A TaqMan ‐ MGB Probe Quantitative PCR Assay Detecting Hematodinium perezi

Abstract

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 ( R 2 = 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 × 10 0 to 5.66 × 10 9 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.

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