Establishment and Application of Real-Time Fluorescence Quantitative PCR Detection Technology for Metschnikowia bicuspidata Disease in Eriocheir sinensis.

Abstract

Metschnikowia bicuspidata causes a "milky disease" in Chinese mitten crab, Eriocheir sinensis, which inflicts significant damage on the breeding industry, but there are no effective drugs for this disease. Precise detection technologies and clarification of transmission routes are now essential to prevent its occurrence. A real-time fluorescent quantitative PCR (qPCR) detection method targeting the mitochondrial cytochrome c oxidase subunit VIA (COX6A) of M. bicuspidata was developed and its sensitivity, specificity, repeatability, and application effectiveness evaluated. There was a robust linear relationship between the qPCR threshold cycle value (Ct) and copy number of the standard with a wide dynamic range. The standard curve had a correlation coefficient (R2) of 0.996, amplification efficiency of 103.092%, and a lower limit of detection sensitivity of 7.6 × 101 copies/µL. The COX6A-qPCR method exhibited high specificity for the detection of M. bicuspidata, with no cross-reactivity. The intra- and inter-group variation coefficients were <1% and 2%, respectively. The qPCR exhibited superior sensitivity compared to existing detection methods, with a positivity rate of 76.67%. The M. bicuspidata content ranged from 1.0 × 101-2.7 × 106 copies/µL. The COX6A-qPCR detection technology exhibited high sensitivity, strong specificity, and excellent repeatability, enabling the accurate quantification of M. bicuspidata.