华支睾吸虫囊蚴RPA检测方法的建立  

Development of RPA assay for the detection of Clonorchis sinensis metacercaria

作　　者：张春玲 邱阳元 郝朔 白雪[1] 刘明远[1] 张藜潇 刘晓雷[1] ZHANG Chun-ling;QIU Yang-yuan;HAO Shuo;BAI Xue;LIU Ming-yuan;ZHANG Li-xiao;LIU Xiao-lei(Key laboratory of Zoonosis Research of Ministry of Education/Institute of Zoonosis,Jilin University,Changchun 130062,China)

机构地区：[1]吉林大学人兽共患病研究所人兽共患病研究教育部重点实验室,吉林长春130062

出　　处：《中国兽医科学》2021年第4期441-445,共5页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2017YFC1601202);中央高校基本科研业务费(2017TD-32)。

摘　　要：为了建立灵敏、快速的鉴别华支睾吸虫诊断方法,本研究根据华支睾吸虫囊蚴的COX-1基因序列设计引物,建立检测华支睾吸虫囊蚴的RPA方法。结果显示:所建立的RPA的灵敏度可达到2.75 ng/μL,而与日本血吸虫、隐孢子虫、阔节裂头绦虫、东方次睾吸虫囊蚴、台湾次睾吸虫囊蚴、异尖线虫和棘颚口线虫均无交叉反应;经条件优化后,可在35℃下5 min内观察到检测结果。另外,在对实际样品检测中,通过添加核酸染料SYBR GreenⅠ,无需PCR仪及紫外凝胶系统的条件下,可以快速地鉴别出华支睾吸虫囊蚴及虫卵。结论,本研究成功地建立了华支睾吸虫囊蚴PRA检测方法,这在食品安全检测中具有重要的应用前景。In order to establish a sensitive and efficient diagnostic method to identify Clonorchis sinensis,in this study,the primers were designed based on COX-1 gene sequence of Clonorchis sinensis metacercaria,and the detection method of recombinase polymerase amplification(RPA) for Clonorchis sinensis metacercaria was been established.The results showed that the sensitivity of RPA reached 2.75 ng/μL and RPA was specificity,and there were no cross-reacts with Schistosoma japonicum,Cryptosporidium,Diphyllobothrium latum,Orientia orientalis,Hypotremes taiwan,Anisakis and Gnathostoma siamense.After optimization,the test results were been observed within 5 minutes at 35 ℃.In addition,with the SYBR Green Ⅰ acid dye addition,the Clonorchis sinensis metacercaria and eggs were been identified rapidly even if with no PCR or UV gel system.Thus,the PRA detection method of Clonorchis sinensis metacercariae was successfully been established in this study,which had important application prospects for food safety detection.

关 键 词：华支睾吸虫 囊蚴 RPA COX-1 特异性 灵敏性 

分 类 号：S852.735[农业科学—基础兽医学]