| 【Objective】A real-time PCR method was developed to detect Acute Hepatopancreas Necrosis Disease
（AHPND）caused by Vibrio parahaemolyticus（VpAHPND）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 VpAHPND,
and a real-time PCR method for the detection of VpAHPND 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 VpAHPND. 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 VpAHPND.【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 VpAHPND in shrimp samples. |
