Development of a TaqMan real-time quantitative PCR assay to detect Metschnikowia bicuspidata in Chinese mitten crab Eriocheir sinensis

Abstract

Milky disease of Chinese mitten crab Eriocheir sinensis caused by Metschnikowia bicuspidata is a novel disease with high mortality. No effective treatment is currently available, but a rapid, accurate detection method is required for the prevention and control of the disease. In this study, the genome-sequencing results of M. bicuspidata and similar species were used for comparative genomic analysis for genes specific to M. bicuspidata. A quantitative PCR (qPCR) detection method for M. bicuspidata was then established using the specific primers and probes designed according to the sequence of a hypothetical protein gene specific to M. bicuspidata. The assay was found to have a high degree of repeatability and reproducibility, with a linear dynamic range (R2 = 0.998) extending over 9 log10 dilutions and a high efficiency (100.7%). Furthermore, the method showed high sensitivity, being able to detect at least 11.3 copies μl-1 of recombinant plasmid, and strong specificity, without any cross-reaction with any of the 9 species of yeast that are closely related to M. bicuspidata or any of 16 species of pathogenic bacteria commonly observed in aquatic animals. The established method was used to examine 138 apparently healthy crabs collected from 22 farms, with 21 samples (15.2%) found to be M. bicuspidata-positive. Thus, the developed qPCR assay is a specific, sensitive, stable, and rapid diagnostic method for the detection and quantification of M. bicuspidata DNA from E. sinensis tissues.