基于dnaJ基因的弧菌PCR检测方法

PCR detection method for Vibrios based on dnaJ gene

  • 摘要: 目的建立一种能扩增dnaJ基因全长的PCR检测方法,为弧菌的快速筛查及种类鉴定提供技术支持.方法以弧菌dnaJ基因为靶基因,在其首尾的保守区域设计一对简并引物,经优化退火温度和引物浓度,建立能扩增弧菌接近全长dnaJ基因的PCR检测方法,并通过特异性试验、灵敏度试验和临床应用评价等验证其适用性.结果优化后的PCR反应体系50.0 μL:2×F8 FastLong PCR MasterMix 25.0 μL,上、下游引物(20 μmol/L)各2.0 μL,DNA模板5.0 μL,灭菌水补足至50.0 μL.扩增程序:94℃预变性3 min;94℃ 10 s,55℃ 15 s,72℃ 15 s,进行35个循环;最后72℃延伸5 min.该PCR检测方法对副溶血弧菌、哈氏弧菌、创伤弧菌、河流弧菌、溶藻弧菌、拟态弧菌、霍乱弧菌和栖黑海弧菌等8种弧菌属细菌具有很强的特异性,对弧菌的最低检出限为102CFU/mL.应用建立的PCR检测方法对51株分离自海水样品的弧菌和32株分离自对虾样品的弧菌进行检测,结果显示全部为阳性,其中,海水样品中包括有副溶血弧菌、溶藻弧菌、创伤弧菌、河流弧菌、哈氏弧菌和栖黑海弧菌,对虾样品中包括副溶血弧菌、溶藻弧菌、创伤弧菌、河流弧菌和哈氏弧菌;经测序及同源性比对分析发现,种间菌株的dnaJ基因序列相似度为79.5%~92.8%,种内菌株间的dnaJ基因序列相似度为98.2%~99.9%.结论基于dnaJ基因建立的弧菌PCR检测方法能扩增出弧菌接近全长dnaJ基因,具有快速便捷、灵敏准确、适应性好等优点,结合测序技术,可为弧菌快速筛查及种类鉴定提供更全面和更准确的技术手段.

     

    Abstract: ObjectiveA 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.MethodTaking 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.ResultThe 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%.ConclusionThe 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.

     

/

返回文章
返回