Multiplex PCR detection of allele on benzimidazole-resistance or -susceptibity in natural populations of Haemonchus contortus

A multiplex PCR was developed to detect benzimidazole-resistance (BZ-R) or -susceptibility (BZ-S) in Haemonchus contortus by amplification with 4 primers of a sequence of the GRU-1 gene of p-tubulin of//, contortus making use of sequence information available in Genbank. The method was based on two allele-non-specific primers and two allele-specific primers. Fl (264 bp) and F3 (799 bp) should be produced in BZ-R, F2 (585 bp) and F3 in BZ-S. With this method, it was found that H. contortus BZ-R strain from Australia showed Fl and F3, and the worm BZ-S strain from Shanghai did F2 and F3. Sequences analysis of the isotype 1 gene of p*-tubulin of BZ-R from Australia and BZ-S from Shanghai showed the code in residue 200 of the gene was respectively TAC and TTC. The LDS0 of Albendazole of the Australian BZ-R strain was 0.54 u,gml"', the Shanghai BZ-S train was only 0.0023 ug-ml' by EHA (Egg Hatch Assay). The multiplex PCR could determinate the genotype of single adult worm or several third stage larvae and was performed on at least 50 ng of genomic DNA. BZ-R H. contortus were not detected in Shihezi and Yining of Xinjiang Region, Wuhe of Anhui Province, Nanjing and Xuzhou of Jiangsu Province. The LD50 of the H. contortus from these locations to Albendazole as determined by EHA varied between 0.0023-0.0032 ug-ml"1. The result indicated that the multiplex PCR could be used to differentiate BZ-R and BZ-S of H. contortus and that the BZ-R situation of H. contortus was not serious in China.