Page 153 - 《渔业研究》2025年第5期

694 渔 业 研 究 第 47 卷

Detection of Metschnikowia bicuspidate based on hyper-branched rolling circle
amplification in the mitten crab (Eriocheir sinensis)

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WEI Hua ，ZHAO Qingru ，TAN Yabing ，LI Jinling ，SUN Xin ，ZHAO Yingying ，
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LI Yingdong ，SUN Na ，LI Xiaodong 1,2,3*
(1. Key Laboratory of Northeast Livestock and Poultry Disease Research, Ministry of Education, School of Animal Science and
Medicine, Shenyang Agricultural University, Shenyang 110866, China;
2. Key Laboratory of Breeding and Reproductive Cultivation of Chinese Mitten Crab, Ministry of Agriculture and
Rural Affairs, Panjin 124000, China;
3. Panjin Guanghe Crab Industry Co., Panjin 124000, China)
Abstract: [Background] Metschnikowia bicuspidate is one of the pathogens of ‘milky disease’ in Eriocheir
sinensis, which is a serious obstacle to the development of aquaculture industry. So far there is no effective drug
treatment. Rapid, efficient and accurate diagnosis is the key to prevent and control the disease. Molecular bio-
logy techniques, such as PCR, have enabled safe and rapid detection of M. bicuspidate compared to traditional
pathogen isolation and culture techniques. But it still requires complex temperature-changing equipment that
makes it difficult to carry out testing at the poolside. [Objective] A new method based on hyper-branched
rolling circle amplification (HRCA) technology has been established for detecting Saccharomyces cerevisiae,
providing a new approach for pool-side detection of yeast pathogens. [Methods] Herein, HRCA was estab-
lished based on the sequence of the 26S rRNA (U44822) of M. bicuspidate, by designing a padlock probe (PLP)
and primers with high specificity. The sensitivity, specificity, reproducibility and application effect of the meth-
od were also evaluated. [Results] The results showed that the optimal reaction system for HRCA was 1 μmol/L
probe concentration, 20 min amplification time, 63 ℃ amplification temperature, and 2 U/mL Bst DNA poly-
merase concentration. The method has a detection range of 0.50−1 000 fmol/L for standard samples, exhibits
high interspecies specificity, enables single base mismatch recognition, and demonstrates good repeatability and
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stability. In actual sample testing, the method had a sensitivity of 4.13×10 pg/μL for detecting DNA from hep-
atopancreas of Chinese mitten crabs and a detection rate of 56.25% for randomly selected Chinese mitten crabs,
consistent with the results obtained by using the qPCR method. [Conclusion] HRCA technique can efficiently,
rapidly and sensitively detect the ‘milky disease’ pathogen of M. bicuspidate under constant temperature, which
is suitable for poolside detection. The study not only provides a new means for the detection of yeast pathogens,
but also provides new technical support for the early diagnosis and prevention of the disease, which has great
potential for application in the field of aquatic pathogen diagnosis.
Key words: hyper-branched rolling circle amplification (HRCA); Metschnikowia bicuspidate; Eriocheir
sinensis; milky disease