基于16S rRNA基因的不同血清型鸭疫里默氏杆菌PCR检测方法的建立

Establishment of the PCR for rapid detection of different serotypes of Riemerella anatipestifer based on the 16S rRNA gene

为建立一种快速诊断鸭疫里默氏杆菌(RA)病的方法,本研究根据NCBI登录的ATCC 11845菌株(CP003388.1)的16S rRNA基因保守序列设计引物,对标准株和疑似感染RA的病料样品进行检测,建立RA PCR检测方法。结果显示,6株RA标准株(包括1、2、10和11血清型)和2个疑似RA感染的病料样品,均能够扩增出698 bp的特异性片段,而鸭大肠杆菌和鸭沙门氏菌扩增均呈阴性。测序分析表明,疑似病料样品中扩增的片段与ATCC 11845菌株相应片段同源性达99.9%。PCR敏感性试验检测显示,其最小检出量为2.3×103cfu/mL;应用该法对10只RA病鸭的组织样品检测显示,脑组织检出率最高,其次为心血、肝脏和脾,而肠和肌肉呈阴性。同时,PCR法对RA的检出率比细菌分离鉴定法高11.7%。本研究建立的基于16S rRNA基因序列的PCR检测方法,能够检测主要流行于浙江地区的4种血清型RA,具有特异、灵敏和快速的优点。

To establish a rapid diagnostic approach of Riemerella anatipestifer disease, the PCR assay was developed for detection of R.anatipestifer with a pair of primers designed according to the conserved region of 16S rRNA sequence of R.anatipestifer ATCC 11845 strain （Acc. No： CP003388.1） in NCBI. The results indicated that a 698 bp specific DNA fragment was able to be amplified from both standard R.anatipestifer strains （including serotype of 1, 2, 10 and 11） and clinical samples with a detection limit of 2.3x103 cfu/mL, but unable to be amplified from E.coli and Salmonella isolates. In addition, Different tissues collected from R.anatipestifer infected ducks were tested by the PCR assay, the results showed that the highest R.anatipestifer detection rate was from cerebral tissue, followed by heart blood, then liver and the spleen, but not detected from intestine and muscle. While, compared with bacterial isolation method, detection rate by PCR assay was 11.7% higher. These results indicated that the PCR assay based on 16S rRNA gene is specific, sensitive and rapid for detection of different serotype R.anatipestifer infection in ducks.