2014-Analytica-Legionella Pneumophila Detection in Water by RRNA (Without PCR)
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Shyam Verma 1 , Jvo Siegrist 2 , Jens Boertz 2 , and Katja Vetter 3 1 Sigma-Aldrich/Supelco, Bellefonte, PA, USA, 2 Sigma-Aldrich/Fluka, Buchs (SG) Switzerland, 3 Scanbec, Bitterfeld-Wolfen, Germany Abstract The current ISO method (ISO 11731:1998; 11731-2 2004) used to detect and identify the Legionella species takes at least 10 days and requires several steps with different media. Therefore, new rapid methods for the detection of Legionella are of great interest. HybriScan ® Legionella kit and HybriScan Legionella pneumophila kit use ribosomal RNA as a modern detection target. The rRNA is greater in number than DNA (no PCR is needed), and is only present in living cells. Therefore, it perfectly suits specific and sensitive detection. Within 2.5 hours, a direct quantitative detection of 10,000 colony forming units (CFU) per liter is easily possible and 1-10 CFU/L can be detected with an enrichment step in BCYE medium. T414055 By using two different probes for detection of microbial rRNA, false-positive results are almost impossible. The quantitative analyis of 39 Legionella positive samples with HybriScan were compared with ISO 11731 (GVPC and BCYE Agar) and no significant difference could be found. HybriScanD Legionella even showed to be more sensitive on low concentrations.. Results 1. T. Leskelä, A. Tilsala-Timisjärvib, J. Kusnetsovc, P. Neubauera, A. Breitenstein, Sensitive genus-specific detection of Legionella by a 16S rRNA based sandwich hybridization assay, J. Microbiol. Meth., 62, pp. 167–179 (2005). 2. ISO 11731, Water quality - Detection and enumeration of Legionella (1998). References Legionella pneumophila Detection in Water by rRNA (without PCR) A rapid and innovative test system for Legionella, especially Legionella pneumophila, in water Legionnaires’ disease is a dangerous and infectious pneumonia which can affect anybody, but those at highest risk are the elderly, sick, smokers, or other people with weak immune systems. The causative organism is the Legionella pneumophila, which easily forms aerosols that can be inhaled, causing infection. The natural source of Legionella pneumophila is all types of water, but particularly water containing algae, rust, sludge, scale, and organic compounds. It is known that Legionella are able to form biofilms inside water pipes where they are protected against antimicrobial treatments. The infected water droplets can be produced in such places as whirlpool spas or air conditioners since these organisms prefer warm environments but can also occur in fresh water such as rivers and ponds. A new sandwich hybridization test was developed in a microtiter plate format which takes less than 2.5 hours and has a sensitivity of 10,000 CFU per liter and 1-10 CFU per liter after an overnight enrichment in BCYE medium. The quantification is possible with this kit using photometric methods. Compared to PCR, this system does not count dead cells, as rRNA from dead cells is depleted within a few hours. Also this technique is more robust (no contamination risk during assay), less expensive, and unaffected by the sample matrix. The rapidity, sensitivity, reliability, robustness, adaptability to sample matrix, and time savings meet today’s analytical microbiology demands. Introduction A new sandwich hybridization test was designed on a 96-well microplate and is done in ~ 2 to 2.5 hours. A positive result is visible to the naked eye but quantification is possible with this kit using photometric methods and standards. Specificity is achieved by targeting conserved or unique rRNA sequences. A biotin-labeled capture probe is used to immobilize the target sequence on a solid support plate (streptavidin-coated microtiter plate). A digoxigenin labeled detection probe provides an enzyme linked optical signal read-out. Detection results from application of anti-DIG- horseradish peroxidase Fab fragments. The bound complex is visualized by horseradish peroxidase substrate TMB. Photometric data are measured at 450 nm and compared with standard solutions. As the detection of Legionella is quite tricky, two additional certified microorganism standards were used as positive controls since they can be used as qualitative and quantitative positive controls (see Table 1). Experimental Table 1. Discs with Certified Reference Test Strains of Legionella Inclusivity in terms of organism and serogroups detected: HybriScanD Legionella: all of the genus Legionella including L. pneumophila HybriScanD Legionella pneumophila: all species of L. pneumophila HybriScanI Legionella pneumophila: all species of L. pneumophila Vitroids ™ Test Strains Origin Strain No. CFU Cat. No. Legionella bozemanll NCTC 11368 50000 RQC02908 Legionella pneumophila ATCC 12821 50000 RQC02008 Figure 1. Principle of the Assay Besides the classical methods, a comparison with three different rapid methods was made. PCR, ELISA and FISH are also methods used for Legionella detection. The differences are shown in Table 2. Table 2. Comparison of Different Technologies for Detection of Beer Spoilage Bacteria Cultivation based method PCR VIT HybriScan Method cultivation based method with optical micro- scopic read out PCR/real-time PCR fluorescence microscopy sandwich hybridization and photometrical signal read out Detection spectrum detection and identification of all beer spoilage microorganisms identification of all relevant Beer spoilage microorganisms possible Lactobacillus sp. and Pediococcus damnosus, Lactobacillus sp. + L.brevis, Pectinatus + Megasphaera cerevisiae identification of all relevant Beer spoilage microorganisms possible Sample preparation selective pre- enrichment enrichment and lysis of bacteria, if necessary pre- enrichment selective pre- enrichment enrichment and lysis of bacteria, if necessary pre- enrichment Time 3 to 7 days 3 hours to 2 days 2 days 3 hours to 2 days Costs per test ca. $1 $13 $17 $3 Detection limit (cfu) 1 1-5 x 10 3 1 x 10 3 1-5 x 10 3 Devices None PCR cycler fluorescence microscope microplate reader Advantages high sensitivity, relatively cheap high sensitivity, quantitative analysis simple detection technology set up, detects only living cells (RNA) rapid and sensitive, qualitative and quantitative detection of living cells, cost efficient analysis Disadvantages time consuming, no detection of non-culturable microbes, labor expensive expensive devices needed, no discrimination between live and dead cells, not officially accepted time consuming, low sample throughput, expensive, not automatable, difficult data analysis, not officially accepted no differentiation of serotypes or subspecies, limited probe design (rRNA target), not officially accepted Figure 2. Comparison of ISO 11731 (purple) versus HybriScanD Legionella (yellow) Comparison of these different technologies is given in Table 2. By comparing HybriScan to PCR, ELISA or FISH, the benefits of this rapid test system become obvious: • Fast, cost-efficient analysis • Inexpensive read-out technology • High sensitivity and specificity Detection of organisms based on ribosomal RNA through HybriScan rapid test system is faster and efficient. The HybriScan can detect much more than Legionella, the development of an assay for a new organism is relatively easy and fast. CFU /mL HybriScan is a trademark of ScanBec GmbH. Vitroids is a trademark of Sigma-Aldrich Co. LLC.
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Detección de Legionella Pneumófila en agua de acuerdo al método de FLUKA Analytical por Sigma-Aldrich.Manual de instrucciones
Transcript of 2014-Analytica-Legionella Pneumophila Detection in Water by RRNA (Without PCR)
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Shyam Verma1, Jvo Siegrist2, Jens Boertz2, and Katja Vetter31Sigma-Aldrich/Supelco, Bellefonte, PA, USA, 2Sigma-Aldrich/Fluka, Buchs (SG) Switzerland, 3Scanbec, Bitterfeld-Wolfen, Germany
AbstractThe current ISO method (ISO 11731:1998; 11731-2 2004) used to detect and identify theLegionella species takes at least 10 days and requires several steps with different media. Therefore, new rapid methods for the detection of Legionella are of great interest. HybriScan Legionella kit and HybriScan Legionella pneumophila kit use ribosomal RNA as a modern detection target. The rRNA is greater in number than DNA (no PCR is needed), and is only present in living cells. Therefore, it perfectly suits specific and sensitive detection. Within 2.5 hours, a direct quantitative detection of 10,000 colony forming units (CFU) per liter is easily possible and 1-10 CFU/L can be detected with an enrichment step in BCYE medium.
T414055
By using two different probes for detection of microbial rRNA, false-positive results are almost impossible. The quantitative analyis of 39 Legionella positive samples with HybriScan were compared with ISO 11731 (GVPC and BCYE Agar) and no significant difference could be found. HybriScanD Legionella even showed to be more sensitive on low concentrations..
Results
1. T. Leskel, A. Tilsala-Timisjrvib, J. Kusnetsovc, P. Neubauera, A. Breitenstein, Sensitive genus-specific detection of Legionella by a 16S rRNA based sandwich hybridization assay, J. Microbiol. Meth., 62, pp. 167179 (2005).
2. ISO 11731, Water quality - Detection and enumeration of Legionella (1998).
References
Legionella pneumophila Detection in Water by rRNA (without PCR)A rapid and innovative test system for Legionella, especially Legionella pneumophila, in water
Legionnaires disease is a dangerous and infectious pneumonia which can affect anybody, but those at highest risk are the elderly, sick, smokers, or other people with weak immune systems.The causative organism is the Legionella pneumophila, which easily forms aerosols that can be inhaled, causing infection. The natural source of Legionella pneumophila is all types of water, but particularly water containing algae, rust, sludge, scale, and organic compounds. It is known that Legionella are able to form biofilms inside water pipes where they are protected against antimicrobial treatments. The infected water droplets can be produced in such places as whirlpool spas or air conditioners since these organisms prefer warm environments but can also occur in fresh water such as rivers and ponds.A new sandwich hybridization test was developed in a microtiter plate format which takes less than 2.5 hours and has a sensitivity of 10,000 CFU per liter and 1-10 CFU per liter after an overnight enrichment in BCYE medium. The quantification is possible with this kit using photometric methods. Compared to PCR, this system does not count dead cells, as rRNA from dead cells is depleted within a few hours. Also this technique is more robust (no contamination risk during assay), less expensive, and unaffected by the sample matrix. The rapidity, sensitivity, reliability, robustness, adaptability to sample matrix, and time savings meet todays analytical microbiology demands.
Introduction
A new sandwich hybridization test was designed on a 96-well microplate and is done in ~ 2 to 2.5 hours. A positive result is visible to the naked eye but quantification is possible with this kit using photometric methods and standards. Specificity is achieved by targeting conserved or unique rRNA sequences. A biotin-labeled capture probe is used to immobilize the target sequence on a solid support plate (streptavidin-coated microtiter plate). A digoxigenin labeled detection probe provides an enzyme linked optical signal read-out. Detection results from application of anti-DIG-horseradish peroxidase Fab fragments. The bound complex is visualized by horseradish peroxidase substrate TMB. Photometric data are measured at 450 nm and compared with standard solutions.As the detection of Legionella is quite tricky, two additional certified microorganism standards were used as positive controls since they can be used as qualitative and quantitative positive controls (see Table 1).
Experimental
Table 1. Discs with Certified Reference Test Strains of Legionella
Inclusivity in terms of organism and serogroups detected:HybriScanD Legionella: all of the genus Legionella including L. pneumophilaHybriScanD Legionella pneumophila: all species of L. pneumophilaHybriScanI Legionella pneumophila: all species of L. pneumophila
Vitroids Test Strains Origin Strain No. CFU Cat. No.
Legionella bozemanll NCTC 11368 50000 RQC02908
Legionella pneumophila ATCC 12821 50000 RQC02008
Figure 1. Principle of the Assay
Besides the classical methods, a comparison with three different rapid methods was made. PCR, ELISA and FISH are also methods used for Legionella detection. The differences are shown in Table 2.
Table 2. Comparison of Different Technologies for Detection of Beer Spoilage Bacteria
Cultivation based method
PCR VIT HybriScan
Method cultivation based method with optical micro-scopic read out
PCR/real-time PCR
fluorescence microscopy
sandwich hybridization and photometrical signal read out
Detection spectrum
detection and identification of all beer spoilage microorganisms
identification of all relevant Beer spoilage microorganisms possible
Lactobacillus sp. and Pediococcusdamnosus, Lactobacillus sp. +L.brevis, Pectinatus+ Megasphaeracerevisiae
identification of all relevant Beer spoilage microorganisms possible
Sample preparation
selective pre-enrichment
enrichment and lysis of bacteria, if necessary pre-enrichment
selective pre-enrichment
enrichment and lysisof bacteria, if necessary pre-enrichment
Time 3 to 7 days 3 hours to 2 days 2 days 3 hours to 2 days
Costs per test ca. $1 $13 $17 $3
Detection limit (cfu)
1 1-5 x 103 1 x 103 1-5 x 103
Devices None PCR cycler fluorescence microscope
microplate reader
Advantages high sensitivity, relatively cheap
high sensitivity, quantitative analysis
simple detection technology set up, detects only living cells (RNA)
rapid and sensitive, qualitative andquantitative detection of living cells, cost efficient analysis
Disadvantages time consuming, no detection of non-culturablemicrobes, labor expensive
expensive devices needed, no discrimination between live and dead cells, not officially accepted
time consuming, low sample throughput, expensive, not automatable, difficult data analysis, not officially accepted
no differentiation of serotypes orsubspecies, limited probe design (rRNAtarget), not officially accepted
Figure 2. Comparison of ISO 11731 (purple) versus HybriScanD Legionella (yellow)
Comparison of these different technologies is given in Table 2. By comparing HybriScan to PCR, ELISA or FISH, the benefits of this rapid test system become obvious: Fast, cost-efficient analysis Inexpensive read-out technology High sensitivity and specificityDetection of organisms based on ribosomal RNA through HybriScan rapid test system is faster and efficient. The HybriScan can detect much more than Legionella, the development of an assay for a new organism is relatively easy and fast.
CFU
/mL
HybriScan is a trademark of ScanBec GmbH.Vitroids is a trademark of Sigma-Aldrich Co. LLC.
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