摘要
为了快速、准确地鉴定鸭疫里默氏杆菌(RA),本实验根据RACH3株16SrRNA和dnaB基因序列设计引物,经引物筛选和PCR反应条件优化后,建立了检测RA的双重PCR方法。结果表明该方法对检测的所有不同血清型RA菌株均能扩增出364bp和627bp两条特异的目的片段,而对鸭致病性大肠杆菌、鸭源禽巴氏杆菌、肠炎沙门氏菌、黄杆菌等对照菌株的扩增结果均为阴性;该PCR方法对细菌HXb2基因组DNA和菌液的检测敏感性分别为17.33ng/mLDNA和2.9×10~3cfu/mL细菌。利用建立的双重PCR检测了RA感染鸭的肝脏和脑组织中的RA,结果显示该方法与细菌分离法符合率分别为100%(9/9)和77.8%(7/9),表明本实验建立的双重PCR方法可用于检测肝脏组织中RA。本研究建立的双重PCR方法既可以对分离的RA疑似菌株进行快速检测和鉴定,也可以直接用于临床样品中RA的检测。
To identify Riemerella anatipestifer rapidly and effectively, a duplex PCR method was established to detect R.anati- pestifer with the primers designed according to the 16S rRNA and dnaB gene sequences of R.anatipestifer strain CH3. The results showed that two specific bands （364 bp and 627 bp） were amplified from all the detected R.anatipestifer strains with different serotypes by the duplex PCR, but no amplification was found from other bacteria, such as avian pathogenic Escherichia coli, avian Pasteurella multocida, Salmonella enteritidis and Flavobacterium johnsoniae. The sensitivity tests showed that the limit detection was 2.9x103 CFU/mL of the bacteria. In addition, R.anatipestifer from the liver and brain tissue samples of infected ducks were detected by the duplex PCR, the results showed that the coincidence of duplex PCR with bacterial isolation was 100% （9/9） and 77.8% （7/9） respectively. It suggested that the duplex PCR could be suitable to detected R.anatipestifer from liver samples. In conclusion, the established duplex PCR could be applied in the clinical detection of R.anatipestifer.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2017年第1期50-53,共4页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金(31272590
31472224)
关键词
鸭疫里默氏杆菌
双重PCR
鉴定
检测
Riemerella anatipestifer
duplex PCR
identification
detection
