直接PCR方法在捻转血矛线虫耐苯丙咪唑类药物检测中的应用

Application of Direct PCR Method in the Detection of Benzimidazole Resistance in Haemonchus contortus

为临床上能快速准确的鉴定捻转血矛线虫对阿苯达唑的耐药性情况,本研究建立了一种快速、灵敏、特异性强、成本低的捻转血矛线虫Ⅰ型β微管蛋白Direct-PCR扩增方法,合成通用引物后,对PCR退火温度进行筛选、进行特异性、敏感性、重复性试验,建立了捻转血矛线虫Ⅰ型β微管蛋白Direct-PCR扩增方法。该方法能特异性的扩增出捻转血矛线虫Ⅰ型β微管蛋白基因中一段385 bp序列,最佳退火温度为60℃,最低检测限度为19 ng。本研究应用建立的Direct-PCR方法和常规PCR方法对32份样品进行Ⅰ型β微管蛋白的提取,结果表明,Direct-PCR扩增阳性率为97%,而常规PCR扩增总阳性率为83%,2种方法差异不显著(P>0.05),但扩增阳性率比常规PCR方法效率高,说明该方法更具有推广和应用价值。该试验成功建立了捻转血矛线虫Ⅰ型β微管蛋白Direct-PCR扩增方法,为捻转血矛线虫耐阿苯达唑的快速诊断提供了一种新的方法。

In order to quickly and accurately identify the drug resistance of Haemonchus contortus to albendazole in clinic,a rapid,sensitive,specific,and low-cost type I-β tubulin of Haemonchus contortus was established by the direct PCR amplification method.After synthesizing general primers,the PCR annealing temperature was screened,and the specificity,sensitivity,and repeatability were tested,the Haemonchus contortus type I-β tubulin was established and amplified. This method could selectively amplify a 385bp DNA fragment of the type I-β tubulin gene of Haemonchus contortus. The optimal annealing temperature was 60℃,and the minimum detection limit was 19 ng. The established direct PCR method and conventional PCR method were used to extract type I-β tubulin from 32 samples. The results showed that the positive rate of direct PCR amplification was 97%,while the total positive rate of conventional PCR amplification was 83%. There was no significant difference between the two methods(P> 0. 05),but the positive rate of amplification was higher than that of the conventional PCR method,indicating that this method had more popularization and application value. The test successfully established a direct PCR amplification method for Haemonchus contortus type I-βtubulin,providing a new method for the rapid diagnosis of albendazole resistance in Haemonchus contortus.