青虾源维氏气单胞菌双重PCR检测方法的建立

根据NCBI已发表的维氏气单胞菌(Aeromonas veronii)促旋酶B亚单位基因gyrB和编码细菌RNA聚合酶σ70因子基因rpoD的保守序列设计引物,建立了一种快速检测青虾源维氏气单胞菌的双重PCR检测方法。结果显示:青虾源维氏气单胞菌gyrB和rpoD基因PCR扩增产物大小分别为815 bp、554 bp,而对嗜水气单胞菌(Aeromonas hydrophila)、弗氏柠檬酸杆菌(Citrobacter freundii)、哈维弧菌(Vibrio harveyi)、副溶血性弧菌(Vibrio parahemolyticus)、需钠弧菌(Vibrio natriegens)、非O1霍乱弧菌(non-O1 Vibrio cholerae)、阴沟肠杆菌(Enterobacter cloacae)、产气肠杆菌(Enterobacter aerogenes)扩增结果皆为阴性;灵敏性试验结果显示该方法检测到的最低菌体DNA量为1.8×10-2 ng/μL,且10份送检样本检测结果与传统细菌分离鉴定结果相一致。结果表明,双重PCR检测方法特异性好、灵敏性高,适用于青虾源维氏气单胞菌的快速检测。

Establishment of a duplex PCR assay for detection of Aeromonas veronii isolated from Macrobrachium nipponense

Primers of the conserved sequence of the helicase B subunit gene gyrB and RNA polymeraseσ70 factor gene rpoD were designed according to their sequence published in NCBI to establish a method for the detection of Aeromonas veronii in Macrobrachium nipponense.A duplex PCR method for the detection was established.The results showed that the PCR amplification products of gyrB and rpoD genes were 815 bp and 554 bp,respectively.Specificity test showed that no specific bands were amplified in Aeromonas hydrophila,Citrobacter freundii,Vibrio harveyi,Vibrio parahemolyticus,Vibrio Natriegens,non-O1 Vibrio cholera,Enterobacter cloacae and Enterobacter aerogenes.The sensitivity test results showed that the minimum amount of bacterial DNA detected by this method was 1.8×10^-2 ng/L,which indicated good sensitivity.The test results of 10 clinical samples were consistent with the traditional bacterial isolation and identification results,which was suitable for clinical testing.The above experiments indicated that the established dual PCR method had good specificity and sensitivity,and was suitable for the rapid detection of A.veronii in Macrobrachium nipponense.