18S rRNA基因巢式PCR-RFLP鉴定吉林、大庆地区断奶前奶牛隐孢子虫分离株

Chracterization of Cryptosporidium spp from preweaned calves of Jilin and Daqing area by 18S rRNA gene nested PCR-RFLP

利用18S rRNA巢式聚合酶链反应(Nested PCR)-限制片段长度多态性(Restriction fragment length polymorphism,RFLP)鉴定吉林、大庆地区牛源隐孢子虫分离株。采取吉林、大庆地区断奶前犊牛粪便,提取DNA后经18S rRNA基因巢式PCR扩增,扩增产物测序后用Blast和MEGA4.0软件进行同源性和系统发育树分析。同时扩增产物分别用SspⅠ、VspⅠ和MboⅡ酶切后进行RFLP分析。通过18S rRNA基因PCR-RFLP分析和测序比对分析表明,吉林分离株包括2种隐孢子虫,分别为C.bovis和C.ryanae。大庆分离株包括3种,分别为C.bovis、C.ryanae和C.andersoni。

Cryptosporidium SPP isolated from feces of preweaned calves in Jilin and Daqing area were identified by 18S rRNA gene nested PCR-RFLP.The genomic DNA of Cryptosporidium was extracted from fecal samples and amplified by using the 18S rRNA gene nested PCR-RFLP assay,and Blast and MEGA4.0 softwares were used to analyze their homology and phylogeny.Meanwhile,their amplified products were digested with restriction enzymes SspⅠ,VspⅠand MboⅡ,respectively.All the digested products were analyzed with RFLP assay.As demonstrated by 18S rRNA gene analysis,the Jilin isolates included C.bovis,and C.ryanae.While the Daqing isolates included C.bovis, C.ryanae and C.andersoni.