巴斯德杆菌属CODEHOP PCR检测方法的建立与初步应用

Establishment and application of CODEHOP PCR assay for detection of Pasteurella spp.in laboratory animals

目的建立巴斯德杆菌的CODEHOP PCR快速检测方法,为实验动物的呼吸道细菌的控制提供参考。方法应用CODEHOP在线简并引物设计工具,比对Genbank中13株巴斯德杆菌的RNA聚合酶β亚基(rpo B)氨基酸序列设计简并引物。对建立CODEHOP PCR方法用21株参考菌株进行特异性和敏感性评价,并应用于实验动物中的巴斯德杆菌检测。结果简并引物Past F6/PastR5扩增标准菌株的目的片段为200 bp左右。能够区分受试的巴斯德杆菌和主要的实验动物呼吸道病原菌。敏感性为0.2 pg/μL^2 pg/μL。在受试的609只实验动物中呼吸道样品中检测出巴斯德杆菌阳性率为19.1%。样品阳性片段经测序验证,准确率为100%。结论所建立的方法具有良好的特异性和敏感性,可用于动物样品中巴斯德杆菌的检测。

Objective We established a rapid detection method of Pasteurella spp. and provided a reference for microbiological quality control of laboratory animal. Methods According to the [3 subunit of bacterial RNA polymerase （rpoB） protein multiple alignments of 13 different Pasteurella spp. published in NCBI. The degenerate primers were designed by CODEHOP designer online. CODEHOP PCR method was applied to detecting Pasteurella spp. after the specificity and sensitivity of the method had been evaluated by 21 reference strains. Results Standard strain amplified fragment were about 200 bp by degenerate primers PastF6/PastR5. The primers are able to distinguish between PasteureUa 5pp. and the other pathognic organisms of laboratory animal respiratory tracts. Sensitivity of this method were 0. 2 pg//xL ~ 2 pg/jxL to different Pasteurella. The PasteureUa positive rate was 19. 1% in 609 animal＇ s respiratory samples. The accuracy of positive results was 100% through verifying by sequenced and blast. Conclusions The established method has good specificity and sensitivity. It can be used to detect Pasteurella spp. in animal samples.