Rapid discrimination of three species of marine Vibrios by using linear-after-the-exponential PCR coupled with pyrosequencing

Aim ：To develop a method based on linear-after-the-exponential-polymerase chain reaction(LATE-PCR) coupled with pyrosequencing for rapid detection of pathogenic marine Vibrios. Methods ：Firstly，LATE-PCR used to detect the target fragments on 16S rRNA gene and outer membrane protein K(OmpK) gene of Vibrios was achieved by carefully designing primers and optimizing reaction conditions.Secondly，the by-products of LATE-PCR were eliminated by a series of enzymatic reactions to reduce interference to pyrosequencing.Finally，the samples were detected directly by pyrosequencing after annealing. Results ：The ideal single chain and enough amount can be obtained for pyrosequencing by 1-2 μL PCR product，and the results were consistent with those of Sanger method.The sequences of the Vibrio samples by pyrosequencing were consistent with GenBank report，and the Vibrio samples can be confirmed through the detection of the target fragment on 16S rRNA gene；and the species of Vibrios can be distinguished by the detection of the target fragment on OmpK gene.We successfully discriminated three Vibrios i. e. Vibrio parahaemolyticus，V.harveyi and V.alginolyticus. Conclusion ：Our method has high sensitivity，simple operation and low cost. It is also suitable for detecting a mass of samples.It is promising that this assay is used in the diagnosis and control of the infection caused by Vibrios and correlative research.