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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 PCR detection system for rapid detection of Cryptosporidium oocysts in order to promote the detection of diarrhea protozoa in human beings and their drinking water. Methods Based on the small subunit of Cryptosporidium SSU rRNA and COWP, primers and probes of nested PCR and fluorescent PCR were designed. The purified genomic DNA of Cryptosporidium parvum Template amplification, respectively, the sensitivity of two detection methods and specific tests; reuse a buffalo farms collected 58 copies of cow dung-like assessment system of value. Results The nested PCR primers and fluorescent PCR primer probes were highly specific. The lowest threshold OD value of nested PCR for Cryptosporidium DNA was 2 pg of Cryptosporidium. Real-time PCR showed the lowest detection threshold was 200 fg Cryptosporidium DNA, the sensitivity is relatively high, the sensitivity of real-time PCR was 1 order of magnitude higher than that of nested PCR (10-fold); one of 58 samples was positive, and the PCR products showed slight sequence analysis Cryptosporidium. Conclusion The specificity and sensitivity of nested PCR and real-time fluorescence PCR in detection of Cryptosporidium are both high and easy to operate, which can provide effective technical means and methods for the diagnosis, treatment and prevention of Cryptosporidium infection. Cows infected with zoonotic C. parvum genetic subtype, with the possibility of zoonotic transmission.