Onchocercal DNA amplification using beta actin gene primers compared with first internal transcribed spacer sequences for monitoring onchocerciasis eradication strategy

Abstract

Ongoing treatment control strategy against onchocerciasis or river blindness will need efficient diagnostic method to evaluate the mass drug administration with ivermectin (Mectizan®). Sole reliance on classical parasitological method of skin snip microscopy for detecting microfilaria has proved less sensitive during post-control period. Detecting any parts of the parasite stages such as antigens, enzymes and nucleic acids (DNA and RNA) is a definitive diagnosis and highly sensitive. This study was to evaluate the diagnostic reliability of the beta actin gene primer pair to confirm its suitability for validating presence or absence of skin microfilaria at post-treatment. DNA extracted from skin snip samples (n=15) from an onchocerciasis mesoendemic area, three from non-endemic, two adult worm fragments and blank wells with only master mix (n=7) were subjected to endpoint polymerase chain reaction (PCR) analysis. Four of the samples had shown reactivity with first internal transcribed spacer (ITS1) primer pair. The amplicons were sequenced and subjected to basic local alignment search tool (BLAST). Out of the 12 amplicons in agarose gel, there were 6 sharp and 6 faint bands of 100bp molecular weight as documented. The sharp bands included 3 ITS1 and one field positive samples, and 2 positive controls. The BLAST analysis showed moderate homology with beta actin with accession number M84916 available in the public GenBank database, and with the positive control sequences. This study has shown that DNA amplification with beta actin gene may be very specific and more sensitive compared with the ITS gene primer sequences.

Keywords: Beta actin, DNA amplification, Onchocerca volvulus, polymerase chain reaction; sequence alignment, skin microfilaria