摘要
目的建立同时检测蓝氏贾第鞭毛虫和微小隐孢子虫的双重荧光定量PCR两步检测方法。方法针对蓝氏贾第鞭毛虫的gdh基因和微小隐孢子虫的cowp基因分别设计特异性引物和TaqMan探针。优化引物和探针浓度后,确定反应体系和反应条件,对其灵敏度、特异性、稳定性和重复性进行评价。通过与金标层析法进行医源性腹泻样本检测的比较评估该方法的实际应用价值。结果该方法能特异地检测蓝氏贾第鞭毛虫和微小隐孢子虫,对其它非目标虫种均不产生扩增曲线,特异性良好。对阳性质粒pMDTM19-T-GIA和pMDTM19-T-CRY同时定量扩增的敏感度分别为45.1 copies/μL和52.8 copies/μL;阳性质粒标准品的标准曲线在109 copies/μL^101 copies/μL之间线性关系良好(R2=0.99),批内和批间重复实验的变异系数均小于5%。该方法与金标层析法具有较好的一致性。结论建立的双重荧光定量PCR两步法可快速、灵敏、特异地同时检测蓝氏贾第鞭毛虫和微小隐孢子虫,具有较好的实际应用价值。
Aimed to establish a two-step duplex fluorescence quantitative PCR method for simultaneous detection of Giardia lamblia and Cryptosporidium parvum. Specific primers and TaqMan probes were designed according to the gdh gene sequence of Giardia lamblia and the cowp gene sequence of Cryptosporidium parvum. Then we constructed the standard plasmids. The reaction system and conditions were determined after optimizing the concentration of the primers and probes. The sensitivity, specificity, stability and repeatability of the method were evaluated. What’s more, we compared this established method with gold immunochromat ographic assay by detecting iatrogenic diarrhea samples suspected of "two insects" infection to evaluate its practical application value. The established method could specifically detect Giardia lamblia and Cryptosporidium parvum, and had no amplification curves for other non-target species, which showed good specificity of the method. The sensitivity of the method for simultaneous quantitative amplification of positive plasmids pMDTM19-T-GIA and pMDTM19-T-CRY was 45.1 copies/μl and 52.8 copies/μl, respectively;the standard curve showed good linearity(R2=0.99) between 109 copies/μL^101 copies/μL. The coefficient of variation for both intra-and inter-assay replicates was less than 5%. At the same time, the coincidence rate between the established method and gold immunochromat ographic assay is 100%. Results of this study suggested that the two-step duplex fluorescence quantitative PCR method not only can be used to detect Giardia lamblia and Cryptosporidium parvum simultaneously, quickly, sensitively and specifically, but also has good practical application value.
作者
李佳
黄达娜
张晓敏
牛丛
万成松
张仁利
LI Jia;HUANG Da-na;ZHANG Xiao-min;NIU Cong;WAN Cheng-song;ZHANG Ren-li(School of Public Health,Southern Medical University,Guangzhou 510515,China;Shenzhen Center for Disease Control and Prevention,Shenzhen 518055,China;Guangdong Province Key Laboratory of Tropical Disease Research,Guangzhou 510515,China)
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2020年第1期32-39,共8页
Chinese Journal of Zoonoses
基金
国家自然科学基金项目（No.81802018）
深圳市三名工程项目（No.SZSM201611064）
深圳市重大科技研发项目（No.20170801001）
广东省热带病研究重点实验室开放课题（No.RDBYJ2018001)~~
