A multiplex real-time fluorescence-based quantitative PCR assay for calf diarrhea viruses

Area of Science:

• Veterinary Virology
• Molecular Diagnostics
• Animal Health

Background:

• Calf diarrhea causes significant economic losses in the cattle industry.
• Several viruses, including bovine torovirus (BToV), bovine enterovirus (BEV), and bovine norovirus (BNoV), are implicated, with limited research and no specific treatments for some.
• Existing vaccines for bovine coronavirus (BCoV), bovine rotavirus (BRV), and bovine viral diarrhea virus (BVDV) do not fully control disease prevalence.

Purpose of the Study:

• To develop a multiplex real-time fluorescence quantitative PCR (qPCR) method for simultaneous detection of six major calf diarrhea viruses.
• To establish an efficient and reliable tool for monitoring the prevalence of BToV, BEV, BNoV, BCoV, BRV, and BVDV in calves.
• To provide a reference for future prevention and control strategies against these viral pathogens.

Main Methods:

• Designed specific primers and probes for key genes of BNoV, BEV, BToV, BCoV, BRV, and BVDV.
• Developed a multiplex real-time fluorescence quantitative PCR assay for simultaneous detection.
• Collected and analyzed 295 fecal and anal swab samples from diarrheic calves in Guangdong, China.

Main Results:

• The multiplex qPCR method demonstrated high efficiency, stability, and sensitivity, with low detectable plasmid levels for each virus.
• The assay showed strong linearity (R² ≥ 0.98) and amplification efficiency (90%-110%) with minimal variation (< 3%).
• In Guangdong, China, prevalence rates were BToV (0.34%), BEV (6.10%), BNoV (0.68%), BCoV (1.36%), BRV (10.85%), and BVDV (2.03%), with BEV and BRV being most prevalent. BToV and BNoV were detected for the first time in this region.

Conclusions:

• Successfully established a sensitive and efficient multiplex real-time qPCR method for detecting six critical bovine viruses.
• The study identified the prevalence of these viruses in Guangdong, China, highlighting BEV and BRV as significant contributors to calf diarrhea.
• The findings provide valuable data for disease surveillance and support the development of targeted control strategies, benefiting the cattle industry.