华支睾吸虫LAMP方法的建立与初步应用  

Establishment and preliminary application of a LAMP assay for the detection of Clonorchis sinensis

作　　者：孙晓敬 马茜 田甜 张磊 刘丽君 姚佳 汪洋 SUN Xiaojing;MA Xi;TIAN Tian;ZHANG Lei;LIU Lijun;YAO Jia;WANG Yang(Department of Pathogen Biology,School of Basic Medical Science,Xi’an Medical University,Xi’an 710021,China)

机构地区：[1]西安医学院基础医学部病原生物学教研室,西安710021

出　　处：《黑龙江畜牧兽医》2024年第13期47-51,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金面上项目(32070069);陕西省科技厅青年基金项目(2021JQ-781);陕西省自然科学基础研究计划青年项目(2024JQYBQN-0176);陕西省教育科学“十四五”规划课题一般项目(SGH21Y0266)。

摘　　要：为了建立华支睾吸虫环介导等温扩增(loop-mediated isothermal amplification,LAMP)快速检测方法,试验利用GenBank中华支睾吸虫(Clonorchis sinensis)的线粒体全基因组设计特异性引物,然后构建LAMP方法,并验证了LAMP方法的特异性和敏感性;以及用粪便镜检(金标准)、PCR和LAMP方法同时检测45份犬粪便样本,评价LAMP方法的检测效果。结果表明:经序列比对,筛选得到华支睾吸虫特异性基因Unit R2,以该基因序列为靶标构建检测方法,优化后的总体积为50μL,Bst3.0 DNA聚合酶的最适用量为1μL,MgSO_(4)的最适浓度为6 mmol/L,最佳反应温度为63℃,最佳反应时间为40 min。构建的LAMP方法可以检测华支睾吸虫整个生命发育周期,实现了对其成虫、嚢蚴及虫卵的全方位覆盖;与其他种类寄生虫无交叉反应;对基因组DNA的最低检测限为10 fg,是PCR方法的100倍;与金标准相比,LAMP方法的特异性为100%(30/30),敏感性为93.33%(14/15)。说明试验建立的华支睾吸虫LAMP方法特异性强、敏感性高、检测结果准确,具有快速检测华支睾吸虫的潜力。In order to establish a rapid detection method for loop-mediated isothermal amplification(LAMP)of Clonorchis sinensis,in the test,the LAMP method was constructed by designing specific primers using the GenBank mitochondrial whole genome of Clonorchis sinensis;then the specificity of the LAMP method was verified and the sensitivity of the detection system was evaluated.45 canine fecal samples were tested simultaneously by fecal microscopy,PCR and LAMP methods to evaluate the accuracy of LAMP.The results showed that Unit R2,a specific gene of Clonorchis sinensis,was screened by sequence comparison;and the assay was constructed using this gene sequence as the target.The optimized total volume was 50μL;the optimal amount of Bst3.0 DNA polymerase was 1μL;the optimal concentration of MgSO4 was 6 mmol/L;the optimal reaction temperature was 63℃,and the optimal reaction time was 40 min.The constructed LAMP method could detect the whole life cycle of Clonorchis sinensis,which could realize the comprehensive coverage of the adult,cysticercus and eggs of Clonorchis sinensis.The method had no cross-reactivity with other species of parasites with a minimum detection limit of 10 fg for genomic DNA,which was 100 times higher than that of PCR;it had a specificity of 100%(30/30)and a sensitivity of 93.33%(14/15)compared with the gold standard.The results suggested that the established LAMP method for Clonorchis sinensis had high specificity,high sensitivity and accurate results,and had the potential for rapid detection of Clonorchis sinensis.

关 键 词：华支睾吸虫 环介导等温扩增(LAMP) 线粒体基因组 核酸 检测 

分 类 号：S852.73+5[农业科学—基础兽医学] R383.2[农业科学—兽医学]