title: A rapid and simplified method of DNA extraction for the detection of Brugia malayi infection in mosquitoes by PCR assay.
abstract: Currently used protocols for the extraction of filarial parasite DNA from mosquito samples are tedious and involve extensive use of expensive and hazardous chemicals. Therefore, in order to arrive at a simple procedure, four different methods (A, B, C and D) were tried for the extraction of DNA from mosquitoes infected with filarial parasite, Brugia malayi. Method D was found to be as efficient as the current procedure for the extraction of DNA from a single microfilaria in pools of 25 mosquitoes and the DNA was suitable for polymerase chain reaction (PCR) amplification, yielding a band of 322 base pairs with primers specific for B. malayi. Method D involved drying and crushing the mosquitoes to a powder, which was homogenized in 100 microl TE buffer, vortexed, boiled for 10 min, centrifuged at 14000 r.p.m. for 10 min, and the supernatant used for the PCR assay. Dot-blot hybridization confirmed the specificity of the PCR amplified fragment. The DNA extracted by this method was stable for about 1 year. When comparing with the standard method, the cost of a single PCR reaction, inclusive of DNA extraction, was reduced by 50% and the hands on time was minimized fivefold. Hence, this simple TE-based method is rapid, safe and also cost-effective in assessing the B. malayi infection in pools of vector mosquitoes.
authors: Vector Control Research Centre (ICMR), Medical Complex, Indira Nagar, 605 006, Pondicherry, India. mosquito@md5.vsnl.net.in
authors: V
authors: V
authors: Vasuki
authors: K P
authors: KP
authors: Patra
authors: S L
authors: SL
authors: Hoti
mesh_terms: D000818:Animals; D017178:Brugia malayi / Q000235:genetics / Q000302:isolation & purification*; D009033:Culicidae / Q000469:parasitology*; D018509:DNA, Helminth / Q000032:analysis / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D018509:DNA, Helminth