The development and evaluation of a single step multiplex PCR method for simultaneous detection of Brugia malayi and Wuchereria bancrofti

A single step novel multiplex polymerase chain reaction (PCR) has been developed for simultaneous detection of human filarial parasites, Brugia malayi and Wuchereria bancrofti, from blood samples and mosquitoes. The primers used were novel and have been tested with the parasite DNA amplifying 188bp (BM) and 129bp (WB) DNA fragments, specific to B. malayi and W. bancrofti, respectively, in a single reaction. The specificity of the PCR product was confirmed by DNA sequencing and slot blot hybridization assay. The test was found highly sensitive for both B. malayi and W. bancrofti by detecting the parasitaemia up to the level of one microfilaria per reaction. The assay was further evaluated on 98 blood samples and 144 mosquito samples collected from filarial endemic areas. The PCR was found to be more efficient in comparison to microscopy by detecting 8% and 5% more filarial parasites in field-collected blood and mosquito samples, respectively. This novel PCR that offers scope for simultaneous detection of both the parasites may be used as a diagnostic tool for the detection of filariasis in population and can be adopted for rapid surveillance and monitoring of mosquitoes for use in the effective control of filariasis.