Use of a simple DNA extraction method for high-throughput detection of filarial parasite Wuchereria bancrofti in the vector mosquitoes.
Molecular xenomonitoring of filariasis is the detection of filarial DNA in mosquitoes by PCR and a useful tool for monitoring transmission. DNA extraction coupled with PCR allows rapid detection of the presence or absence of the filarial parasite in vector mosquitoes compared to traditional method of manual dissection of the mosquito and observation for parasite under a microscope. A Tris-EDTA (TE) buffer-based boiling method of DNA extraction developed earlier by us was employed and explored for its suitability in the detection of Wuchereria bancrofti DNA in pools of Culex quinquefasciatus mosquitoes in real-time PCR assay. In this preliminary study, 1,000 laboratory-reared C. quinquefasciatus were made into 40 pools, each containing 25 mosquitoes spiked with 2mf. DNA from the first 20 pools was extracted using Qiagen DNeasy blood and tissue kit as standard, and the other 20 pools were subjected to TE buffer-based boiling method of DNA extraction. When the results (Ct values) obtained for DNA samples extracted by TE buffer-based boiling method were compared with that of the DNA samples extracted by the standard Qiagen method, they were found to be highly concordant without any significant difference (P = 0.9). Besides being cost- and time-effective, this protocol was found useful in extracting filarial DNA from two other mosquito genus Aedes and Anopheles, species of which have been reported as important vectors of W. bancrofti in other endemic regions of the world. Thus, TE buffer-based boiling method of DNA extraction is useful for the high-throughput detection of W. bancrofti in vector mosquitoes.
Vector Control Research Centre, ICMR, Indra Nagar, Medical Complex, Puducherry 605 006, India. vvasuki@yahoo.com
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Vasuki
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Subramanian
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Hoti
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Jambulingam
D000330:Aedes / Q000469:parasitology; D000818:Animals; D000852:Anopheles / Q000469:parasitology; D003465:Culex / Q000469:parasitology*; D018509:DNA, Helminth / Q000235:genetics / Q000302:isolation & purification*; D004199:Disease Vectors; D004772:Entomology / Q000379:methods*; D010274:Parasitology / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D013048:Specimen Handling / Q000379:methods*; D014958:Wuchereria bancrofti / Q000235:genetics / Q000302:isolation & purification*
D018509:DNA, Helminth