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A Sensitive and Broad-Spectrum Real-Time Reverse Transcription Quantitative PCR Assay for the Detection of Bovine
Caixia Wang1, Haoyang Yu1, Bin Zhang2
1Animal Inspection and Quarantine Institute, Chinese Academy of Quality and Inspection & Testing, Beijing 100176, China.
Background:
Bovine coronavirus (BCoV) is an economically significant pathogen that causes respiratory and enteric infections in cattle and wild ruminants. Although multiple diagnostic methods are available, there is still an urgent need for a sensitive real-time reverse transcription quantitative PCR (RT-qPCR) assay capable of detecting continuously emerging novel strains of BCoV.
Objective:
This study aimed to develop a sensitive and broad-spectrum RT-qPCR assay for the efficient detection of BCoV.
Methods:
Following optimization and comparison of two primer-probe sets targeting the M gene, an RT-qPCR assay was established. Using Decaro's method as a reference, gradient dilutions of transcribed RNA solutions and BCoV-derived cDNA were prepared to test sensitivity, repeatability, and reproducibility. Additionally, four other bovine viruses were used to evaluate specificity. Finally, the diagnostic sensitivity and specificity of both assays were analyzed using 46 field samples.
Results:
A sensitive and broad-spectrum RT-qPCR assay was developed in this study. Its analytical sensitivity was 10 copies/μL with transcribed RNA as the template, comparable to that of Decaro's method. While the cDNA of an emerging Chinese viral isolate was used as the template, the analytical sensitivity was one order of magnitude higher than that of Decaro's method. Reproducibility testing revealed intra-assay coefficients of variation (CV) ranging from 1.75 to 3.56%, and inter-assay CV values between 3.13 and 4.91%. When evaluating the diagnostic sensitivity and specificity with 46 field samples, our assay exhibited higher diagnostic sensitivity compared to Decaro's method, a difference explained by two mutations within the primer and probe regions of Decaro's method.
Conclusion:
A highly sensitive and broad-spectrum RT-qPCR assay was successfully developed for the detection of BCoV, especially for the detection of emerging novel strains.
Highlights:
The RT-qPCR assay developed in this study exhibited high sensitivity, particularly in detecting BCoV field strains and newly emerging variants circulating in recent years.
