Establishment and Application of Real-Time Fluorescence Quantitative PCR Detection Technology for IMetschnikowia bicuspidata/I Disease in IEriocheir sinensis/I

• Published in: Journal of fungi (Basel) 2023-07, Vol.9 (8)
• Main Authors: Xing, Yuenan, Chen, Ye, Feng, Chengcheng, Bao, Jie, Li, Xiaodong, Jiang, Hongbo
• Format: Article
• Language: English
• Subjects: Aquaculture industry; Fluorescence; Polymerase chain reaction
• ISSN: 2309-608X; 2309-608X
• DOI: 10.3390/jof9080791

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 (R[sup.2]) of 0.996, amplification efficiency of 103.092%, and a lower limit of detection sensitivity of 7.6 × 10[sup.1] 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