南方农业学报2018，Vol.49Issue(6)：1228-1234,7.DOI:10.3969/j.issn.2095-1191.2018.06.27
基于dnaJ基因的弧菌PCR检测方法
PCR detection method for Vibrios based on dnaJ gene
摘要
Abstract
[Objective]A PCR detection method that could amplifiy full length of dnaJ gene was developed in order to provide technical support for rapid detection and identification of Vibrio species.[Method]Taking dnaJ gene of Vibrio as target gene,a pair of degenerate primers were designed on the beginning and the end conserved domain. Then a PCR de-tection method that could amplify almost full length dnaJ gene of Vibrios was developed following optimization of annea-ling temperature and primers concentrations. The specificity and sensitivity tests,detection for clinical samples were car-ried out to evaluate the applicability of this method.[Result]The optimized PCR amplification reactions 50.0 μL:2×F8 FastLong PCR MasterMix 25.0 μL included 2.0 μL for upstream primer and downstream primer,5.0 μL of the template and nuclease free water to a final volume of 50.0 μL. PCR amplification was carried out as follows: 3 min initial denatu-ration step at 94℃,followed by 35 cycles of 94℃for 10 s,55℃for 15 s,and 72℃for 15 s,with a final extension step of 5 s at 72℃. The method showed strong specificity for V. parahaemolyticus,V. harveyi,V. vulnificus,V. fluvialis ,V. al-ginolyticus,V. mimicus,V. cholerae and V. ponticus,and its detection sensitivity can reach 102 CFU/mL. Detected by the PCR method established,the results of 51 Vibrios strains isolated from seawater samples and 32 Vibrios strains isolated from Penaeus vannamei samples were positive. The 51 Vibrios strains isolated from seawater samples contained strains V. parahaemolyticus,V. harveyi,V. vulnificus,V. fluvialis,V. alginolyticus and V. Ponticus ;and 32 Vibrios strains isolated from P. vannamei samples contained V. parahaemolyticus,V. harveyi,V. vulnificus,V. fluvialis and V. alginolyticus. Se-quencing analysis and homology alignment of above 83 Vibrios showed that interspecific dnaJ gene sequence similarities reached 79.5%-92.8%,and intraspecies dnaJ gene sequence similarities reached 98.2%-99.9%.[Conclusion]The method developed base on dnaJ gene in this study can amplify almost full length dnaJ gene of Vibrios,and is fast,convenient, sensitive,accurate and adaptable.It can provide all-around and rapid detection of Vibrios and identification of Vibrio spe-cies along with sequencing analysis.关键词
弧菌/dnaJ基因/PCR检测/鉴定Key words
Vibrios/dnaJ gene/PCR detection/identification分类
农业科技引用本文复制引用
陈静,韦信贤,童桂香,黄鸾玉,黎小正,吴祥庆,黄国秋,谢宗升,熊建华,谭红连..基于dnaJ基因的弧菌PCR检测方法[J].南方农业学报,2018,49(6):1228-1234,7.基金项目
广西创新驱动发展专项项目(桂科AA17204081-4) （桂科AA17204081-4）
广西自然科学基金项目(2015GXNSFBA139069) （2015GXNSFBA139069）
广西水产畜牧兽医局科技推广应用项目(桂渔牧科201633022) （桂渔牧科201633022）
广西直属公益性科研院所基本科研业务费专项项目(GXIF-2016-10) （GXIF-2016-10）
