农业生物技术学报2013，Vol.21Issue(4)：498-504,7.DOI:10.3969/j.issn.1674-7968.2013.04.016
兔支气管败血波氏杆菌TaqMan荧光定量PCR检测方法的建立与应用
Development and Applification of TaqMan Fluorescence Quantitative PCR Assay to Detect Rabbit Bordetella Bronchiseptica
摘要
Abstract
A specific, sensitive and rapid TaqMan fluorescence quantitative PCR was established for testing Rabbit Bordetella bronchiseptica. In present study, a pair of primers and probes were designed from target gene of virulence factors CyaA of Bordetella bronchiseptica. Amplified PCR product was cloned and sequenced, the results showed that the homology was 100% compared with the reference sequence published in GenBank. The positive recombinant plasmids were served as quantitative detection of standards to establish standard curve. The detectable quantity of Bordetella bronchiseptica genomic DNA was 0.32 pg, which was 25 times sensitivity compared with common PCR, there was no cross reaction with common clinical bacteria by TaqMan fluorescence quantitative PCR. The 45 suspected samples were detected by TaqMan fluorescence quantitative PCR or routine PCR. The positive detection rates were 75.5% and 66.7%, respectively, the coincidence rate was 88.2%. The results showed that the TaqMan fluorescent quantitative Rabbit Bordetella bronchiseptica detection method was specificity, sensitivity and reproducibility. The present study provides an effective sensitive and reproducible method to diagnose and control Rabbit Bordetella bronchiseptica in further research.关键词
兔支气管败血波氏杆菌/CyaA/荧光定量PCR/检测Key words
Rabbit Bordetella bronchiseptica/ CyaA/ TaqMan fluorescence quantitative PCR/ Detection引用本文复制引用
钱微,刘燕,肖琛闻,韦强,季权安,鲍国连,姚火春..兔支气管败血波氏杆菌TaqMan荧光定量PCR检测方法的建立与应用[J].农业生物技术学报,2013,21(4):498-504,7.基金项目
国家兔产业技术体系岗位专家项目(No.CARS-44-C-2)、浙江省重大项目(No.2011C12028)和浙江省行业公益项目(No.2011C22013) （No.CARS-44-C-2）
