Detection of amplified Wuchereria bancrofti DNA in mosquitoes with a nonradioactive probe

Abstract

A technique to identify Wuchereria bancrofti larvae in mosquito vectors with an enzyme-labeled DNA probe is described. To overcome the low sensitivity of nonradioactive detection methods, analyte DNA was amplified by polymerase chain reaction (PCR). Oligonucleotide primers were used to amplify W. bancrofti-specific DNA fragments of 380 and 650 bp, respectively. Parasite DNA in mosquito extracts was isolated free of inhibitors of the PCR by hybridization to a biotinylated DNA fragment (IWb 67), which hybridizes to DNA from most filarial species, followed by absorption of the resulting DNA hybrids onto avidin-coated acrylic beads. PCR-amplified DNA was detected with a biotin-labeled W. bancrofti-specific repeat DNA (IWb 35) coupled to avidin-alkaline phosphatase and the chemiluminescent substrate, AMPPD. The DNA equivalent of less than one larva can be detected by this method in mosquito extracts. The sensitivity of detection was comparable to that of radioactive probes and the assay is suitable for field application in endemic countries.