To establish a multiplex qRT-PCR method for detecting calf diarrhea disease, aiming to simultaneously and rapidly detect multiple pathogens that cause diarrhea in calves.

This study searched for conserved gene sequences of various pathogens causing diarrhea in calves as target genes, designing specific primers and probes, constructing plasmid standards, optimizing primer concentrations and annealing temperatures, and testing their sensitivity, specificity, and clinical sample validation.

A multiplex qRT-PCR method for detecting calf diarrhea disease was successfully established using 103 copies/μL plasmid as the standard.The reaction system consisted are 10 μL 2 × Fast-Start Universal SYBR Green Master, 0.5 μL upstream primer, 0.5 μL downstream primer, 1 μL template, 8 μL sterilized ultrapure water, and a total reaction volume of 20 μL. The reaction conditions are: 95 ℃ for 3 minutes; 94 ℃ for 20 seconds, 56 ℃ for 15 seconds, 72 ℃ for 30 seconds, 36 cycles; 72 ℃ for 5 minutes. The detection sensitivity is 1 copy/μL, and there is no cross-reactivity with other pathogens. The intra-group and inter-group coefficients of variation are both less than 2%. Among 315 clinical fecal samples of diarrheal calves, a total of 248 are detected as positive (78.73%), and 42 are positive for mixed infections (13.33%), which is completely consistent with the positive detection rate of the single PCR detection method.

This study successfully established a multiplex qRT-PCR method capable of simultaneously detecting six pathogens responsible for calf diarrhea. The method exhibits a detection sensitivity of up to 1 copy/μL for plasmid DNA, shows no cross-reactivity with other common pathogens, and achieves a 100% concordance rate with single PCR method in the detection of 315 clinical samples.