摘要
为建立支气管败血波氏杆菌SYBR GreenⅠ荧光定量PCR检测方法,本研究根据波氏菌属毒力基因DNT设计一对特异性引物,通过优化,确定最佳反应条件,建立了支气管败血波氏杆菌荧光定量PCR检测方法。并对其进行敏感性、特异性、重复性试验以及临床样品的检测。以支气管败血波氏杆菌标准质粒为模板,建立标准曲线结果显示:标准品浓度在1.13×10~8拷贝/μL～1.13×10~2拷贝/μL范围内具有良好的线性关系,相关数R^2=0.997;该方法的灵敏度可达1.13×10~1拷贝/μL。建立的荧光定量PCR方法与副猪嗜血杆菌、猪链球菌、金黄色葡萄球菌等12种细菌和伪狂犬病毒、猪瘟病毒等4种病毒无交叉反应,具有良好的特异性;批内变异系数(CV)均小于2%,批间变异系数均小于4%,具有良好的稳定性。对临床样品的检测结果显示该方法检测的阳性率为57.7%,与普通PCR的符合率为93.24%。本研究建立的方法可以用作支气管败血波氏杆菌感染的早期诊断、快速检测与定量分析。
To develop rapid and specific assay for Bordetella bronchiseptical detection, a SYBR GreenⅠbased real-time PCR method was established with a pair of primers designed according to the sequence of dermonecrotic toxingene （DNT） of B.bronch- iseptical. Initially, a recombinant plasmid containing DNT gene was constructed and served as a standard template to generate the standard curve. The standard curve produced a linear relationship between Ct value and concentrations of the standard template at a range of 1.13×10^8copies/μL to 1.13×10^2copies/μL, R^2=0.997. Inaddition, the specificity and sensitivity of the method was analyzed through optimum reaction conditions. The specificity assay showed no amplification of 12 kinds of bacteria including Haemophilus parasuis, Streptococcus suis, Pasteurella multocidaetc and 4 kinds of viruses such as pseudorabies virus, classical swine fever virus etc, except B.bronchiseptical. and the sensitivity of this method was 1.13×101copies/μL. The reproducibility test showed that the variation coefficients of intra-and inter-assay were less than 3% and 4%, respectively. For the detection of clinical samples, the consistent rate between conventional PCR and the real-time PCR （57.7% positive rate） 93.24%. Therefore, the established real-time PCR method is a rapid, specificand sensitive method for the detection B.bronchiseptical.
作者
尹清清
德西措姆
罗怡琳
邬旭龙
张鹏飞
杨泽晓
姚学萍
王印
YIN Qing-qing;DEXI Cuo-mu;LUO Yi-lin;WU Xu-long;ZHANG Peng-fei;YANG Ze-xiao;YAO Xue-ping;WANG Yin(College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130,China;Key Laboratory of Animal Disease and Human Health of Sichuan Province,Chengdu 611130,China;ZuogongAnimal Husbandry Station,Tibet Autonomous Region,Changdu 854400,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2018年第10期917-921,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
国家"十二五"国家科技支撑计划(2013BAD12B04)
