重组酶介导的核酸等温扩增荧光法快速检测日本血吸虫感染性钉螺  

Rapid detection of Schistosoma japonicum-infected snails with recombinaseaided isothermal amplification assay

作　　者：李婷 刘燕红 赵松[1] 熊春蓉[1] 董萱 张键锋[1] 李伟[1] 应清界 杨坤[1] LI Ting;LIU Yan-Hong;ZHAO Song;XIONG Chun-Rong;DONG Xuan;ZHANG Jian-Feng;LI Wei;YING Qing-Jie;YANG Kun(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 GeneTechnology Co., Ltd., 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

出　　处：《中国血吸虫病防治杂志》2019年第2期109-114,120,共7页Chinese Journal of Schistosomiasis Control

基　　金：江苏省医学重点人才项目(ZDRCA2016056);江苏省卫生计生委科研课题(X201802)

摘　　要：目的建立重组酶介导的核酸等温扩增荧光(Recombinase aided amplification,RAA)法快速检测日本血吸虫感染性钉螺技术,并探索钉螺样品的最佳处理方式。方法将钉螺样本分成3组,每组均含7个亚组,其中6个亚组分别为50只阴性钉螺中混合1、2、3、4、5、10只日本血吸虫感染性钉螺,1个亚组为100只阴性钉螺中混合1只感染性钉螺。3组钉螺分别采用压碎去壳核酸试剂盒提取法、压碎去壳核酸粗提法和直接压碎带壳核酸粗提法进行处理,并分别进行荧光RAA与PCR法检测,对检测结果进行比较。结果建立了荧光RAA检测技术,工作温度为39℃,30 min内即可完成日本血吸虫感染性钉螺检测。钉螺群体样本采用压碎去壳核酸试剂盒提取法处理后,荧光RAA法最低可检测到100只阴性钉螺中混合1只感染性钉螺,PCR法最低可检测到50只阴性钉螺中混合1只感染性钉螺;采用压碎去壳核酸粗提法处理后,荧光RAA法最低可检测到100只阴性钉螺中混合1只感染性钉螺,PCR法最低可检测到50只阴性钉螺中混合3只感染性钉螺;采用直接压碎带壳核酸粗提法处理后,荧光RAA法最低可检出50只阴性钉螺中混合10只感染性钉螺,PCR法仅可检测出50只阴性钉螺中混合10只感染性钉螺。结论成功建立了钉螺群体样本中日本血吸虫感染性钉螺快速检测的荧光RAA法,该方法具有快速、灵敏、操作简便等特点;压碎去壳核酸粗提法为钉螺样品的最优处理方式。Objective To develop a florescent recombinase-aided amplification(RAA) assay for rapid detection of Schistosoma japonicum-infected Oncomelania snails and explore the optimal method for treatment of snail samples. Methods Snail samples were divided into 3 groups, and each group consisted of 7 subgroups. There were 50 uninfected snails mixed with 1, 2, 3, 4,5 and 10 infected snails in the 6 subgroups, respectively, and the remaining subgroup contained 100 uninfected snails mixed with1 infected snails. DNA was extracted from snails in the three groups using a genomic DNA extraction kit following snail crushing and snail shells removal, crude nucleic acid extraction assay following snail crushing and snail shells removal, and crude nucleic acid extraction assay following direct snail crushing with snail shells preserved, and subjected to florescent RAA and PCR assays. The detection results were compared between the two assays. Results A florescent RAA assay was developed, which completed the detection of S. japonicum-infected snails at 39 ℃ within 30 min. Following DNA extraction from mass snail samples with a genomic DNA extraction kit following snail crushing and snail shells removal, the lowest detection limit of the florescent RAA assay was one infected snail mixed in 100 uninfected snails, while the lowest detection limit of PCR assay was one infectedsnail mixed in 50 uninfected snails. Following DNA extraction using crude nucleic acid extraction method following snail crushing and snail shells removal, the lowest detection limit of the florescent RAA assay was one infected snail mixed in 100 uninfected snails, while the lowest detection limit of PCR assay was 3 infected snails mixed in 50 uninfected snails. Following DNA extraction with a crude nucleic acid extraction assay following direct snail crushing with snail shells preserved, the lowest detection limit of the florescent RAA assay was 10 infected snails mixed in 50 uninfected snails, while the lowest detection limit of PCR assay was 10 infected snails

关 键 词：日本血吸虫 钉螺 重组酶介导的核酸等温扩增技术 荧光检测 

分 类 号：R383.24[医药卫生—医学寄生虫学]