基于重组酶介导等温扩增技术的广州管圆线虫核酸检测方法的建立  

Establishment of a recombinase-aided isothermal amplification assay for nucleic acid detection of Angiostrongylus cantonensis

作　　者：张强 丁昕 刘燕红 刘剑峰[1] 徐祥珍[1] 应清界 戴洋[1] 曹俊[1,3] ZHANG Qiang;DING Xin;LIU Yan-Hong;LIU Jian-Feng;XU Xiang-Zhen;YING Qing-Jie;DAI Yang;CAO Jun(National Health Commission Key Laboratory of Parasitic Disease Control and Prevention,Jiangsu Provincial Key Laboratory on Parasite and Vector Control Technology,Jiangsu Institute of Parasitic Diseases,Wuxi 214064,China;Jiangsu Qitian Gene Tech-nology Co.,Ltd.,China;Public Health Research Center,Jiangnan University,China)

机构地区：[1]国家卫生健康委员会寄生虫病预防与控制技术重点实验室、江苏省寄生虫与媒介控制技术重点实验室、江苏省血吸虫病防治研究所,无锡214064 [2]江苏省奇天生物科技有限公司 [3]江南大学公共卫生研究中心

出　　处：《中国血吸虫病防治杂志》2020年第4期350-354,共5页Chinese Journal of Schistosomiasis Control

基　　金：江苏省“科教强卫工程”项目(ZDXKA2016016);江苏省属公益类科研院所自主科研项目(BM2018020);江苏省青年医学人才项目(QNRC2016622)。

摘　　要：目的建立一种基于重组酶介导等温扩增技术(RAA)的广州管圆线虫核酸检测方法。方法选择广州管圆线虫内转录间隔区1(ITS1)基因序列作为靶基因序列,设计、合成并筛选出特异性最强的引物和探针,构建用于广州管圆线虫核酸检测的基础及荧光RAA检测方法。分别以含检测靶基因片段序列的不同拷贝数重组质粒以及不同浓度广州管圆线虫基因组DNA为模板进行焚光RAA扩增,评价其检测敏感性;分别以广州管圆线虫、曼氏血吸虫、似蚓蛔线虫、华支睾吸虫、细粒棘球绦虫、十二指肠钩口线虫及小管福寿螺和藁杆双脐螺螺体基因组DNA为模板进行荧光RAA扩增,评价其检测特异性。结果成功建立了用于广州管圆线虫核酸检测的荧光RAA法,该方法可在37℃20min内对广州管圆线虫特异性DNA片段实现实时扩增。以含靶基因片段序列的不同拷贝数重组质粒和不同浓度广州管圆线虫基因组DNA为模板时,该法最低检出限分别为10拷贝/μL重组质粒和100 pg/μL基因组DNA;以曼氏血吸虫、似蚓蛔线虫、华支睾吸虫、细粒棘球绦虫、十二指肠钩口线虫及小管福寿螺和藁杆双脐螺螺体基因组DNA为模板,检测结果均为阴性。结论成功建立了一种可用于广州管圆线虫核酸检测的荧光RAA法,其检测简便快速,具备较好的敏感性和特异性。Objective To establish a recombinase-aided isothermal amplification(RAA) assay for the nucleic acid detection of Angiostrongylus cantoneusis.Methods The internal transcribed spacer-1(ITS1) gene sequence of A.cautonensis was used as the detection target sequence,and the specific primers and probes were designed and synthesized,followed by screening of the primers and probes with the highest specificity,to establish the basic and fluorescent RAA assay for nucleic acid detection of A.cantonensis.The sensitivity of the fluorescent RAA assay was evaluated by using the target gene fragment sequence-contained recombinant plasmids at various copy numbers and the genomic DNA from A.cantonensis as the template DNA samples,and the specificity of the fluorescent RAA assay was evaluated by using the genomic DNA from A.cantonensis,Schistosoma mansoni, Ascaris lumbricoides, Clonorchis sinensis,Echinococcus granulosus and Ancylostoma duodeuale,as well as Pomacea canaliculata and Biomphalaria straminea snail tissues as the template DNA samples.Results A fluorescent RAA assay was successfully established for nucleic acid detection of A.cantonensis,which achieved real-time amplification of the specific DNA fragment of A.cantonensis within 20 min at 37 ℃.By using the target gene fragment sequence-contained recombinant plasmids at various copy numbers and the genomic DNA from A.cantonensis as the DNA templates,the lowest detection limits of the fluorescent RAA assay were 10 copies/μL of recombinant plasmids and 100 pg/μL of genomic DNA,respectively.The fluorescent RAA assay was negative for detection of the genomic DNA from A.cantonensis, S.mansoui, A.lumbricoides, C.sinensis,E.granulosus,A.duodenale, and P.canaliculata and B.straminea snail tissues.Conclusion A simple,rapid fluorescent RAA assay has been successfully established,which has a high sensitivity and specificity for the nucleic acid detection of A.cantonensis.

关 键 词：广州管圆线虫 等温扩增 重组酶 核酸检测 检测效能 

分 类 号：R383.19[医药卫生—医学寄生虫学]