Establishment of a PCR-pyrosequencing method for the rapid detection of three marine vibrios and the investigation on 16S rRNA genotyping of Vibrio vulnificus / 临床检验杂志

ABSTRACT

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 whA gene of V.vulnificus and the toxR genes of V.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 sequence 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 whA 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.