in South Korea carrying Shiga toxin-producing Escherichia colirdquo FEMS Immunology and MedicalMicrobiology vol 56 no 1 pp 41ndash47 2009

[7] M A Karmali V Gannon and J M Sargeant ldquoVerocytotoxin-producing Escherichia coli (VTEC)rdquo Veterinary Microbiologyvol 140 no 3-4 pp 360ndash370 2010

[8] B Soborg S G Lassen L Muller et al ldquoA verocytotoxin-producing E coli outbreak with a surprisingly high risk ofhaemolytic uraemic syndrome Denmark September-October2012rdquo Euro Surveillance vol 18 no 2 2013

[9] T Dallman G P Smith B OrsquoBrien et al ldquoCharacterizationof a verocytotoxin-producing enteroaggregative Escherichia coliserogroupO111H21 strain associatedwith a household outbreakin Northern Irelandrdquo Journal of Clinical Microbiology vol 50no 12 pp 4116ndash4119 2012

[10] F Scheutz E M Nielsen J Frimodt-Moslashller et al ldquoCharacteris-tics of the enteroaggregative Shiga toxinverotoxin-producingEscherichia coli O104 H4 strain causing the outbreak ofhaemolytic uraemic syndrome in Germany May to June 2011rdquoEurosurveillance vol 16 no 24 2011

[11] R Stephan and L E Hoelzle ldquoCharacterization of shigatoxin type 2 variant B-subunit in Escherichia coli strains fromasymptomatic human carriers by PCR-RFLPrdquo Letters in AppliedMicrobiology vol 31 no 2 pp 139ndash142 2000

[12] K T Lim C S Teh and K L Thong ldquoLoop-mediated isother-mal amplification assay for the rapid detection of Staphylococcusaureusrdquo BioMed Research International vol 2013 Article ID895816 5 pages 2013

[13] N A Nasr H M Al-Mekhlafi A Ahmed M A Roslan andA Bulgiba ldquoTowards an effective control programme of soil-transmitted helminth infections among Orang Asli in ruralMalaysiamdashpart 2 knowledge attitude and practicesrdquo ParasitVectors vol 6 article 28 2013

[14] R Ngui Y A Lim S C Chow J A de Bruyne and C KLiam ldquoPrevalence of bronchial asthma among Orang Asli inPeninsular Malaysiardquo Medical Journal of Malaysia vol 66 no1 pp 27ndash31 2011

[15] A K Mahdy J Surin A Mohd-Adnan K L Wan andY A Lim ldquoMolecular characterization of Giardia duodenalisisolated from Semai Pahang Orang Asli (Peninsular Malaysiaaborigines)rdquo Parasitology vol 136 no 11 pp 1237ndash1241 2009

[16] A A Rahim Y Y Lee N A Majid et al ldquoHelicobacterpylori infection among Aborigines (the Orang Asli) in thenortheastern region of Peninsular Malaysiardquo The AmericanJournal of TropicalMedicine andHygiene vol 83 no 5 pp 1119ndash1122 2010

[17] Y Hara-Kudo J Nemoto K Ohtsuka et al ldquoSensitive andrapid detection of Vero toxin-producing Escherichia coli usingloop-mediated isothermal amplificationrdquo Journal of MedicalMicrobiology vol 56 no 3 pp 398ndash406 2007

[18] Y Shao X He E M Harrison et al ldquomGenomeSubtractor aweb-based tool for parallel in silico subtractive hybridizationanalysis of multiple bacterial genomesrdquo Nucleic Acids Researchvol 38 no 2 pp W194ndashW200 2010

6 The Scientific World Journal

[19] W S Ho L K Tan P T Ooi C C Yeo and K L ThongldquoPrevalence and characterization of verotoxigenic-Escherichiacoli isolates from pigs in Malaysiardquo BMC Veterinary Researchvol 9 article 109 2013

[20] A Yano R Ishimaru and R Hujikata ldquoRapid and sensitivedetection of heat-labile I and heat-stable I enterotoxin genes ofenterotoxigenic Escherichia coli by Loop-Mediated IsothermalAmplificationrdquo Journal of Microbiological Methods vol 68 no2 pp 414ndash420 2007

[21] J Hill S Beriwal I Chandra et al ldquoLoop-mediated isothermalamplification assay for rapid detection of common strains ofEscherichia colirdquo Journal of Clinical Microbiology vol 46 no 8pp 2800ndash2804 2008

[22] X Zhao Y Li L Wang et al ldquoDevelopment and applicationof a loop-mediated isothermal amplification method on rapiddetection Escherichia coli O157 strains from food samplesrdquoMolecular Biology Reports vol 37 no 5 pp 2183ndash2188 2010

[23] A T Maurelli R E Fernandez C A Bloch C K Rode andA Fasano ldquolsquoBlack holesrsquo and bacterial pathogenicity a largegenomic deletion that enhances the virulence of Shigella sppand enteroinvasive Escherichia colirdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 95 no7 pp 3943ndash3948 1998

[24] G M Pupo R Lan and P R Reeves ldquoMultiple independentorigins of Shigella clones of Escherichia coli and convergentevolution of many of their characteristicsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 97 no 19 pp 10567ndash10572 2000

[25] G E Sims and S H Kim ldquoWhole-genome phylogeny ofEscherichia coliShigella group by feature frequency profiles(FFPs)rdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 108 no 20 pp 8329ndash8334 2011

[26] M J van den Beld and F A Reubsaet ldquoDifferentiation betweenShigella enteroinvasive Escherichia coli (EIEC) and noninvasiveEscherichia colirdquo European Journal of Clinical Microbiology ampInfectious Diseases vol 31 no 6 pp 899ndash904 2012

[27] QHu J Tu XHan Y ZhuCDing and S Yu ldquoDevelopment ofmultiplex PCR assay for rapid detection of Riemerella anatipes-tifer Escherichia coli and Salmonella enterica simultaneouslyfrom ducksrdquo Journal of Microbiological Methods vol 87 no 1pp 64ndash69 2011

[28] K L Thong M Y Lai C S J Teh and K H ChualdquoSimultaneous detection of methicillin-resistant Staphylococcusaureus Acinetobacter baumannii Escherichia coli Klebsiellapneumoniae and Pseudomonas aeruginosa by multiplex PCRrdquoTropical Biomedicine vol 28 no 1 pp 21ndash31 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 The Scientific World Journal

Table 1 PCR and LAMP results for the samples obtained from indigenous individuals which were previously detected with different parasiticinfections

ID AGE Parasitic infection PCR LAMP (E coli)Generic E coli VT producer

PG 122 8 Giardia + +PG 201 12 Giardia Entamoeba coli minus minus

PG 31 10 Giardia minus +PB 111 73 Giardia + +PB 145 5 Giardia + + VT1VT2PM 192 7 Giardia + +PM 252 7 Giardia Entamoeba coli + +PM 312 8 Giardia + +PM 531 10 Giardia minus minus

PM 532 9 Giardia + +PM 541 10 Giardia + +SP 111 6 Giardia + +SP 73 1 Giardia + +DB 117 15 Giardia + + VT1DB 21 30 Giardia Trichuris Ascaris Entamoeba coli Iodamoeba + +BP 15 4 Giardia Trichuris + +BP 142 10 Giardia Trichuris Ascaris + +SB 33 11 Giardia + + VT1DK 104 7 Giardia Trichuris + +DK 173 6 Giardia TrichurisHookworm Entamoeba coli + +DK 176 13 Giardia Trichuris Entamoeba coli + +DK 1910 8 Giardia Trichuris Ascaris + +DK 271 10 Giardia Trichuris + +DK 291 7 Giardia Trichuris + +UK 152 32 Giardia + +UK 155 4 Giardia Trichuris Ascaris + +UK 173 7 Giardia Ascaris + +UK 62 45 Giardia Ascaris + +UK 65 13 Giardia Ascaris + + VT1UL 132 5 Giardia Trichuris Ascaris + + VT1UL 33 12 Giardia Trichuris AscarisHookworm minus minus

UL 44 1 Giardia + +UK 153 9 Giardia Ascaris + +UK 171 32 Giardia Ascaris minus minus

UK 63 18 Giardia + +PP 11 58 Giardia Trichuris Ascaris + +PS 73 11 Giardia + +NT 44 13 Giardia Trichuris Ascaris + +NT 62 30 Giardia Ascaris + + VT2NT 63 8 Giardia Ascaris Entamoeba coli + +PG 27 17 GiardiaHookworm + +PG 262 43 Giardia + +JJ 72 36 Giardia + +JJ 114 7 Giardia + + VT1JJ 115 5 Giardia + + VT1SW 53 10 Giardia + +SW 101 31 Giardia + +SW 112 1 Giardia + +SW 143 4 Giardia E coli + +SW 163 32 GiardiaHookworm + +

The Scientific World Journal 5

conditions of the indigenous populations in Malaysia giventhe fact that these communities are afflicted with parasiticinfections not only as reported previously [14 15] but alsoas a reservoir for VTEC

In this study all the volunteers were infected by differentparasites such as Giardia Trichuris Ascaris HookwormEntamoeba coli and Iodamoeba There is no statistical evi-dence that showed correlation between parasitic infectionand VTEC colonization Our results also did not suggest anygroups (single or multiple parasitic infections) that are proneto VTEC colonization Interestingly we found that E coliwas hardly detected in the stool samples containing multipleparasites as in the first run of LAMP assay (sample PM 252DB 21 BP 15 BP 142 DK 173 DK 176 DK 1910 DK 271UK 155 UL 132 NT 62 NT 63 and SW 163) Reasonsthat influenced the sensitivity of this LAMP assay will befurther investigated Meanwhile the 4 samples which werenot detected with E coli might be due to recent antibiotictreatments

5 Conclusion

We have successfully developed a LAMP assay that canspecifically detect generic E coli and further performed incombination with the LAMP assay that detect verocytotoxin-related gene Our study suggests the high risk of indigenouscommunity for carriage and excretion of VTEC Due to thehigh specificity and sensitivity of the generic E coli LAMPassay developed in this study we proposed the application ofthis assay in food and animals as an alternative approach forrapid and more specific detection of E coli

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank University of Malaya for support andfacilities This study was funded by Higher Impact ResearchGrant (UMC6251HIRMOHE02) andGrant GA 013-2013(Molecular Diagnostics of Bacterial Pathogens)

References

[1] R E Ley D A Peterson and J I Gordon ldquoEcological andevolutionary forces shaping microbial diversity in the humanintestinerdquo Cell vol 124 no 4 pp 837ndash848 2006

[2] J C Clemente L K Ursell L W Parfrey and R Knight ldquoTheimpact of the gut microbiota on human health an integrativeviewrdquo Cell vol 148 no 6 pp 1258ndash1270 2012

[3] R Burcelin E Luche M Serino and J Amar ldquoThe gutmicrobiota ecology a new opportunity for the treatment ofmetabolic diseasesrdquo Frontiers in Bioscience vol 14 no 13 pp5107ndash5117 2009

[4] H Tlaskalova-Hogenova R Stpankova H Kozakova et alldquoThe role of gut microbiota (commensal bacteria) and themucosal barrier in the pathogenesis of inflammatory and

autoimmune diseases and cancer contribution of germ-freeand gnotobiotic animalmodels of human diseasesrdquoCellular andMolecular Immunology vol 8 no 2 pp 110ndash120 2011

[5] O Tenaillon D Skurnik B Picard and E Denamur ldquoThepopulation genetics of commensal Escherichia colirdquo NatureReviews Microbiology vol 8 no 3 pp 207ndash217 2010

[6] S Hong K H Oh S H Cho et al ldquoAsymptomatic healthyslaughterhouse workers in South Korea carrying Shiga toxin-producing Escherichia colirdquo FEMS Immunology and MedicalMicrobiology vol 56 no 1 pp 41ndash47 2009

[7] M A Karmali V Gannon and J M Sargeant ldquoVerocytotoxin-producing Escherichia coli (VTEC)rdquo Veterinary Microbiologyvol 140 no 3-4 pp 360ndash370 2010

[8] B Soborg S G Lassen L Muller et al ldquoA verocytotoxin-producing E coli outbreak with a surprisingly high risk ofhaemolytic uraemic syndrome Denmark September-October2012rdquo Euro Surveillance vol 18 no 2 2013

[9] T Dallman G P Smith B OrsquoBrien et al ldquoCharacterizationof a verocytotoxin-producing enteroaggregative Escherichia coliserogroupO111H21 strain associatedwith a household outbreakin Northern Irelandrdquo Journal of Clinical Microbiology vol 50no 12 pp 4116ndash4119 2012

[10] F Scheutz E M Nielsen J Frimodt-Moslashller et al ldquoCharacteris-tics of the enteroaggregative Shiga toxinverotoxin-producingEscherichia coli O104 H4 strain causing the outbreak ofhaemolytic uraemic syndrome in Germany May to June 2011rdquoEurosurveillance vol 16 no 24 2011

[11] R Stephan and L E Hoelzle ldquoCharacterization of shigatoxin type 2 variant B-subunit in Escherichia coli strains fromasymptomatic human carriers by PCR-RFLPrdquo Letters in AppliedMicrobiology vol 31 no 2 pp 139ndash142 2000

[12] K T Lim C S Teh and K L Thong ldquoLoop-mediated isother-mal amplification assay for the rapid detection of Staphylococcusaureusrdquo BioMed Research International vol 2013 Article ID895816 5 pages 2013

[13] N A Nasr H M Al-Mekhlafi A Ahmed M A Roslan andA Bulgiba ldquoTowards an effective control programme of soil-transmitted helminth infections among Orang Asli in ruralMalaysiamdashpart 2 knowledge attitude and practicesrdquo ParasitVectors vol 6 article 28 2013

[14] R Ngui Y A Lim S C Chow J A de Bruyne and C KLiam ldquoPrevalence of bronchial asthma among Orang Asli inPeninsular Malaysiardquo Medical Journal of Malaysia vol 66 no1 pp 27ndash31 2011

[15] A K Mahdy J Surin A Mohd-Adnan K L Wan andY A Lim ldquoMolecular characterization of Giardia duodenalisisolated from Semai Pahang Orang Asli (Peninsular Malaysiaaborigines)rdquo Parasitology vol 136 no 11 pp 1237ndash1241 2009

[16] A A Rahim Y Y Lee N A Majid et al ldquoHelicobacterpylori infection among Aborigines (the Orang Asli) in thenortheastern region of Peninsular Malaysiardquo The AmericanJournal of TropicalMedicine andHygiene vol 83 no 5 pp 1119ndash1122 2010

[17] Y Hara-Kudo J Nemoto K Ohtsuka et al ldquoSensitive andrapid detection of Vero toxin-producing Escherichia coli usingloop-mediated isothermal amplificationrdquo Journal of MedicalMicrobiology vol 56 no 3 pp 398ndash406 2007

[18] Y Shao X He E M Harrison et al ldquomGenomeSubtractor aweb-based tool for parallel in silico subtractive hybridizationanalysis of multiple bacterial genomesrdquo Nucleic Acids Researchvol 38 no 2 pp W194ndashW200 2010

6 The Scientific World Journal

[19] W S Ho L K Tan P T Ooi C C Yeo and K L ThongldquoPrevalence and characterization of verotoxigenic-Escherichiacoli isolates from pigs in Malaysiardquo BMC Veterinary Researchvol 9 article 109 2013

[20] A Yano R Ishimaru and R Hujikata ldquoRapid and sensitivedetection of heat-labile I and heat-stable I enterotoxin genes ofenterotoxigenic Escherichia coli by Loop-Mediated IsothermalAmplificationrdquo Journal of Microbiological Methods vol 68 no2 pp 414ndash420 2007

[21] J Hill S Beriwal I Chandra et al ldquoLoop-mediated isothermalamplification assay for rapid detection of common strains ofEscherichia colirdquo Journal of Clinical Microbiology vol 46 no 8pp 2800ndash2804 2008

[22] X Zhao Y Li L Wang et al ldquoDevelopment and applicationof a loop-mediated isothermal amplification method on rapiddetection Escherichia coli O157 strains from food samplesrdquoMolecular Biology Reports vol 37 no 5 pp 2183ndash2188 2010

[23] A T Maurelli R E Fernandez C A Bloch C K Rode andA Fasano ldquolsquoBlack holesrsquo and bacterial pathogenicity a largegenomic deletion that enhances the virulence of Shigella sppand enteroinvasive Escherichia colirdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 95 no7 pp 3943ndash3948 1998

[24] G M Pupo R Lan and P R Reeves ldquoMultiple independentorigins of Shigella clones of Escherichia coli and convergentevolution of many of their characteristicsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 97 no 19 pp 10567ndash10572 2000

[25] G E Sims and S H Kim ldquoWhole-genome phylogeny ofEscherichia coliShigella group by feature frequency profiles(FFPs)rdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 108 no 20 pp 8329ndash8334 2011

[26] M J van den Beld and F A Reubsaet ldquoDifferentiation betweenShigella enteroinvasive Escherichia coli (EIEC) and noninvasiveEscherichia colirdquo European Journal of Clinical Microbiology ampInfectious Diseases vol 31 no 6 pp 899ndash904 2012

[27] QHu J Tu XHan Y ZhuCDing and S Yu ldquoDevelopment ofmultiplex PCR assay for rapid detection of Riemerella anatipes-tifer Escherichia coli and Salmonella enterica simultaneouslyfrom ducksrdquo Journal of Microbiological Methods vol 87 no 1pp 64ndash69 2011

[28] K L Thong M Y Lai C S J Teh and K H ChualdquoSimultaneous detection of methicillin-resistant Staphylococcusaureus Acinetobacter baumannii Escherichia coli Klebsiellapneumoniae and Pseudomonas aeruginosa by multiplex PCRrdquoTropical Biomedicine vol 28 no 1 pp 21ndash31 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World Journal 5

conditions of the indigenous populations in Malaysia giventhe fact that these communities are afflicted with parasiticinfections not only as reported previously [14 15] but alsoas a reservoir for VTEC

In this study all the volunteers were infected by differentparasites such as Giardia Trichuris Ascaris HookwormEntamoeba coli and Iodamoeba There is no statistical evi-dence that showed correlation between parasitic infectionand VTEC colonization Our results also did not suggest anygroups (single or multiple parasitic infections) that are proneto VTEC colonization Interestingly we found that E coliwas hardly detected in the stool samples containing multipleparasites as in the first run of LAMP assay (sample PM 252DB 21 BP 15 BP 142 DK 173 DK 176 DK 1910 DK 271UK 155 UL 132 NT 62 NT 63 and SW 163) Reasonsthat influenced the sensitivity of this LAMP assay will befurther investigated Meanwhile the 4 samples which werenot detected with E coli might be due to recent antibiotictreatments

5 Conclusion

We have successfully developed a LAMP assay that canspecifically detect generic E coli and further performed incombination with the LAMP assay that detect verocytotoxin-related gene Our study suggests the high risk of indigenouscommunity for carriage and excretion of VTEC Due to thehigh specificity and sensitivity of the generic E coli LAMPassay developed in this study we proposed the application ofthis assay in food and animals as an alternative approach forrapid and more specific detection of E coli

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank University of Malaya for support andfacilities This study was funded by Higher Impact ResearchGrant (UMC6251HIRMOHE02) andGrant GA 013-2013(Molecular Diagnostics of Bacterial Pathogens)

References

[1] R E Ley D A Peterson and J I Gordon ldquoEcological andevolutionary forces shaping microbial diversity in the humanintestinerdquo Cell vol 124 no 4 pp 837ndash848 2006

[2] J C Clemente L K Ursell L W Parfrey and R Knight ldquoTheimpact of the gut microbiota on human health an integrativeviewrdquo Cell vol 148 no 6 pp 1258ndash1270 2012

[3] R Burcelin E Luche M Serino and J Amar ldquoThe gutmicrobiota ecology a new opportunity for the treatment ofmetabolic diseasesrdquo Frontiers in Bioscience vol 14 no 13 pp5107ndash5117 2009

[4] H Tlaskalova-Hogenova R Stpankova H Kozakova et alldquoThe role of gut microbiota (commensal bacteria) and themucosal barrier in the pathogenesis of inflammatory and

autoimmune diseases and cancer contribution of germ-freeand gnotobiotic animalmodels of human diseasesrdquoCellular andMolecular Immunology vol 8 no 2 pp 110ndash120 2011

[5] O Tenaillon D Skurnik B Picard and E Denamur ldquoThepopulation genetics of commensal Escherichia colirdquo NatureReviews Microbiology vol 8 no 3 pp 207ndash217 2010

[6] S Hong K H Oh S H Cho et al ldquoAsymptomatic healthyslaughterhouse workers in South Korea carrying Shiga toxin-producing Escherichia colirdquo FEMS Immunology and MedicalMicrobiology vol 56 no 1 pp 41ndash47 2009

[7] M A Karmali V Gannon and J M Sargeant ldquoVerocytotoxin-producing Escherichia coli (VTEC)rdquo Veterinary Microbiologyvol 140 no 3-4 pp 360ndash370 2010

[8] B Soborg S G Lassen L Muller et al ldquoA verocytotoxin-producing E coli outbreak with a surprisingly high risk ofhaemolytic uraemic syndrome Denmark September-October2012rdquo Euro Surveillance vol 18 no 2 2013

[9] T Dallman G P Smith B OrsquoBrien et al ldquoCharacterizationof a verocytotoxin-producing enteroaggregative Escherichia coliserogroupO111H21 strain associatedwith a household outbreakin Northern Irelandrdquo Journal of Clinical Microbiology vol 50no 12 pp 4116ndash4119 2012

[10] F Scheutz E M Nielsen J Frimodt-Moslashller et al ldquoCharacteris-tics of the enteroaggregative Shiga toxinverotoxin-producingEscherichia coli O104 H4 strain causing the outbreak ofhaemolytic uraemic syndrome in Germany May to June 2011rdquoEurosurveillance vol 16 no 24 2011

[11] R Stephan and L E Hoelzle ldquoCharacterization of shigatoxin type 2 variant B-subunit in Escherichia coli strains fromasymptomatic human carriers by PCR-RFLPrdquo Letters in AppliedMicrobiology vol 31 no 2 pp 139ndash142 2000

[12] K T Lim C S Teh and K L Thong ldquoLoop-mediated isother-mal amplification assay for the rapid detection of Staphylococcusaureusrdquo BioMed Research International vol 2013 Article ID895816 5 pages 2013

[13] N A Nasr H M Al-Mekhlafi A Ahmed M A Roslan andA Bulgiba ldquoTowards an effective control programme of soil-transmitted helminth infections among Orang Asli in ruralMalaysiamdashpart 2 knowledge attitude and practicesrdquo ParasitVectors vol 6 article 28 2013

[14] R Ngui Y A Lim S C Chow J A de Bruyne and C KLiam ldquoPrevalence of bronchial asthma among Orang Asli inPeninsular Malaysiardquo Medical Journal of Malaysia vol 66 no1 pp 27ndash31 2011

[15] A K Mahdy J Surin A Mohd-Adnan K L Wan andY A Lim ldquoMolecular characterization of Giardia duodenalisisolated from Semai Pahang Orang Asli (Peninsular Malaysiaaborigines)rdquo Parasitology vol 136 no 11 pp 1237ndash1241 2009

[16] A A Rahim Y Y Lee N A Majid et al ldquoHelicobacterpylori infection among Aborigines (the Orang Asli) in thenortheastern region of Peninsular Malaysiardquo The AmericanJournal of TropicalMedicine andHygiene vol 83 no 5 pp 1119ndash1122 2010

[17] Y Hara-Kudo J Nemoto K Ohtsuka et al ldquoSensitive andrapid detection of Vero toxin-producing Escherichia coli usingloop-mediated isothermal amplificationrdquo Journal of MedicalMicrobiology vol 56 no 3 pp 398ndash406 2007

[18] Y Shao X He E M Harrison et al ldquomGenomeSubtractor aweb-based tool for parallel in silico subtractive hybridizationanalysis of multiple bacterial genomesrdquo Nucleic Acids Researchvol 38 no 2 pp W194ndashW200 2010

6 The Scientific World Journal

[19] W S Ho L K Tan P T Ooi C C Yeo and K L ThongldquoPrevalence and characterization of verotoxigenic-Escherichiacoli isolates from pigs in Malaysiardquo BMC Veterinary Researchvol 9 article 109 2013

[20] A Yano R Ishimaru and R Hujikata ldquoRapid and sensitivedetection of heat-labile I and heat-stable I enterotoxin genes ofenterotoxigenic Escherichia coli by Loop-Mediated IsothermalAmplificationrdquo Journal of Microbiological Methods vol 68 no2 pp 414ndash420 2007

[21] J Hill S Beriwal I Chandra et al ldquoLoop-mediated isothermalamplification assay for rapid detection of common strains ofEscherichia colirdquo Journal of Clinical Microbiology vol 46 no 8pp 2800ndash2804 2008

[22] X Zhao Y Li L Wang et al ldquoDevelopment and applicationof a loop-mediated isothermal amplification method on rapiddetection Escherichia coli O157 strains from food samplesrdquoMolecular Biology Reports vol 37 no 5 pp 2183ndash2188 2010

[23] A T Maurelli R E Fernandez C A Bloch C K Rode andA Fasano ldquolsquoBlack holesrsquo and bacterial pathogenicity a largegenomic deletion that enhances the virulence of Shigella sppand enteroinvasive Escherichia colirdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 95 no7 pp 3943ndash3948 1998

[24] G M Pupo R Lan and P R Reeves ldquoMultiple independentorigins of Shigella clones of Escherichia coli and convergentevolution of many of their characteristicsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 97 no 19 pp 10567ndash10572 2000

[25] G E Sims and S H Kim ldquoWhole-genome phylogeny ofEscherichia coliShigella group by feature frequency profiles(FFPs)rdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 108 no 20 pp 8329ndash8334 2011

[26] M J van den Beld and F A Reubsaet ldquoDifferentiation betweenShigella enteroinvasive Escherichia coli (EIEC) and noninvasiveEscherichia colirdquo European Journal of Clinical Microbiology ampInfectious Diseases vol 31 no 6 pp 899ndash904 2012

[27] QHu J Tu XHan Y ZhuCDing and S Yu ldquoDevelopment ofmultiplex PCR assay for rapid detection of Riemerella anatipes-tifer Escherichia coli and Salmonella enterica simultaneouslyfrom ducksrdquo Journal of Microbiological Methods vol 87 no 1pp 64ndash69 2011

[28] K L Thong M Y Lai C S J Teh and K H ChualdquoSimultaneous detection of methicillin-resistant Staphylococcusaureus Acinetobacter baumannii Escherichia coli Klebsiellapneumoniae and Pseudomonas aeruginosa by multiplex PCRrdquoTropical Biomedicine vol 28 no 1 pp 21ndash31 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 The Scientific World Journal

[19] W S Ho L K Tan P T Ooi C C Yeo and K L ThongldquoPrevalence and characterization of verotoxigenic-Escherichiacoli isolates from pigs in Malaysiardquo BMC Veterinary Researchvol 9 article 109 2013

[20] A Yano R Ishimaru and R Hujikata ldquoRapid and sensitivedetection of heat-labile I and heat-stable I enterotoxin genes ofenterotoxigenic Escherichia coli by Loop-Mediated IsothermalAmplificationrdquo Journal of Microbiological Methods vol 68 no2 pp 414ndash420 2007

[21] J Hill S Beriwal I Chandra et al ldquoLoop-mediated isothermalamplification assay for rapid detection of common strains ofEscherichia colirdquo Journal of Clinical Microbiology vol 46 no 8pp 2800ndash2804 2008

[22] X Zhao Y Li L Wang et al ldquoDevelopment and applicationof a loop-mediated isothermal amplification method on rapiddetection Escherichia coli O157 strains from food samplesrdquoMolecular Biology Reports vol 37 no 5 pp 2183ndash2188 2010

[23] A T Maurelli R E Fernandez C A Bloch C K Rode andA Fasano ldquolsquoBlack holesrsquo and bacterial pathogenicity a largegenomic deletion that enhances the virulence of Shigella sppand enteroinvasive Escherichia colirdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 95 no7 pp 3943ndash3948 1998

[24] G M Pupo R Lan and P R Reeves ldquoMultiple independentorigins of Shigella clones of Escherichia coli and convergentevolution of many of their characteristicsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 97 no 19 pp 10567ndash10572 2000

[25] G E Sims and S H Kim ldquoWhole-genome phylogeny ofEscherichia coliShigella group by feature frequency profiles(FFPs)rdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 108 no 20 pp 8329ndash8334 2011

[26] M J van den Beld and F A Reubsaet ldquoDifferentiation betweenShigella enteroinvasive Escherichia coli (EIEC) and noninvasiveEscherichia colirdquo European Journal of Clinical Microbiology ampInfectious Diseases vol 31 no 6 pp 899ndash904 2012

[27] QHu J Tu XHan Y ZhuCDing and S Yu ldquoDevelopment ofmultiplex PCR assay for rapid detection of Riemerella anatipes-tifer Escherichia coli and Salmonella enterica simultaneouslyfrom ducksrdquo Journal of Microbiological Methods vol 87 no 1pp 64ndash69 2011

[28] K L Thong M Y Lai C S J Teh and K H ChualdquoSimultaneous detection of methicillin-resistant Staphylococcusaureus Acinetobacter baumannii Escherichia coli Klebsiellapneumoniae and Pseudomonas aeruginosa by multiplex PCRrdquoTropical Biomedicine vol 28 no 1 pp 21ndash31 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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