微小隐孢子虫种特异性基因片段的克隆及诊断引物的确定

Cloning of a Species-specific Gene Fragment from Cryptosporidium parvum and the Development of Diagnostic PCR Primers

目的 寻找用于诊断微小隐孢子虫病的特异性引物。 方法 在RAPD分析过程中获得了 1条微小隐孢子虫 [Cryptosporidium parvum (C .parvum ) ]种特有 712bp的基因片段 ,将该片段分离、纯化、克隆和测序 ,据此序列合成一对特异性引物FF。用引物FF扩增美国 2株C .parvum和中国 4株C .parvum ,并用该引物与Morgan( 1996 )发表的C .parvum种特异性诊断引物 0 2 1作对照 ,检测镜检C .parvum卵囊阴性的兔粪样 35份和人粪样 5 5份。 结果 引物FF扩增 6株C .parvum均能产生预计 6 0 3bp的片段 ,引物FF和引物 0 2 1粪样检测结果完全一致 ,其敏感性是可检出 1个卵囊的DNA。 结论 获得的引物FF特异性强 ,敏感性高 ,可用于微小隐孢子虫病的诊断。

Objective To develop a pair of diagnostic PCR primers for Cryptosporidium parvum . Methods A species-specific gene fragment of C.parvum was obtained through RAPD analysis. After the fragment was isolated,purified,cloned and sequenced,a pair of primers FF was designed and synthesised based on the sequence. With the primers, the anticipated fragment in size of 603 bp was amplified by PCR from 2 American strains and 4 Chinese strains of C.parvum . The samples of 35 rabbits feces and 55 human feces were detected by PCR with primers FF and 021,the latter was a species-specific diagnostic primer reported by Morgan. Results All six strains amplified by the primers FF showed same detection rate with 021. Sensitivity test indicated that DNA of 1 oocyst per gram of feces could be detected by the PCR. Conclusion The primers FF showed high specificity and sensitivity, and can be used for diagnosing Cryptosporidium parvum infection.