杀鲑气单胞菌杀鲑亚种和杀日本鲑亚种PCR检测方法的建立

Development of a PCR Method to Detect the Aeromonas salmonicida subsp.salmonicida and Aeromonas salmonicida subsp.masoucida

杀鲑气单胞菌(Aeromonas salmonicida)是一种重要的鱼类致病菌,可以感染多种海淡水鱼类。杀鲑气单胞菌包括5个亚种,目前常用的生理生化特征和16S rDNA序列分析方法很难实现亚种的快速精确区分。为实现杀鲑气单胞菌亚种的快速鉴定和检测,针对我国常见的杀鲑气单胞菌杀鲑亚种(A.salmonicida subsp.salmonicida)和杀日本鲑亚种(A.salmonicida subsp.masoucida),本研究开发了其特异性的PCR检测方法。根据Gene Bank已公布的杀鲑气单胞菌基因组信息,选择杀鲑亚种phoB基因和杀日本鲑亚种LOC111476736基因作为目标基因,根据其序列设计特异性引物,进一步对PCR反应的退火温度、引物浓度、dNTPs浓度、Mg^(2+)浓度和酶浓度5个方面进行了优化,并测试了该方法的特异性、敏感性和应用效果。结果显示,2对引物分别可以扩增出杀鲑气单胞菌杀鲑亚种522 bp的phoB特异性基因片段和杀日本鲑亚种515 bp的LOC111476736特异性基因片段。杀鲑亚种特异性引物最适退火温度为64℃,10μmol/L引物、2 mmol/L dNTPs、25 mmol/L MgSO4和1 U/μL KOD酶的最适添加量分别为1.5、2、1.5和0.5μL。杀日本鲑亚种特异性引物最适退火温度为64℃,10μmol/L引物、2 mmol/L dNTPs、25 mmol/L MgSO4和1 U/μL KOD酶的最适添加量分别为0.75、1、1.5和0.5μL。以鳗弧菌(Vibrio anguillarum)、美人鱼发光杆菌(Photobacterium damselae)、杀鱼爱德华氏菌(Edwardsiella piscicida)、杀鲑气单胞菌其他亚种等14种其他水产病原菌或常见环境菌为模板进行PCR检测,均无特异性条带。该方法对杀鲑气单胞菌杀鲑亚种的检测灵敏度为12.8 CFU/反应(菌体)或17.6 fg/反应(DNA),对杀鲑气单胞菌杀日本鲑亚种的检测灵敏度为23.8 CFU/反应(菌体)或27.2 fg/反应(DNA)。利用杀鲑气单胞菌杀鲑亚种和杀日本鲑亚种分别对大菱鲆(Scophthalmus maximus)进行人工感染实验,感染后取病鱼组织进行PCR检测,结果显示,本方法可以从感染后的大菱鲆中分别检测到相应病原。综上所述,本研究建立了杀鲑气单胞菌杀鲑亚种和杀日本鲑亚种的特异性PCR检测方法,该检测方法为杀鲑气单胞菌杀鲑亚种和杀日本鲑亚种的流行病调查和快速诊断提供了支撑。

Aeromonas salmonicida is an important pathogen that can infect a variety of marine and freshwater fish.There are five subspecies of Aeromonas salmonicida:A.salmonicida subsp.salmonicida,A.salmonicida subsp.smithia,A.salmonicida subsp.achromogenes,A.salmonicida subsp.masoucida,and A.salmonicida subsp.pectinolytica.Traditionally,the detection of A.salmonicida has been based on 16S rRNA sequencing and physiological and biochemical characterization,but it is difficult to identify the subspecies using these methods.Outer membrane protein(A-layer protein,VapA),which is encoded by the vapA gene and regulated by the luxS gene,is an important secretion protein of A.salmonicida.It is involved in bacterial self-agglutination induction,macrophage phagocytosis resistance,and provides protection against chemicals such as antibiotics and disinfectants.In addition,the vapA gene is also an effective molecular marker for the identification of A.salmonicida subspecies,however,gene sequencing and phylogenetic analysis are required for subspecies determination.To establish an accurate and sensitive rapid detection of A.salmonicida subspecies,in this study we tried to establish a specific PCR method for A.salmonicida subsp.salmonicida and A.salmonicida subsp.masoucida identification.Based on genome analysis,the phoB and LOC111476736 genes were used as molecular markers for PCR amplification with specific primers designed according to the sequences in the GenBank database.The target gene was amplified using the genomic DNA of A.salmonicida subsp.salmonicida or A.salmonicida subsp.masoucida,and the method was optimized to improve the efficiency and accuracy of distinguishing these two subspecies from other pathogens in aquaculture.First,the annealing temperature,primer concentration,dNTPs concentration,Mg2+concentration,and enzyme dosage of the PCR system were optimized to improve the sensitivity of the detection method.The results showed that the primers could amplify the phoB gene fragment of 522 bp and the LOC111476736 gene fragment of 515 bp.The optimal annealing temperature of specific primers for A.salmonicida subsp.salmonicida was 64℃,and the optimal volume of 10μmol/L primers,2 mmol/L dNTPs,25 mmol/L MgSO4,and 1 U/μL enzyme were 1.5μL,2.0μL,1.5μL,and 0.5μL(25μL reaction system),respectively.The optimum annealing temperature of specific primers for A.salmonicida subsp.masoucida was 64℃,and the optimum volume of 10μmol/L primers,2 mmol/L dNTPs,25 mmol/L MgSO4,and 1 U/μL enzyme were 0.75μL,1.00μL,1.50μL,and 0.50μL(25μL reaction system),respectively.The sensitivity of the detection method was determined using a gradient diluted A.salmonicida subsp.salmonicida ATCC33658 bacterin as the template,and the target band could not be amplified when the bacterin concentration was lower than 12.8 CFU/reaction.The detection limit of A.salmonicida subsp.salmonicida based on the phoB gene sequence established in this study was 12.8 CFU/reaction.With DNA as the template,when the concentration of the DNA template was lower than 17.6 fg/reaction,the target band could not be amplified.Thus,the detection limit of specific primers based on the phoB gene sequence for A.salmonicida subsp.salmonicida was 17.6 fg/reaction.When the gradient dilution of A.salmonicida subsp.masoucida ATCC27013 bacterin was used as the template,the target band could not be amplified when the bacterin concentration was lower than 23.8 CFU/reaction.Thus,the detection limit of the method for A.salmonicida subsp.masoucida,based on the LOC111476736 gene sequence was 23.8 CFU/reaction.With DNA of ATCC27013 as the template,when the concentration of the DNA template was lower than 27.2 fg/reaction,the target band could not be amplified.Thus,in this study,the detection limit of DNA for A.salmonicida subsp.masoucida using specific primers designed according to the LOC111476736 gene sequence was 27.2 fg/reaction.The specificity of the detection method using specific primers based on the phoB and LOC111476736 genes was also determined in this study.Aquaculture pathogens or environmental bacteria,such as Vibrio anguillarum,Photobacterium damselae,Edwardsiella piscicida,Escherichia coli,Aeromonas hydrophila,Vibrio harveyi,Aeromonas encheleia,Streptococcus parauberis,Streptococcus iniae,Streptococcus dysgalactiae,and Bacillus subtilis,were used as templates for specificity testing.No specific products were found for any of the other pathogens tested.The specific PCR products could only be amplified from the bacterins of A.salmonicida subsp.salmonicida or A.salmonicida subsp.masoucida.We also tested the application of detection methods using an experimentally infected turbot as a model.Turbot was infected with A.salmonicida subsp.salmonicida strain ASS20200608XZ11L or A.salmonicida subsp.masoucida strain ASM20160705RZ6S by intramuscular injection.All turbot died within 7 days post-infection,and the liver,spleen,and kidney of moribund fish were used as templates.The results showed that the established method could accurately detect A.salmonicida subsp.salmonicida or A.salmonicida subsp.masoucida in the turbot,without nonspecific amplification in the tissues of the healthy turbot.In conclusion,we established a specific PCR method to detect two subspecies of A.salmonicida,and these methods could be used as effective tools for investigating the epidemiology of A.salmonicida.