焦磷酸测序快速检测3种海洋性弧菌的方法学建立及创伤弧菌16S rRNA基因分型的研究

Establishment of a PCR-pyrosequencing method for the rapid detection of three marine vibrios and the investigation on 16S rRNA genotyping of Vibrio vulnificus

目的建立一种海洋致病性弧菌的快速诊断方法,为海洋性弧菌感染的临床检测构建新的技术平台。方法分别针对创伤弧菌vvh A,副溶血弧菌和溶藻弧菌的toxR基因设计扩增引物和测序引物,并PCR扩增特异性DNA片段,制备单链模板,进行焦磷酸测序,得到的碱基序列经NCBI比对验证;对创伤弧菌16S rRNA基因进行分型。结果创伤弧菌的扩增引物和测序引物均表现出较好的特异性,4株创伤弧菌均扩增出167 bp大小的DNA片段,并且测序结果与NCBI比对达到100%匹配度,而其他菌株未见扩增条带,测序结果为阴性;11株副溶血弧菌和溶藻弧菌均分别扩增出105 bp和134 bp大小的DNA片段,并且经测序得到与预期相符的DNA碱基序列。4株创伤弧菌中1株属于16S rRNA-A型,其余3株均属于16S rRNA-B型。结论本研究建立的PCR-焦磷酸测序方法是一种短核苷酸序列实时测定的新方法,具有高通量、精确、简单易行的优点,适用于海洋性致病菌及陆地感染细菌的快速精准鉴定。

Objective To establish a rapid diagnostic method for the detection of marine vibrios, and then construct a new technology platform for the clinical diagnosis of marine vibrio infection. Methods A pair of PCR primers and a sequencing primer based on the vvhA gene of V. vulnificus and the toxR genes of E parahemolyticus and V. alginolyticus were designed respectively, and then the specific DNA fragments were amplified. Next, the single-stranded DNA templates were prepared for pyrosequencing. The obtained base se- quence was validated by NCBI alignment. In addition, the 16S rRNA genotyping of V. vulnificus was also performed. Results The PCR primers and sequencing primer of V. vulnificus showed good specificity, and a 167 bp DNA fragment was amplified from 4 strains of V. vulnificus. The pyrosequencing results completely matched with the vvhA gene sequence of V. vulnificus. Meanwhile, the control strains were negative. A 105 bp DNA fragment and a 134 bp DNA fragment were amplified from 11 strains of V. parahemolyticus and V. alginolyticus, respectively, and the pyrosequencing results were consistent with the expected sequence. In addition, one of 4 strains of V. vulnificus was identified as 16S rRNA-A type, and the other 3 as 16S rRNA-B type. Conclusion The PCR-pyrosequencing method established in this study is a new method for the real-time detection of short nucleotide sequences. It has some advantages such as high throughput, high precision and simple operation, and may be applied to the fast and accurate identification of marine and terrestrial pathogenic bacteria.