PCR-RFLP鉴定隐孢子虫种类研究

Cryptosporidium Species from Dairy Cow and Poultry Identified by PCR-restriction Fragment Length Polymorphism

为快速、准确鉴别人畜隐孢子虫种类,建立了巢式PCR扩增隐孢子虫18SrRNA基因的特殊区域,扩增片段测序结果表明:安徽牛源分离株(Cryptosporidiummuris)781bp;北京鸡源分离株(C.baileyi)776bp;北京牛源分离株(C.muris)781bp;河南牛源分离株(C.muris)725bp;长春牛源分离株(C.muris)776bp;宁夏鸡源分离株(C.baileyi)725bp,该片段位于18SrRNA全序列271～1103bp之间。使用Ssp 限制性内切酶消化发现C.muris产生418～420bp和305～363bp两个片段,C.baileyi产生544～545bp和185～231bp两个片段。所检测的6个分离株可以显著区分为C.muris和C.baileyi两个种,所建立的PCR-RFLP可以有效鉴别隐孢子虫种类。

Nested PCR was developed to amplify specific sequence of 18S rRNA gene for identifying Cryptosporidium species from animals rapidly and accurately in this experiment. The isolates amplified successfully in the secondary PCR is as follow:Anhui isolate(Cryptosporidium muris) from dairy cow,Beijing isolate(C.muris) from dairy cow, Beijing isolate(C.baileyi) from chicken,Henan isolate(C.muris) from dairy cow, Changchun isolate(C.muris) from dairy cow,Ningxia isolate(C.baileyi) from chicken and their PCR products were 781 bp, 781 bp, 776 bp, 725 bp, 776 bp, 725 bp respectively. The amplified fragment was between 271～1 103 bp in 18S rRNA gene.The secondary PCR products was digested with SspⅠ,C.muris generated two visible bands of 418～420 bp and (305～)363 bp, whereas C.baileyi generated two visible bands of 544～545bp and 185～231 bp. 6 isolates were differentiated significantly 2 species. The developed PCR-RFLP in this experiment can identify Cryptosporidium species accurately.