PCR-焦磷酸测序检测副溶血性弧菌研究

Detection of Vibrio parahaemolyticus by PCR-pyrosequencing

中文关键词:  副溶血性弧菌  聚合酶链式反应  焦磷酸测序  DNA碱基序列  toxR基因

英文关键词:Vibrio parahaemolyticus  polymerase chain reaction (PCR)  pyrosequencing  DNA base sequence  toxR gene

中文摘要:

      目的 建立副溶血性弧菌(Vibrio parahaemolyticus,VP )的PCR-焦磷酸测序方法。方法 根据副溶血性弧菌toxR基因设计扩增引物和测序引物, 先用PCR特异性地扩增目的片段, 再制备单链模版, 最后用焦磷酸测序法检测DNA碱基序列, 检测的序列为CCAAGGATTCACAGCAGAAGCCACAGGTGCTTTT, 则判断为副溶血性弧菌。结果 扩增引物和测序引物表现出良好的特异性。PCR扩增结果: 20株VP均扩增出大小为137 bp的DNA片段, 而创伤弧菌等对照菌株未扩增出DNA条带。焦磷酸测序结果: 20株副溶血性弧菌均测出与预期相符的DNA碱基序列, 而其他对照菌株未测出DNA碱基序列或检测结果与测序引物后的序列不匹配。VP标准菌株ATCC 17802发生A-T突变, 密码子CCU变成CCA, 而两个密码子都是脯氨酸的密码子。结论 该方法特异性高,是快速检测VP有效手段。

英文摘要:

      Objective To establish a method for the detection of Vibrio parahaemolyticus(VP) by polymerase chain reaction(PCR)-pyrosequencing. Methods Amplification primers and sequencing primers were designed according to the toxR gene of VP. Then, the target fragment was amplified specifically by PCR and single-stranded DNA template was obtained from the PCR products. Finally, the base sequence was detected by pyrosequencing under the guidance of the sequencing primers. Vibrio parahaemolyticus could be confirmed when the target fragment CCAAGGATTCACAGCAGAAGCCACAGGTGCTTTT was detected. Results PCR primers and sequencing primers showed a good specificity. PCR results showed that 20 VP trains could be amplified to 137 bp DNA fragments, while Vibrio vulnificus and other control strains could not. Meanwhile, pyrosequencing results showed that 20 VP were consistent with the expected results, while the other control strains could not be detected the sequences or the test results did not match the sequences of sequencing primers. In addition, VP standard strain ATCC 17802 appeared A-T mutation. As a result, codon CCU became the CCA, both of which are proline codon. Conclusion This method showed a good specificity, and it could be used for the detection of VP rapidly.