隐孢子虫PCR检测体系的建立及其应用

Establishment and application of PCR method for detection of Cryptosporidium

目的建立快速检测隐孢子虫卵囊的PCR检测体系,以期在人群和食品及其饮用水腹泻原虫监测中推广应用。方法以隐孢子虫小亚基SSU r RNA和卵囊壁蛋白(COWP)为靶基因,设计巢式PCR和荧光PCR的引物和探针;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,分别进行两种检测方法的敏感性和特异性试验;再用某水牛养殖场采集的58份牛粪样考核检测体系的应用价值。结果设计的巢式PCR引物和荧光PCR引物探针特异性都很高,巢式PCR对隐孢子虫DNA的检测最低OD阈值为2 pg隐孢子虫DNA;实时荧光PCR显示最低检测阈值为200 fg隐孢子虫DNA,灵敏性都比较高,实时荧光PCR的灵敏性比巢式PCR高1个数量级(10倍);58份检测样本中,有一份为阳性样本,PCR产物经序列分析显示为微小隐孢子虫。结论巢式PCR和实时荧光PCR检测隐孢子虫的特异性和灵敏性均高,操作简便,能为隐孢子虫感染的诊治和预防提供有效的技术手段和方法依据。奶牛感染人兽共患微小隐孢子虫基因亚型,具有人兽共患传播的可能性。

Objective To establish a quick PCR method for detection of Cryptosporidium,to supply for diarrhea parasite monitoring and food and drinking water monitoring in a large sample population. Methods According to the specific sequence of small sub-unit rRNA（SSU rRNA）and Cryptosporidium outer wall protein（COWP）gene of Cryptosporidium in GenBank,the primers and fluorogenic probe of PCR were designed. With the separation and purification of Cryptosporidium parvum egg genomic DNA as a template for amplification,the sensitivity and specificity of the assays were determined. Fifty-eight cattle fecal samples collected from a farm were examined by using the methods. Results The designed primers could amplify the DNA,and the probes had high specificity and stability. Cryptosporidium DNA of nested PCR detection minimum OD threshold was 2 pg Cryptosporidium DNA. The detection limit of the real-time PCR was 200 fg/μL,and the sensitivity of real-time PCR was 10 times higher than that of nested PCR. One fecal sample was positive,and the DNA sequences were identical with SSU rRNA gene of C. parvum. Conclusion The PCR and real-time PCR are rapid,simple methods with high sensitivity and specificity,and they provide an efficient way for diagnosis,treatment and control of Cryptosporidium infections. The cow infected with tiny Cryptosporidium gene subtype has the risk of person-animal transmission.