A rapid and simplified method of DNA extraction for the detection of Brugia malayi infection in mosquitoes by PCR assay.
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.
Vasuki
V
V
Vector Control Research Centre (ICMR), Medical Complex, Indira Nagar, 605 006, Pondicherry, India. mosquito@md5.vsnl.net.in
Patra
K P
KP
Hoti
S L
SL
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*
D018509:DNA, Helminth