diagnosis and characterization of leishmania species using pcr-rflp

8/22/2019 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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http://localhost/var/www/apps/conversion/tmp/scratch_10/restriction.exe


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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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http://localhost/var/www/apps/conversion/tmp/scratch_10/pcr.exe


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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

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