An epitope-specific PCR test for diagnosis of Leishmania donovani infections.
The carboxy-terminal region of recombinant heat shock protein 70 of Leishmania donovani has been shown to have an immunodominant epitope. We designed a PCR assay using a new set of primers encompassing the gene sequence from 457bp to 927bp, which amplified a segment of the L. donovani genome in a species-specific manner. The assay was sensitive enough to detect 0.5pg of parasite DNA, which increased 10-fold (0.05pg) when an internal probe (583-609bp) was used in the Southern blot. The assay was able to detect parasite DNA from the liver and spleen of L. donovani-infected hamsters as well as lesion aspirates from parasitologically confirmed post-kala-azar dermal leishmaniasis (PKDL) and bone marrow aspirates from visceral leishmaniasis (VL) patients. No amplification was seen with axenically cultured promastigotes of L. infantum, L. tropica, L. major, L. mexicana, L. aethiopica or other intracellular organisms such as Entamoeba histolytica, Mycobacterium tuberculosis, Plasmodium vivax or human peripheral blood mononuclear cells. This PCR provides a specific tool for the diagnosis of VL and PKDL with simultaneous species identification.
Arora
Sunil K
SK
Department of Immunopathology, Post Graduate Institute of Medical Education and Research, Chandigarh 160012, India. skarora_in@yahoo.com
Gupta
Sachin
S
Bhardwaj
Suvercha
S
Sachdeva
Naresh
N
Sharma
N L
NL
D000818:Animals; D015139:Blotting, Southern; D006224:Cricetinae; D018604:Epitope Mapping / Q000379:methods; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D006801:Humans; D016056:Immunodominant Epitopes / Q000032:analysis*; D007893:Leishmania donovani / Q000145:classification* / Q000235:genetics; D007898:Leishmaniasis, Visceral / Q000175:diagnosis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods; D013045:Species Specificity
D018840:HSP70 Heat-Shock Proteins; D016056:Immunodominant Epitopes