Objective A real-time PCR method was developed to detect Acute Hepatopancreas Necrosis Disease (AHPND)caused by Vibrio parahaemolyticus(Vp_AHPND)for the rapid detection of such disease.

Method A pair of specific primers and fluorogenic-labeled TaqMan probe were designed according to the conservative sequence of Vp_AHPND, and a real-time PCR method for the detection of Vp_AHPND was established by optimizing the reaction system and conditions. The standard curve was made with recombinant plasmids as standard products, and the specificity, sensitivity, repeatability and clinical application test were carried out.

Result The method had a wide quantitative range from 2.3×101 to 2.3×107 copies/μL and had linear relationship in its standard curve. The real-time PCR method had a high sensitivity with the detection limit as low as to 2.3 copies/μL for the purified recombinant plasmids of PMD-18T-pirB, and the entire detection could be completed within 1 h for a single sample. It also had a high specificity in detecting DNA of Vp_AHPND. The variation coefficients among cycle thresholds(Ct)of the repeatability test were 0.45%-0.69%. Comparing with the nested PCR recommended by OIE, the TaqMan probe real-time PCR had a same test rate of Vp_AHPND.

Conclusion The TaqMan probe real-time PCR method was highly specific, sensitive and repeatable, which could be used for the early diagnosis of acute and latent infections of Vp_AHPND in shrimp samples.