针对隐孢子虫和贾第鞭毛虫双重实时荧光PCR检测方法的建立.docVIP

隐孢子虫和贾第鞭毛虫双重实时荧光PCR检测方法的建立 廖立珊1,2，肖娜3，孙洁1,2，阮周曦1,2，林庆燕1,2，吕建强1,2，曹琛福1,2，曾少灵1,2，张彩虹1,2，陶虹1,2，刘建利1,2，唐金明1,2，杨俊兴1,2，花群义1,2，秦智锋1,2* (1.深圳出入境检验检疫局，广东，深圳市，518001； 2.深圳市外来有害生物质检测技术研发重点实验室，广东深圳，518045； 3.深圳市盐田区疾病预防控制中心，广东，深圳市，518001） 摘要：本研究根据隐孢子虫和贾第鞭毛虫相对保守序列,设计两对引物及其相应的Taqman探针,通过对引物和探针浓度、Taq 酶以及反应条件等优化筛选后，建立了能同时检测隐孢子虫和贾第鞭毛虫的双重实时荧光PCR方法。所建立的隐孢子虫和贾第鞭毛虫双重荧光PCR检测方法的灵敏度为 10-9，相当于46.1copies/μL，重复性良好，特异度实验未发现有非特异性扩增。结论本研究建立的双重荧光定量RT-PCR技术可以快速、准确地检测隐孢子虫和贾第鞭毛虫。? 关键词：隐孢子虫，贾第鞭毛虫，双重实时荧光PCR Development of Duplex fluorescence quantitative PCR for Cryptosporidium and Giardia in water samples LIAO Li-shan1,2,XIAO Na3,SUN Jie1,2, RUAN Zhou-xi1,2, LIN Qing-yan1,2, LV Jian-qiang1,2, CAO Chen-fu1,2, ZENG Shao-ling1,2, ZHANG Cai-hong1,2,TAO Hong1,2 ,LIU Jian-li1,2,TANG Jin-ming1,2, YANG Jun-xing1,2, HUA Qun-yi1,2, QIN Zhi-feng1,2 （1. Shenzhen Entry-Exit Inspection Quarantine Bureau，Shenzhen，Guangdong，518001, China； 2．Shenzhen Key Laboratory of Inspection Research Development of Alien Pests Shenzhen，Guangdong，518045, China； 3. Yantian District of Shenzhen City Center for Disease Control and Prevention Shenzhen，Guangdong，518001, China) Abstract：According to conservative sequences of Cryptosporidium and Giardia lamblia,two sets of primers and Taq-man probes were designed．After optimization of reaction conditions, we set up a double fluorescent real-time PCR method to detect Cryptosporidium and Giardia lamblia.The sensitivity of Cryptosporidium and Giardia lamblia dual fluorescence PCR detection method was 10-9,equivalent to 46.1 copies/ul,reproducibility was good, and high specificity.The detection system based on real-time PCR could be utilized to rapidly and sensitively detect Cryptosporidium and Giardia lamblia． Key words：Cryptosporidium parvum；Giardia lamblia；Double real time fluorescence PCR. 隐孢子虫(Cryptosporidium parvum)是一种全球普遍存在的人兽共患寄生原虫，可引起隐孢子虫病。寄生于人体的种主要是微小隐孢子虫, 该虫是机会致病原虫, 但也是一种重要的腹泻病原。蓝氏贾第鞭毛虫(Giardia lamblia)简称贾第鞭毛虫，是一种寄生于人类和动物肠道的原生动物。寄生于人体小肠、胆囊，主要在十二指肠，可引起腹痛、腹泻和吸收不良等症状，引起贾第虫病（Giardia