Comparative efficacy of AS-PCR and RFLP-PCR techniques for detection of benzimidazole resistance in Haemonchus contortus of sheep*

Benzimidazole (BZ) resistance in Haemonchus contortus is due to the mutation at 200 codon of the β-tubulin isotype I gene which substitute phenylalanine (Phe) into tyrosine (Tyr) amino acid. A study was conducted to assess the comparative efficacy of allele specific-PCR (AS-PCR) and restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) techniques for diagnosis of benzimidazole resistance in the β-tubulin isotype I gene of H. contortus. A total of 162 adult male H. contortus were collected from various locations of Rajasthan and analyzed for BZ resistance. The genotypes of parasites for BZ resistance were assigned as rr (homozygous resistant), rS (heterozygous) and SS (susceptible) types by both the techniques. The genotypic frequencies of different genotypes (rr, rS and SS) were found very significantly different (P < 0.001) among genotypes. Overall, the prevalence of BZ resistant allele (r) was found 87% and 86% using AS-PCR and RFLP-PCR technique, respectively. The results indicated that both techniques are rapid and sensitive, and can be used at large scale for diagnosis of BZ resistance in H. contortus. However, RFLP-PCR is an easy, highly reproducible and less expensive than allele specific-PCR.