IMS-qPCR检测水源性微小隐孢子虫方法的建立

中国寄生虫学与寄生虫病杂志 ›› 2013, Vol. 31 ›› Issue (3): 4-180-184.

IMS-qPCR检测水源性微小隐孢子虫方法的建立

高姗姗1,2，吴绍强3，罗静1，王承民1，张敏1，赵宝华2，何宏轩1 *

1 中国科学院动物研究所动物生态与保护生物学院重点实验室和国家野生动物疫病研究中心，北京 100101；2 河北师范大学生命科学学院，石家庄 050016；3 中国检验检疫科学研究院动物检疫研究所，北京 100123

出版日期:2013-06-30 发布日期:2013-07-17

Development of an IMS-qPCR Method for Detection of Cryptosporidium parvum in Water

GAO Shan-shan1,2, WU Shao-qiang3, LUO Jing1, WANG Cheng-min1, ZHANG Min1, ZHAO Bao-hua2, HE Hong-xuan1 *

1 National Research Center for Wildlife Diseases, Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101，China；2 College of Life Science, Hebei Normal University, Shijiazhuang 050016，China；3 Institute of Animal Quarantine, Chinese Academy of Inspection and Quarantine, Beijing 100123, China

Online:2013-06-30 Published:2013-07-17

摘要/Abstract

摘要： 目的将免疫磁珠分离技术（IMS）和荧光探针定量PCR（qPCR）相结合，建立水源性微小隐孢子虫卵囊的检测方法。方法以抗微小隐孢子虫卵囊表面抗原Cp23单克隆抗体包被链霉素磁珠，制备特异免疫磁珠，根据卵囊回收率优化分离和富集卵囊条件。根据微小隐孢子虫核糖体DNA小亚基（18S rDNA）基因（登录号AB513881.1）序列设计引物和荧光标记探针，以分离纯化后的微小隐孢子虫卵囊基因组DNA为模板进行扩增，将扩增产物克隆至Peasy-T1载体。经筛选鉴定后，将重组质粒倍比稀释至104~108 copy/μl，荧光探针定量PCR检测，并绘制标准曲线。用荧光探针定量PCR分别检测微小隐孢子虫、贝氏隐孢子虫、刚地弓形虫、犬隐孢子虫和大肠埃希菌基因组DNA，判断该方法的特异性；检测100~108 copy/μl重组质粒，判断该方法的敏感性。对采自河北某奶牛养殖场的50份污水样品，分别采用免疫荧光分析法（IFA）和IMS-qPCR检测，并比较分析检测结果。结果磁珠与Cp23单克隆抗体的最佳孵育浓度为20 ng/ml，最佳孵育时间为30 min，最佳捕获时间为30 min，卵囊的回收率>95%。PCR扩增产物长度为272 bp，重组质粒经酶切和测序鉴定正确。荧光探针定量PCR结果显示，重组质粒拷贝数与Ct值之间呈现良好的线性关系，相关系数r2=0.996 1。特异性和敏感性检测结果显示，仅微小隐孢子虫有扩增条带，最低10 copy/μl微小隐孢子虫卵囊18S rDNA重组质粒可被检测到。以IFA检测结果为金标准，50份水样的检测结果显示，IMS-qPCR的特异性为100%（18/18），敏感性为93.8%（30/32）。结论 IMS-qPCR可用于检测水源性微小隐孢子虫卵囊。

关键词: 微小隐孢子虫, 免疫磁珠, 荧光探针定量PCR, 核糖体DNA小亚基

Abstract: Objective To develop a detection method for Cryptosporidium parvum oocysts from water samples, which combined immunomagnetic separation （IMS） with Taqman real-time PCR （qPCR）. Methods Conditions of sepa-ration and enrichment of IMS method by using specific streptavidin magnetic beads coated with monoclonal antibodies Cp23 directed against C. parvum oocysts were optimized. Special primers of PCR and Taqman probes were designed referring to the 18S rDNA gene of C. parvum（GenBank Accession No. AB513881.1）. The conserved genes were amplified from genomic DNA of C. parvum, and then cloned into Peasy-T1 vector. Tenfold dilutions of positive plasmids（104-108 copy/μl） were used to construct a standard curves by Taqman qPCR. The specificity of the assay was determined using genomic DNA of C. baileyi, Toxoplasma gondii, C. canis and Escherichia coli. The sensitivity of this assay was evaluated by analyzing 10-fold serially dilutions of plasmids ranging from 100 to 108 copy/μl. Both IMS-qPCR assay and indirect immunofluorescent-antibody assay（IFA） were applied to detect 50 water samples collected from the dairy cattle farms in Hebei. Results The optimal incubation concentration and time of antibody Cp23 were 20 ng/ml and 30 min, respectively, and the catching time was 30 min, the recovery rate was more than 95%. PCR product was 272 bp, and identified by restriction enzyme digestion and nucleotide sequencing. There was a good linear relationship between the standard plasmids and Ct value（correlation r2=0.996 1） of the Taqman qPCR. No cross-reactivity was observed with C. baileyi, T. gondii, C. canis and E. coli. The sensitivity of C. parvum-specific assay was 10 copy/μl. Compared with IFA as golden standard method, the specificity and sensitivity of IMS-qPCR for 50 water samples was 100%（18/18） and 93.8%（30/32）, respectively. Conclution The IMS-qPCR assay can be used to specifically detect C. parvum oocysts in water samples.

Key words: Cryptosporidium parvum, Immunomagnetic-bead, Taqman real-time PCR, 18S rDNA gene

高姗姗1,2，吴绍强3，罗静1，王承民1，张敏1，赵宝华2，何宏轩1 *. IMS-qPCR检测水源性微小隐孢子虫方法的建立[J]. 中国寄生虫学与寄生虫病杂志, 2013, 31(3): 4-180-184.

GAO Shan-shan1,2, WU Shao-qiang3, LUO Jing1, WANG Cheng-min1, ZHANG Min1,. Development of an IMS-qPCR Method for Detection of Cryptosporidium parvum in Water[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2013, 31(3): 4-180-184.

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