Multiplex reverse transcriptase pcr assay for the detection of infective stage wuchereria bancrofti and its vector culex quinquefasciatus

There is currently a global program attempting to eliminate lymphatic filariasis (LF) by administering drugs to affected communities with the goal of interrupting transmission of the parasite. At present the global programme for the elimination of lymphatic filariaisis (GPELF) uses indirect human measures to evaluate the success of its primary goal, the interruption of transmission. An infective stage (L3) detection assay provides a more direct measure of transmission risk and may be useful as a sensitive and non-invasive method for monitoring GPELF programs. In this study, we have developed a multiplex reverse transcription polymerase chain reaction (RT-PCR) assay for the simultaneous detection of infective stage (L3) Wuchereria bancrofti and the vector species Culex quinquefasciatus. The assay could detect a single L3 in an optimum pools size of 25 mosquitoes when each of the two sets of L3 specific primers (WbL31 and WbL32) were used, indicating the respective diagnostic bands of 203 bp and 111 bp, along with an amplicon of 500 bp for the vector. The detection potential of the assay in terms of sensitivity and specificity of the primer WbL3-1 was 85% and 100%, whereas the same for the primer WbL3-2 was 95% and 100% respectively based on the decoded results of the assessment of 20 coded samples and the variation in the sensitivity was insignificant. This assay may be useful as a non-invasive surveillance tool for early detection of LF resurgence following suspension of MDA, if the detection potential of the assay would be evaluated on larger number of field caught mosquitoes.