A TaqMan-MGB Probe Quantitative PCR Assay Detecting Hematodinium perezi.

Hematodinium perezi, a pathogenic dinoflagellate, is one of major epidemiological agents that lead to severe losses of cultured marine crustaceans in China. This study aimed to develop a novel, sensitive and specific detection method qualified for early surveillance and control of the disease caused by H. perezi. The present study established a TaqMan-MGB probe quantitative PCR (qPCR) method, targeting the first internal transcribed spacer 1 (ITS 1) region of H. perezi by optimising reaction components. A high correlation coefficient (R² = 0.9996) was obtained in a standard curve with a 103.4% efficiency. No amplification was observed for the host's genome and pathogens other than H. perezi in the TaqMan-MGB probe qPCR assays, showing high specificity to H. perezi. When using the plasmid standard DNA as templates, the detection limit of the qPCR method was determined to be 5.66 copies/reaction and 10 times more sensitive than the conventional PCR. The TaqMan-MGB probe qPCR assays exhibited high repeatability, and the intra- and inter-assay coefficients of variation (CV) ranged from 0.11% to 2.25% over a wide dynamic range of detection from 5.66 × 10⁰ to 5.66 × 10⁹ copies of targeting gene. The application was also validated on clinical samples, including those with low infection with H. perezi. This novel one-step TaqMan-MGB probe qPCR provides an option for surveillance and epidemiological investigations of H. perezi infection, with an advantage at the early infection stage.