Diagnosis and Characterization of Leishmania Species Using PCR-RFLP
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Transcript of Diagnosis and Characterization of Leishmania Species Using PCR-RFLP
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DIAGNOSISANDCHARACTERIZATIONOF
LEISHMANIASPECIESUSINGPCR-RFLP
Prepared by: Hercolanium
Supervised by: Dr. N. H.
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INTRODUCTION
In Jordan there are several species ofLeishmania;
Leishmania infantum, Leishmania tropica, and
Leishmania major.
Leishmania tropica is the major species ofLeishmaniaparasite in Jordan but recent
revolutions in Syria and other countries resulted in
the migration of high number of refugees and with
them came new species mainlyLeishmania majorfrom Syria.
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The problem with that is the prediction of anL.
majoroutbreak that might take place several
years from now.
Identification ofLeishmaniaspecies will be so
important to decide the best treatment.
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DIAGNOSIS
To minimize the predicament there should be a gold standard to diagnose
patients if the need calls for it. Several methods are available:
Traditional diagnostic methods:
1. Microscopic Examination
Examination of intracellular amastigotes from Giemsa-stained lesionbiopsy smears (CL), or lymph node, bone marrow aspiration (VL)
It depends on skilled clinicians, has low sensitivity, and the
procedures are invasive in the case of VL.
2. Culture
Growth of promastigotes in days to weeks
NNN medium is the medium of choice
Sensitivity is considerably mediocre 5
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Microscopy
Culture
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CONT
Serological Diagnosis
1. Indirect Fluorescence Antibody (IFA)
2. ELISA
Both are not suitable for field conditions
3. Direct Agglutination Test (DAT)
4. rK39
5. Fast Agglutination Screening Test (FAST)
Both DAT and rK39 dipsticks are easy to use,
require minimal technological expertise or lab. Set-up and are ideal for field and lab. Use.
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CONT
rK39 is an abbreviation for a Recombinant Kinesin-related protein of 39 kDa
that is apart of a kinesis-related protein ofLeishmania chagasi which is
conserved withinLeishmania donovani complex, and antibody against it from
cutaneous and mucocotaneous leishmaniasis cannot be detected.
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CONT
PCR and Leishmaniasis:
1. There has been a lot or research has been done and over 400
articles on it have been published
2. High-copy-number target genes are chosen
rRNA genes
ITS1 Region of rRNA genes
Kinetoplast DNA minicircles
Mini-exon genes
Other genes
Gp63 gene
Hsp70 gene
Cysteine proteinase gene 10
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CONT kDNA minicircle gene:
An excellent target for a
sensitive and rapid detection
methods Present at thousands of
copies per cell
Show the highest
sensitivity by PCR
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CONT
Mini-exon (spliced leader) gene repeat
Involved in the transsplicing process of nuclear mRNA
Present 100-200 tandemly repeated copies per nuclear genome
Sequence variation of non-transcribed spacer was observed
between individual species
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CONT
ITS(Internal Transcribed Spacer) refers to a piece of non-functional
RNA situated between structural ribosomal RNAs (rRNA) on a
common precursor transcript.
Read from 5' to 3', this polycistronic rRNA precursor transcript
contains the 5' external transcribed sequence (5' ETS), 18S rRNA,
ITS1, 5.8S rRNA, ITS2, 28S rRNA and finally the 3'ETS.
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During rRNA maturation, ETS and ITS pieces are excised and as non-
functional maturation by-productsrapidly degraded. Genes
encoding ribosomal RNA and spacers occur in tandem repeats that are
thousands of copies long, each separated by regions of non-
transcribed DNA termed intergenic spacer(IGS) or non-transcribed
spacer(NTS).
Sequence comparison of the ITS region is widely used in taxonomy
and molecular phylogeny because:
a) It is easy to amplify even from small quantities of DNA (due to
the high copy number of rRNA genes).b) It has a high degree of variation even between closely related
species (due to the relatively low evolutionary pressure acting
on such non-functional sequences).14
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SPECIESIDENTIFICATION PCR-RFLP
ITS1 of SSUrRNA gene;
restrictionenzymeHaeIII
Mini-exongene;
restrictionenzymeEae I
DNA sequencingof PCR products
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RFLP
Stands for RestrictionFragment LengthPolymorphism
Takes advantage ofdifferences in DNA betweenindividuals that result in
different fragments whendigested with restrictionenzymes
3 fragments
2 fragments
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How many fragments wi l l
result when each of theseal leles are digested w ith
DdeI?
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RFLP
To see RFLP, DNA is digested
with the appropriate restriction
enzymes and run on an agarose
gel.
A Southern Blot is performed to
complete the analysis.
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SOUTHERNBLOTTING
A method to visualize specificsegments of DNAusually a
particular gene.
Uses radioactive probes thatbind to the specific DNAsegments
Ex. When testing for the
hemoglobin alleles, the probewould bind to these regions
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POLYMERASECHAINREACTION
PCR allows scientists
to amplifysmall,specific segments of
DNA = make millions
of copies of segment
Allows foramplification at an
exponential rate
DNA Replication in a
test tube
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FILTERPAPERPCR
A new method for obtaining of samples from fields is Filter
paper; in which the clinician impress a filet paper on the
lesion.
Filter papers were then allowed to air-dry, and 6-mm punches
were obtained and stored in 1.5-mL Eppendorf tubes
containing 700 mL 100% ethanol for qualitative PCR testing.
To avoid contamination between specimens, the single-hole
punch was used on clean filterpaper 10 times, then immersed
and washed in soapy water for 10 min, allowed to air-dry, andthen cleaned again with 2 separate isopropyl alcohol wipes.
Sensitivity and specificityof filterpaper PCR were 92.3% 23
Specimen collection using the Fisher brand 7-cm filter paper lesion impression
method.A, cleansing of an ulcer suspected to be cutaneous
leishmaniasis with topical antiseptic;B, preparation of the filter paper for
specimen collection; C, application of the filter paper to the ulcer base; D,evidence of tissue fluid and ulcer exudates wicked onto the filter paper
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REFERENCES
Hijjawi N.S., Atoum M, Kanani K, Rasheed M, Abdel-dayem M, Irhimeh M.
R. Molecular diagnosis and identification of differentLeishmaniaspecies for
Jordanian cutaneous leishmaniasis patients. Unpublished
Phramongkutklao College of Medicine; Saovanee Leelayoova, Mathirut
Mungthin, Suradej Siripattanapipong, and Tawee Naaglor
Hajjaran H, Vasigheh F, Mohebali M, Rezaei S, Mamishi S, Charedar S. Direct
diagnosis ofLeishmaniaspecies on serosity materials punctured from
cutaneous leishmaniasis patients using PCR-RFLP. J Clin Lab Anal.2011;25(1):204. doi: 10.1002/jcla.20377
Van Thiel P-P A.M., Van Gool T., Faber W.R., Leenstra T., Kager P.A.
Variation in Clinical Presentation and Genotype of CausativeLeishmania
majorStrain in Cutaneous Leishmaniasis in North and South Afghanistan. Am
J Trop Med Hyg. 2011 July 1; 85(1): 6063.
Toz SO, Nasereddin A, Ozbel Y, Ertabaklar H, Culha G, et al. Leishmaniasis in
Turkey: molecular characterization ofLeishmaniafrom human and canine
clinical samples. Trop Med Int Health. 2009;14:14011406
Some internet sites26