鸭疫里默氏杆菌16S rRNA和dnaB基因的双重PCR检测方法的建立

发布时间：2018-05-06 03:22

本文选题：鸭疫里默氏杆菌 + 双重PCR　；参考：《中国预防兽医学报》2017年01期

【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。
[Abstract]:In order to identify RIA quickly and accurately, a double PCR method for the detection of RA was established based on the sequence of 16SrRNA and dnaB genes of RACH3 strain. The primers were screened and the conditions of PCR reaction were optimized. The results showed that the method could amplify two specific target fragments of 364bp and 627bp to all the RA strains of different serotypes, but to duck pathogenic Escherichia coli, Pasteurella duckling, Salmonella enteritidis. The results of amplification of the control strains were negative, and the sensitivity of the PCR method to the detection of bacterial HXb2 genomic DNA and bacterial fluid was 17.33ng/mLDNA and 2.9 脳 10~3cfu/mL, respectively. The double PCR was used to detect RAA in liver and brain tissues of ducks infected with RA. The results showed that the coincidence rates of this method with bacterial isolation method were 100 / 9 and 77.8 / 7 / 9, respectively. The results showed that the double PCR method established in this experiment could be used to detect RAA in liver tissue. The dual PCR method developed in this study can be used for rapid detection and identification of suspected strains of RA in clinical samples as well as for the detection of RA in clinical samples.
【作者单位】：山东农业大学动物科技学院;中国农业科学院上海兽医研究所;
【基金】：国家自然科学基金(31272590、31472224)
【分类号】：S852.61

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