多重PCR检测捻转血矛线虫苯并咪唑类药物抗性等位基因

 利用GenBank发表的捻转血矛线虫β微管蛋白基因组DNA序列设计2对引物,建立检测捻转血矛线虫苯并咪唑类药物抗性的多重PCR方法,利用该方法检测我国不同地区捻转血矛线虫对苯并咪唑类药物的抗性,并用生物学检测验证。结果表明,建立的多重PCR方法在抗性检测上具有很强的特异性,澳大利亚抗性虫株只有F1和F3条带,上海敏感虫株只有F2和F3片段;序列分析证实抗性虫株编码β微管蛋白第200位氨基酸的密码子为TAC,敏感虫株为TTC。并且该法具有很高的灵敏性,检测基因组DNA的最低量为50 ng。生物学检测,澳大利亚抗性虫株丙硫苯咪唑LD50达0.54 ?g·ml-1,上海敏感虫株的LD50为0.0023 μg·ml-1,与多重PCR结果相符,说明多重PCR可以用于捻转血矛线虫苯并咪唑类药物抗性的检测。利用该技术检测源于新疆石河子、伊宁,安徽五河,江苏南京、徐州等地的虫株发现,这些地理株均表现为敏感型,丙硫苯咪唑LD50在0.0023～0.0032 ?g·ml-1之间,提示我国捻转血矛线虫苯并咪唑类药物抗性尚不严重。

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.