Laboratory evaluation of Ssp I PCR assay for the detection of Wuchereria bancrofti infection in Culex quinquefasciatus.
OBJECTIVE
There is a need to delimit the areas of filariasis transmission in view of the Filariasis Elimination Programme launched in India. Infection rate in vectors is an important parameter in determining transmission and it is conventionally assessed by dissection and microscopy. A PCR assay based on Ssp I repeats of Wuchereria bancrofti has shown potential in the detection of infection in vectors. The aim of the present study was to evaluate the specificity and sensitivity of this assay on W. bancrofti and its vector, Culex quinquefasciatus, prevalent in India.


METHODS
The DNA from pools of C. quinquefasciatus to which W. bancrofti microfilariae (mf) were added, was extracted by lysing with 0.1 M NaOH and 0.2 per cent sodium dodecyl sulphate (SDS), followed by silica absorption in the presence of guanidinium thiocyanate. The PCR assay of the DNA samples was carried out using NV-1 and NV-2 primers and the species specific SspI band was visualized on agarose gels stained with ethidium bromide.


RESULTS
The Ssp I PCR assay was found to be highly species specific, as it did not detect the DNA of a closely related filarial parasite, Brugia malayi. The assay detected as little as 0.04 pg of W. bancrarofti DNA. Minimum number of parasite detectable in pools of mosquitoes was 1 mf. A pool size of 50 mosquitoes was found to be optimum for the PCR assay.


CONCLUSIONS
The Ssp I PCR assay was found to be highly specific and sensitive in detecting filarial parasite in pools of mosquitoes and therefore has potential application in rapid assessment of transmission of filariasis.
Hoti
S L
SL
Vector Control Research Centre (ICMR), Pondicherry, India.
Vasuki
V
V
Patra
K P
KP
Hariths
V R
VR
Ravi
G
G
Sushma
S
S
D000818:Animals; D003465:Culex / Q000469:parasitology*; D005368:Filariasis / Q000175:diagnosis*; D018840:HSP70 Heat-Shock Proteins; D016133:Polymerase Chain Reaction / Q000379:methods*; D011494:Protein Kinases; D012091:Repetitive Sequences, Nucleic Acid; D012097:Repressor Proteins; D029702:Schizosaccharomyces pombe Proteins; D014958:Wuchereria bancrofti / Q000235:genetics*
D018840:HSP70 Heat-Shock Proteins; C111935:PSS1 protein, S pombe; D012097:Repressor Proteins; D029702:Schizosaccharomyces pombe Proteins; D011494:Protein Kinases