摘要
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。
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.
作者
刘晓丹
肖自东
孙威
李席席
周一凡
张晓君
LIU Xiao-dan;XIAO Zi-dong;SUN Wei;Li Xi-xi;ZHOU Yi-fan;ZHANG Xiao-jun(College of Animal Science and Technology,Yangzhou University,Yangzhou 225009,Jiangsu,China)
出处
《淡水渔业》
CSCD
北大核心
2020年第1期76-81,共6页
Freshwater Fisheries
基金
江苏省现代农业产业技术体系建设项目02(青虾产业技术体系)
国家自然科学基金(31972830)
扬州大学‘青蓝工程’项目
