CAJ | 학술논문

根据哈维氏弧菌(Vibro harveyi)溶血素基因vhhP2的保守序列设计特异性引物,建立了SYBR Green I实时定量PCR检测哈维氏弧菌的方法。构建含vhhP2基因的重组质粒作为标准品,进行SYBR Green I实时定量PCR,在Tm 为60℃时,扩增产物的熔解曲线仅有一个特异峰,扩增所得标准曲线为 y=-3.331x+37.48,相关系数为0.998,扩增效率为1,最低可检测到7个拷贝。实验结果表明该检测技术具有较高的特异性、敏感性和重复性,对哈维氏弧菌病的快速诊断和流行病学调查有重要意义。
근거합유씨호균(Vibro harveyi)용혈소기인vhhP2적보수서렬설계특이성인물,건립료SYBR Green I실시정량PCR검측합유씨호균적방법。구건함vhhP2기인적중조질립작위표준품,진행SYBR Green I실시정량PCR,재Tm 위60℃시,확증산물적용해곡선부유일개특이봉,확증소득표준곡선위 y=-3.331x+37.48,상관계수위0.998,확증효솔위1,최저가검측도7개고패。실험결과표명해검측기술구유교고적특이성、민감성화중복성,대합유씨호균병적쾌속진단화류행병학조사유중요의의。
Vibrio harveyi is an important pathogenic bacterium of most marine aquaculture animals that has caused sig-nificant losses within the aquaculture industry. The pathogenicity of V. harveyi is influenced by quorum sensing, mean-ing that population density plays a role in determining the outcome of an infection. Thus, there is a need to develop a method to detect the density of Vibrio harveyi. We designed a pair of specific primers based on the V. harveyi spe-cies-specific vhhP2 gene to establish a SYBR Green I real-time fluorescence quantitative PCR detection method. A 151 bp gene fragment was amplified from the chromosomal DNA of V.harveyi from different sources. The primers did not cross react with nine other bacteria species using conventional PCR, suggesting the primer pair has good intra-species specificity and inter-species commonality. A recombinant plasmid containing the vhhP2 gene of V. harveyi was con-structed and used to construct the standard curve. The standard curve for the Ct values and initial template was repre-sented by the formula:y=-3.331x+37.48. The correlation coefficient was 0.998 and the amplification efficiency was 1.00, indicating that there was a good linear relationship between initial templates and Ct values. The melting curve had only one specific peak at an annealing temperature of 60℃. The detection limit of the assay was seven copies per reac-tion, which is 10 000 times more sensitive than that of conventional polymerase chain reaction (PCR). The results of intra-and inter-assay variability tests demonstrated that the method was highly reproducible. Our results suggest that this SYBR Green I real-time PCR assay may be used for the rapid and accurate detection of V. harveyi from infected aquaculture species. This will allow early diagnosis of V. harveyi infection and improve the efficacy of disease preven-tion and surveillance programs.