Establishment and application of six-plex fluorescent quantitative PCR assay for the detection of calf diarrhea pathogens.

Calf diarrhea is a common clinical disease, causing seriously economic losses to the cattle breeding industry. Bovine coronavirus (BCoV), Bovine rotavirus (BRV), Bovine viral diarrhea virus (BVDV), Escherichia coli (E. coli), Salmonella and Clostridium perfringens (C. perfringens) are common pathogens causing calf diarrhea, Diarrhea in calves caused by these six pathogens is clinically similar and most of them have conditions such as mixed or secondary infections. However, the commonly used method of detecting these six pathogens one by one at present is time-consuming and laborious, which seriously restricts the timely and effective prevention and control of calf diarrhea in production practice. This study established a rapid, efficient, and sensitive multiplex fluorescence quantitative PCR detection method, comprising two independent triplex reactions, for the six pathogens causing calf diarrhea. The results show that the drawn standard curve has a good linear relationship, and R2 is greater than 0.990. There is no amplification of other common pathogens in calves. The coefficient of variation (CV) of intra-group repetition and inter-group repetition ranged from 0.17 % to 0.70 %. By testing 932 clinical rectal swab samples of diarrheal calves and comparing them with commercial kits, the results showed that the multiplex fluorescence quantitative PCR detection method established in this study had the characteristics of high specificity and high sensitivity. In conclusion, the detection method established in this study can be used for large-scale detection of clinical samples and is of great applied value for the prevention and treatment of calf diarrhea.