安氏隐孢子虫ITS-1基因PCR检测方法的建立  

Establishment of PCR assay for detection of ITS-1 gene in Cryptosporidium andersoni

作　　者：彭昊[1,2] 李军[1] 陶立[1] 陈泽祥[1] 唐林生[2] 魏晓瑞[2] 马春霞[1] 禤雄标[1] 胡帅[1] 许力干[1] 谢永平[1] 杨威[1] 

机构地区：[1]广西兽医研究所/广西畜禽疫苗新技术重点实验室,南宁530001 [2]广西大学动物科技学院,南宁530005

出　　处：《南方农业学报》2012年第5期701-704,共4页Journal of Southern Agriculture

基　　金：公益性行业(农业)科研专项项目(201103008);广西科研基本业务费专项项目(桂科专项12-2);广西水产畜牧兽医局资助项目(桂渔牧财[2011]52号)

摘　　要：【目的】建立针对安氏隐孢子虫的PCR快速检测方法,为安氏隐孢子虫的检测和分子流行病学调查提供技术支撑。【方法】根据GenBank中安氏隐孢子虫的ITS-1基因序列设计1对特异性引物,建立基于ITS-1基因的PCR方法,并通过特异性试验、敏感性试验、临床检测以及与经典巢式PCR检测方法进行比较等,验证其可行性。【结果】基于ITS-1基因的PCR检测方法能扩增出安氏隐孢子虫的特异条带,而对猪源隐孢子虫、贝氏隐孢子虫、微小隐孢子虫、牛泰勒氏虫、牛巴贝斯虫、伊氏锥虫、食道口线虫的扩增结果均为阴性;将测序结果进行BLAST比对,发现其与GenBank中安氏隐孢子虫的同源性均在99%以上。敏感性最低可检测每克牛粪中5个卵囊;用于检测广西不同地区的1613份牛粪样品时,与经典巢式PCR检测方法相比,发现两者的阳性检出率均为11.72%,且吻合率达100%。【结论】安氏隐孢子虫ITS-1基因PCR检测方法具有快速、敏感、特异的特点,适用于安氏隐孢子虫的检测和分子流行病学调查分析。[Objective]The aim of this research was to develop an swiftly efficient PCR detection assay for Cryptosporidium andersoni to provide technical support for investigation of molecular epidemiology. [ Method ]A pair of specific primer was designed using published ITS-1 gene sequence and a PCR assay system was developed to conduct specificity test, sensitivity test, clinical detection and conventional nest PCR. [Result]The results showed that the specific band was only amplified from C. andersoni using this PCR assay, but not amplified from C. suis, C. bailey, C. parvum, Theileria sergentl, Babesia boris, Trypanosoma evansi and Oesophagostomum. The BLAST comparative result revealed that these sequences had above 99% homology with the some sequences published in GenBank. The sensitivity of the assay was 5 oocysts per gram in cattle feces. Total 1613 dairy fecal samples were collected from different areas of Guangxi and tested by the newly developed PCR assay and conventional nest PCR, for which the positive detection rate was both recorded as 11.72%. It was proved that the efficiency of these two methods was 100%. [Conclusion]The established PCR assay was suitable for detection of ITS-1 geue in C. andersoni and molecular epidemics investigation because of its rapidness, sensitivity and specificity.

关 键 词：安氏隐孢子虫 ITS-1基因 PCR检测 巢式PCR 

分 类 号：S852.723[农业科学—基础兽医学]