RT-PCR assay for the detection of infective (L3) larvae of lymphatic filarial parasite, Wuchereria bancrofti, in vector mosquito Culex quinquefasciatus.

Abstract

BACKGROUND & OBJECTIVES: Periodic monitoring of vector population for infection and infectivity rates is central to the evaluation of the filariasis elimination strategies in endemic areas to monitor the success of MDA and also to establish endpoints for intervention. The main objective of this study was to develop a RT-PCR assay, based on L3 stage-specific primers to detect the presence of infective stage larvae of filarial parasite, Wuchereria bancrofti in the vector Culex quinquefasciatus. MATERIAL & METHODS: Subtracted probe development technique was employed for the identification of infective stage (L3) specific genes. The subtracted cDNA was labeled by non-radioisotopic method and used for screening cDNA library of L3 stage larvae of W. bancrofti constructed in UniZap XR. Recombinants were probed and identified from the library. The inserts of the recombinant clones were purified and sequenced. Primers were designed based on the sequence information of three recombinant clones for detecting L3 larvae of W. bancrofti in the vector by RT-PCR assay. Preliminary laboratory evaluation was carried out to assess the sensitivity and specificity of WbL31 RT-PCR assay. RESULTS: cDNA library of L3 stage of W. bancrofti constructed in UniZap XR vector, constituted 5 x 10(5) phages with 80-90% recombinant phages and the size of inserts varied from 0.1 to 1.0 kb. When subtracted cDNA was random prime labeled and used for screening cDNA library of L3 stage of W. bancrofti constructed in UniZap XR, 18 clones were identified from the library. Three genes were found up-regulated in the L3 stage, out of which WbL31 (cuticular collagen) was found to be useful in detecting L3 larvae of W. bancrofti in the vector by RT-PCR assay with high specificity and sensitivity (98-100%). CONCLUSION: Present paper marks first report on the development of an infective stage-specific RT-PCR assay (WbL31 RT-PCR assay) to detect L3 stage W. bancrofti in the vector. This assay will have potential application in assessing the transmission of infection and hence in decision-making related to elimination programme.