Design of a multiplex quantitative reverse transcription‐PCR system to simultaneously detect 16 pathogens associated with bovine respiratory and enteric diseases

Bovine respiratory disease (BRD) and bovine enteric disease (BED) are two major diseases in cattle, resulting in severe economic losses in the dairy and beef industries. The two major diseases are associated with several factors such as viruses, bacteria, the health condition of the host and environmental factors. We aimed to design a new efficient diagnostic method, which rapidly detect causative pathogens, minimizing economic loss due to BRD and BED. We designed a multiplex quantitative reverse transcription‐PCR (qRT‐PCR) system for the simultaneous diagnosis of 16 pathogens, including 12 viruses and 4 bacteria related to BRD and BED, based on single qRT‐PCR assays in previous studies. The designed multiplex qRT‐PCR was highly sensitive and has minimal detection levels which will be no different from those of single qRT‐PCR. Moreover, the multiplex qRT‐PCR could more efficiently detect the causative pathogens than conventional RT‐PCR in test using a part of BRD and BED clinical samples. Furthermore, our data revealed that the multiplex qRT‐PCR had high performance in its specificity and reproducibility tests. Our system can effectively detect multiple BRD or BED related pathogens from each animal while testing several clinical samples via the multiplex qRT‐PCR. It is more time‐, cost‐ and labour‐efficient than other diagnostic methods. Rapid detection of infected animals from the herd using our system will greatly contribute to infection control and prompt treatment in field.