黄颡鱼源维氏气单胞菌Aha和gyrB基因双重PCR检测方法的建立

Establishment of a duplex PCR assay based on Aha and gyrB genes for detection of Aeromonasveronii

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。

To develop a rapid method to detect pathogenic Aeromonas veronii in Pelteobagrus fulvidraco, a duplex PCR assay was established with two pairs of primers designed according to the Aha and gyrB of A.veronii strain RCl10724. The results showed that two specific fragments about 419 bp and 745 bp were amplified from the genomic DNA extracted from A.veronii by the duplex PCR, which no cross-reactions with other bacteria such as K1ebsiella pneumonia, Edwadsiella tarda, Vibrioparahe molyticus, Vibrio metschnikovii, Pseudomonas fluorescens, Staphylococcus aureus, Flavobacterium cloumnare, A.hydrophila, A.schuberti, Streptococcus agalactiae. The detection limits of the method were 6.6 × 10^3 ng/μL for A.veronii ATCC35624 and 3.2×10^2 cfu/mL for RC110724, respectively. Using the assay to detect 30 clinical samples, the results showed that the coincidence between bacterial isolation method and the duplex PCR was 100%. Meanwhile, the strains Rc110724 DNA were detected in the tissues from liver, spleen, kindey and brain of P.fulvidraco affected by the A.veronii strain RC110724, especially in liver and spleen. In conclusion, the established duplex PCR in this study was specific, sensitive for the epidemiological surveillance of A.veronii infection in P.fulvidraco and other fish.