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为建立一种快速、准确检测黄颡鱼源维氏气单胞菌(A.veronii)的方法,本研究根据A.veronii菌株RC110724黏附素基因(Aha)和促旋酶B亚单位基因(gyrB)序列设计引物,经条件优化建立了A.veronii的双重PCR检测方法。结果显示该方法可以同时扩增出A.veronii 419 bp和745 bp两条特异性的片段,而对嗜水气单胞菌、迟缓爱德华菌、副溶血性弧菌、麦氏弧菌、荧光假单胞菌、金黄色葡萄球菌、柱状黄杆菌、肺炎克雷伯氏菌、无乳链球菌、舒伯特气单胞菌扩增结果为阴性;该方法对A.veronii标准株ATCC35624和RC110724基因组DNA和菌体检测下限分别为6.6×10~(-3)ng/μL和3.2×10~2cfu/mL。利用建立的双重PCR方法对30份临床样品进行检测,结果与细菌传统分离鉴定符合率为100%。同时,双重PCR可以检出菌株RC110724人工感染的黄颡鱼肝、脾和肾组织中的细菌DNA,对肝和脾脏组织的样品检测效果最好。本研究建立的双重PCR检测方法特异性好,具有较高的检测灵敏性,可用于黄颡鱼等A.veronii感染病例的快速诊断和流行病学监测。
In order to establish a rapid and accurate method for the detection of A.veronii, the present study was based on the gene sequences of the A. veronii strain RC110724, the adhesin gene (Aha) and the gyrase B subunit gene ) Primers were designed and double-PCR was established by A.veronii. The results showed that this method can simultaneously amplify two specific fragments of 419 bp and 745 bp of A.veronii, but not for A. hydrophila, B. edwardsii, Staphylococcus aureus, Klebsiella pneumonia, Klebsiella pneumoniae, Streptococcus agalactiae, Aeromonas sobrensis amplification results were negative; the method of A.veronii standard strains ATCC35624 and RC110724 genome The detection limits of DNA and mycelia were 6.6 × 10 ~ (-3) ng / μL and 3.2 × 10 ~ 2cfu / mL, respectively. A total of 30 clinical samples were tested by double PCR method. The results were in line with the traditional bacterial isolation and identification of 100%. At the same time, double PCR could detect the bacterial DNA in the liver, spleen and kidney tissues of artificial infected catfish strain RC110724. The detection of liver and spleen samples was the best. The double PCR assay established in this study has good specificity and high detection sensitivity and can be used for the rapid diagnosis and epidemiological surveillance of A.veronii infection such as yellow catfish.