Oligonucleotide based magnetic bead capture of Onchocerca volvulus DNA for PCR pool screening of vector black flies.
BACKGROUND
Entomological surveys of Simulium vectors are an important component in the criteria used to determine if Onchocerca volvulus transmission has been interrupted and if focal elimination of the parasite has been achieved. However, because infection in the vector population is quite rare in areas where control has succeeded, large numbers of flies need to be examined to certify transmission interruption. Currently, this is accomplished through PCR pool screening of large numbers of flies. The efficiency of this process is limited by the size of the pools that may be screened, which is in turn determined by the constraints imposed by the biochemistry of the assay. The current method of DNA purification from pools of vector black flies relies upon silica adsorption. This method can be applied to screen pools containing a maximum of 50 individuals (from the Latin American vectors) or 100 individuals (from the African vectors).


RESULTS
We have evaluated an alternative method of DNA purification for pool screening of black flies which relies upon oligonucleotide capture of Onchocerca volvulus genomic DNA from homogenates prepared from pools of Latin American and African vectors. The oligonucleotide capture assay was shown to reliably detect one O. volvulus infective larva in pools containing 200 African or Latin American flies, representing a two-four fold improvement over the conventional assay. The capture assay requires an equivalent amount of technical time to conduct as the conventional assay, resulting in a two-four fold reduction in labor costs per insect assayed and reduces reagent costs to $3.81 per pool of 200 flies, or less than $0.02 per insect assayed.


CONCLUSIONS
The oligonucleotide capture assay represents a substantial improvement in the procedure used to detect parasite prevalence in the vector population, a major metric employed in the process of certifying the elimination of onchocerciasis.
Centro de Biotecnología Genómica, Instituto Politécnico Nacional, Reynosa, Tamaulipas, México.
Hemavathi
H
Gopal
Hassan K
HK
Hassan
Mario A
MA
Rodríguez-Pérez
Laurent D
LD
Toé
Sara
S
Lustigman
Thomas R
TR
Unnasch
D000818:Animals; D018509:DNA, Helminth / Q000235:genetics / Q000302:isolation & purification*; D004772:Entomology / Q000379:methods*; D005260:Female; D008280:Magnetics; D008403:Mass Screening / Q000379:methods; D008863:Microspheres; D009841:Oligonucleotides / Q000235:genetics; D017181:Onchocerca volvulus / Q000235:genetics / Q000302:isolation & purification*; D010274:Parasitology / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012843:Simuliidae / Q000469:parasitology*
D018509:DNA, Helminth; D009841:Oligonucleotides