PCR detection method for Vibrios based on dnaJ gene

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.