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首页> 外文期刊>Parasitology >Molecular detection of benzimidazole resistance in Haemonchus contortus using real-time PCR and pyrosequencing.
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Molecular detection of benzimidazole resistance in Haemonchus contortus using real-time PCR and pyrosequencing.

机译：使用实时荧光定量PCR和焦磷酸测序技术对捻转血矛线虫的苯并咪唑抗性进行分子检测。

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摘要

Benzimidazoles (BZ) are widely used to treat parasitic nematode infections of humans and animals, but resistance is widespread in veterinary parasites. Several polymorphisms in beta-tubulin genes have been associated with BZ-resistance. In the present study, we investigated beta-tubulin isotype 1 sequences of 18 Haemonchus contortus isolates with varying levels of resistance to thiabendazole. The only polymorphism whose frequency was significantly increased in the resistant isolates was TTC to TAC at codon 200. Real-time PCR (using DNA from 100 third-stage larvae, L3s) and pyrosequencing (from DNA from 1000-10 000 L3s) were used to measure allele frequencies at codon 200 of these isolates, producing similar results; drug sensitivity decreased with increasing TAC frequency. Pyrosequencing was also used to measure allele frequencies at positions 167 and 198. We showed that such measurements are sufficient to assess the BZ-resistance status of most H. contortus isolates. The concordance between real-time PCR and pyrosequencing results carried out in different laboratories indicated that these tools are suitable for the routine diagnosis of BZ-resistance in H. contortus. The molecular methods were more sensitive than the 'egg hatch test', and less time-consuming than current in vivo- or in vitro-anthelmintic resistance detection methods. Thus, they provide a realistic option for routine molecular resistance testing on farms.
机译：苯并咪唑（BZ）广泛用于治疗人和动物的寄生线虫感染，但耐药性在兽医寄生虫中广泛存在。 β微管蛋白基因中的几种多态性已与BZ耐药性相关。在本研究中，我们调查了18种弯曲变形杆菌的β-微管蛋白同型1序列，这些菌株对噻菌灵的抗药性不同。在抗性分离物中，频率显着增加的唯一多态性是密码子为200的TTC到TAC。使用实时PCR（使用来自100个第三级幼虫的L3s的DNA）和焦磷酸测序（来自1000-10 000 L3s的DNA）。测量这些分离株密码子200的等位基因频率，产生相似的结果;随着TAC频率的增加，药物敏感性降低。焦磷酸测序也可用于测量位置167和198处的等位基因频率。我们显示，这些测量足以评估大多数弯曲杆菌分离株的BZ抗性状态。在不同的实验室中进行的实时PCR和焦磷酸测序结果之间的一致性表明，这些工具适合于对锥虫嗜血杆菌抗BZ的常规诊断。分子方法比“卵孵化测试”更灵敏，并且比目前的体内或体外驱虫抗药性检​​测方法耗时少。因此，它们为农场的常规分子抗性测试提供了一个现实的选择。

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