Abstract: The gyrB gene, which encodes the B subunit protein of DNA gyrase, is a single copy gene and has conserved regions for PCR primers. A pair of specific primers target to the gyrB gene of V. parahaemolyticus was designed, and a SYBR green I-based real-time PCR for V. parahaemolyticus detection was established. The PCR primers could amplify 285-bp gene fragment from chromosomal DNA of V. parahaemolyticus, and no positive reaction was detected in 8 other pathogenic bacteria using conventional PCR. In addition, the results of melting curve analysis showed only a specific peak with a melting temperature (Tm) of 90 ℃, and no primer-dimers peak was observed. These findings indicated that the PCR primers had high specificity. Both geometric growth and plateau phases were observed in PCR amplification curves. Analysis of standard curves revealed excellent correlation between the number of copies (in the range of 2.06 × 108 to 2.06 × 103) and PCR threshold cycle (Ct) with a correlation coefficient of 0.992 (R2 ＝0.992). It took only 4－5 h (from nucleic acid extraction to analysis of results) to detect samples by the method. Therefore, SYBR green-based I real-time PCR had the advantages of higher sensitivity and ease of operation over traditional methods and could be used for inspection and quarantine of import and export commodities, food safety detection, and diagnostic studies and molecular epidemic survey of aquatic animal diseases caused by V. parahaemolyticus.

Key words: Vibrio parahaemolyticus, gyrB gene, SYBR green I, real-time PCR