Development of real-time RT-PCR systems for detection and quantitation of bovine enteric viral pathogens

Abstract

The enteric viruses in animals are responsible for severe and devastating losses to the livestock owners with a profound negative impact on animal, health, welfare, and productivity. These viruses are usually transmitted via the feco-oral route and primarily infect the digestive tract of the humans, bovines and different mammals as well as birds. Some of the important enteric viruses in ruminants are: Rotavirus A (RVA), Peste des petits virus (PPRV), Norovirus (NV), Bovine corona virus (BoCV) and Bluetongue virus (BTV). In the present study, sensitive, specific and reliable TaqMan probe-based RT-qPCRs were developed and standardized for the rapid detection and quantification of enteric viruses from fecal samples. The assays result in efficient amplification of the RVA, BTV and BoCV RNA with a limit of detection (LoD) of 5, 5 and 4 copies, respectively, which is 1000 times more sensitive than the traditional gel-based RT-PCR. The reproducibility of each assay was satisfactory, thus allowing for a sensitive and accurate measurement of the viral RNA load in clinical samples. In conclusion, real time PCR developed for these viruses are highly specific and sensitive technique for the detection of diarrheic viral pathogens of cattle and buffalo.

Keywords:

• Real-time
• TaqMan-based RT-qPCR
• Rotavirus (RVA)
• Bovine coronavirus (BoCV)
• Bluetongue virus (BTV)

Acknowledgement

The authors are thankful to the Department of Animal Biotechnology, College of Veterinary and Animal Sciences, Lala Lajpat Rai University of Animal Sciences for providing financial, technical, logistic support and human resource for the smooth conduction of work. Authors also wish to appreciate the work of all the researchers whose works are referenced in the present research.

Competing interests

The authors have no relevant financial or non-financial interests to disclose.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The authors would like to acknowledge funding support from state scheme entitled ‘Development of molecular diagnostics for important diseases of livestock, poultry and pets’. Scheme no. 121 C (a) ABT-2 Dev work. The authors would also like to thank the LUVAS administration and various staff of Animal Husbandry Department of Haryana for their direct or indirect help.