贝氏隐孢子虫PCR检测试剂盒的研制  

Development of a PCR detection kit for Cryptosporidium baileyi in poultry

作　　者：杨建伟[1] 李国清[1] 刘霞[1] 张媛媛[1] 徐前明[1] 

机构地区：[1]华南农业大学兽医学院,广州510642

出　　处：《中国人兽共患病学报》2009年第3期241-244,共4页Chinese Journal of Zoonoses

基　　金：广东省科技计划项目(2006B20801006)

摘　　要：目的研制禽类贝氏隐孢子虫的PCR检测试剂盒。方法根据贝氏隐孢子虫18SrRNA和ITS-1序列,选取遗传标记位点最多的一段核苷酸序列作为特异PCR引物的靶序列,设计、合成了一对针对贝氏隐孢子虫的特异性引物(YJWF:5’-ACTGATATACAATACCACGG-3’和YR:5’-GCGGATAAAGTTCAGGTTC-3’),对PCR反应条件进行了一系列优化,并分别对试剂盒的特异性、敏感性、稳定性、重复性和保存期等进行了系列研究。结果该试剂盒能特异性地扩增出贝氏隐孢子虫基因组中相应片段,与安氏隐孢子虫,微小隐孢子虫,柔嫩艾美尔球虫等相关原虫基因组DNA无交叉反应;最低能检测到3.48个卵囊;反复冻融50次对试剂盒没有影响;室温条件下至少可保存4个月。结论该试剂盒的各项指标达到了贝氏隐孢子虫的检测要求。To develop a PCR kit for the detection of Cryptosporidium baileyi in poultry, a portion of 18S rRNA and ITS-1 sequence of C. baileyi was chosen as the target DNA for designing the species-specific primers（YJWF5＇-ACTGATAT-ACAATACCACGG-3＇ and YR： 5＇-GCGGATAAAGTTCAGGTTC-3＇）. The PCR amplification conditions were optimized, and a series of tests were conducted in the specificity, sensitivity, stability, reproducibility, and stored period of the kit, respectively. This kit was specific and there was no cross-reaction with other related protozoa, such as C. andersoni, C.parvum, Eimeria tenella and so on. It was able to detect the presence of C. baileyi in poultry as low as 3.48 oocysts. Repeated frozen-thaw for 50 times had no influence on the activity of the kit. The kit worked well after being stored at room temperature for at least four months. The results indicated that the kit is suitable for the detection of C. baileyi in poultry.

关 键 词：贝氏隐孢子虫 18S RRNA ITS-1 PCR 试剂盒 

分 类 号：R832.3[医药卫生—航空、航天与航海医学]