副溶血弧菌与嗜水气单胞菌双重PCR检测方法的建立

Establishment of a Dual PCR for Vibrio parahaemolyticus and Aeromonas hydrophila

为建立一种可精准、快捷鉴别检测副溶血弧菌(VP)及嗜水气单胞菌(AH)的方法,根据GenBank公布的VP Tlh基因及AH aerA基因保守区序列,设计合成两对特异性引物,通过优化反应体系及反应程序,成功建立了一种可同时检测VP与AH的双重PCR方法,并开展了特异性及敏感性试验。结果显示:VP Tlh和AH aerA基因扩增条带大小分别为500、760 bp;反应体系中Tlh与aerA基因引物最优浓度分别为7.5、10.0 nmol/L,反应程序中最佳退火温度为56.8℃;该方法对VP、AH的最低可检出细菌浓度均为1.2×10^(3)CFU/mL;对地衣芽孢杆菌、微小杆菌、巨大芽孢杆菌、蜡阳芽孢杆菌、普通变形杆菌、维氏气单胞菌、布氏柠檬酸杆菌、豚鼠气单胞菌、霍乱弧菌、拟态弧菌、简氏气单胞菌等其他非目标菌均无交叉反应,仅对VP和AH呈特异性阳性扩增。综上,本研究建立了一种可快速鉴别诊断VP、AH的双重PCR检测方法,其特异性强、灵敏度高,可为VP、AH等细菌性病原引发的水生动物疫病的快速诊断、监测及有效防控提供技术支持。

In order to establish an accurate and rapid method for identifying and detecting Vibrio parahaemolyticus(VP)and Aeromonas hydrophila(AH),based on the conserved region sequences of VP Tlh and AH aerA genes published by GenBank,two pairs of specific primers were designed and synthesized,followed by optimization of the reaction system and procedure,a dual PCR assay was established for simultaneously detecting VP and AH,and tested for its specificity and sensitivity.The results showed that the amplified bands of VP Tlh and AH aerA genes were 500 and 760 bp,respectively;the optimal concentrations of the primers were 7.5 and 10.0 nmol/L,respectively in the reaction system,and the optimal annealing temperature was 56.8℃in the reaction procedure;the minimum detectable bacterial concentrations for VP and AH by this method were both 1.2×10^(3)CFU/mL;it failed to crossly react with Bacillus licheniformis,Microbacillus,Bacillus megaterium,Bacillus cereus,Proteus vulgaris,Aeromonas viridis,Citrobacter brucei,Aeromonas guinea-pig,Vibrio cholerae,Vibrio mimicus,Aeromonas jeanensis,but with a specific positive amplification for VP and AH.In conclusion,a dual PCR assay with strong specificity and high sensitivity was established for rapid differential diagnosis of VP and AH,which could technically support rapid diagnosis,monitoring and effective prevention and control of aquatic animal diseases caused by bacterial pathogens such as VP and AH.