Development of a nested PCR assay for specific detection of Metschnikowia bicuspidata infecting Eriocheir sinensis

Abstract

In recent years, the “milky disease” caused by Metschnikowia bicuspidata has seriously affected the Eriocheir sinensis culture industry. Discovering and blocking the transmission route has become the key to controlling this disease. The existing polymerase chain reaction (PCR) detection technology for M. bicuspidata uses the ribosomal DNA (rDNA) sequence, but low sensitivity and specificity lead to frequent false detections. We developed a highly specific and sensitive nested PCR method to detect M. bicuspidata, by targeting the hyphally regulated cell wall protein (HYR) gene. This nested HYR-PCR produced a single clear band, showed no cross-reaction with other pathogens, and was superior to rDNA-PCR in specificity and sensitivity. The sensitivity of nested HYR-PCR (6.10 × 101 copies/μL) was greater than those of the large subunit ribosomal RNA gene (LSU rRNA; 6.03 × 104 copies/μL) and internal transcribed spacer (ITS; 6.74 × 105 copies/μL) PCRs. The nested HYR-PCR also showed a higher positivity rate (71.1%) than those obtained with LSU rRNA (16.7%) and ITS rDNA (24.4%). In conclusion, we developed a new nested HYR-PCR method for the specific and sensitive detection of M. bicuspidata infection. This will help to elucidate the transmission route of M. bicuspidata and to design effective management and control measures for M. bicuspidata disease.

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

“…Nested PCR has a higher sensitivity and specificity than conventional PCR; however, its operation is complex and time-consuming. Additionally, it cannot quantify the extent of pathogen infection [ 13 ]. qPCR technology possesses not only high sensitivity, good specificity, and accuracy, but also ease of operation and implementation, rendering it widely applicable for the detection of aquatic pathogens [ 19 ].…”

“…The COX6A gene primers demonstrated robust specificity and produced clear bands during sample analysis. Currently, the primers designed for LSU rRNA and its ITS region have the potential to amplify template DNA from other pathogens, resulting in a target band of Staphylococcus aureus appearing at the same location as M. bicuspidata [ 13 ]. The presence of other microorganisms in environmental samples or individuals may result in erroneous detection, and thus necessitating the sequencing of the target band to confirm M. bicuspidata identity.…”

“…Conventional PCR primers were designed based on the 26S rDNA and 5.8S-ITS rDNA sequences of M. bicuspidata [ 11 , 12 ]. The previously published outer primers PN1/PN2 and inner primers PN3/PN4, which were designed based on the HYR cell wall protein gene, were used as primers for the nested PCR [ 13 ] ( Table 1 ).…”

“…Conventional PCR primers were designed based on the 26S rDNA and 5.8S-ITS rDNA sequences of M. bicuspidata [11,12]. The previously published outer primers PN1/PN2 and inner primers PN3/PN4, which were designed based on the HYR cell wall protein gene, were used as primers for the nested PCR [13] (Table 1). The standard PCR reaction procedure was as follows: initial denaturation at 95 • C for 10 min, followed by denaturation at 95 • C for 1 min, annealing at 55 • C for 45 s, and extension at 72 • C for 1 min, for 35 cycles, and then the final extension at 72 • C for an additional 10 min.…”

“…However, the accurate diagnosis of crabs with mild initial infection levels is hindered by low sensitivity, thereby impeding disease prevention and treatment efforts. The previously reported nested PCR detection technology, which is highly sensitive and specific, is time-consuming and does not enable the direct quantification of M. bicuspidata in Chinese mitten crabs [ 13 ].…”