Specific Detection of Bovine Coronavirus N Protein with TaqMan Probe qRT-PCR

Jin-Jing Geng, Zhuan-di Gong, Qyong-yi Li, Xiao-yun Shen, Suo-cheng Wei

Abstract


Background:  Bovine Coronavirus (BCoV) can cause acute diarrhea in newborn calves and adult cattle. BCoV infection may cause losses to production by reduced weight gain, reduced milk yield. Several methods have been applied to detect and diagnose BCoV. However, each assay has its deficiency. Currently, real-time quantitative PCR (qRT-PCR) has been utilized to identify and quantify many viral pathogens since it is a highly sensitive. However, the technical assay varies due to normalization control of the signal with an internal standard, typically a housekeeping gene. The main objective of the present study to establish a novel TaqMan probe real-time PCR (qRT-PCR) for detecting BCoV.

Materials, Methods & Results:  The present study was aimed to establish a novel TaqMan probe real-time PCR (qRT-PCR) for detecting bovine coronaviruses (BCoV), and also to develop a diagnostic protocol which simplifies sample collection and processing. One pair of specific primers, one pair of universal primers and a TaqMan probe were designed from the known sequences of conserved nucleocapsid (N) protein of BCoV. Reaction systems of TaqMan qRT-PCR were optimized including concentrations of the primers and probe as well as annealing temperatures. Prior to optimizing the assay, the recombinant plasmids of pMD18-T-BCoV-N were successfully constructed to make standard curves. The sensitivity, specificity and reproducibility were evaluated on the TaqMan qRT-PCR, respectively. A total of 321 feces specimens collected from diarrheic calves were detected with this assay. The results showed the optimized reaction conditions for qRT-PCR were 14.5 μM/L primers, 19.5 μM/L probes and 45.0°C annealing temperatures. The established TaqMan qRT-PCR assay could specially detect BCoV without detecting any other viruses. Its minimum detection limit was 4.72 × 101 copies/μL. However, universal PCR could detect only 4.72 × 103 copies/μL. Its sensitivity was 100-fold stronger than universal PCR. In conclusion, this TaqMan qRT-PCR had excellent specificity, sensitivity and stability with a 100-fold sensitivity stronger than universal PCR. Minimum detection limit was 4.72 × 101 copies/μL. This method was a cost-effective method to diagnose diarrhea and distinguish pathogens in dairy farms.

Discussion:  In this study, the authors developed a quantitative real-time PCR (qRT-PCR) in this study based on the TaqMan probe of BCoV. This TaqMan qRT-PCR assay selected and used one pair of specific primers (BCoV-qF/BCoV-qR) and a specific TaqMan probe (BCoV-probe) targeting the conserved nucleocapsid (N) gene. The specificity of primers and probes was validated with Primer-BLAST. The specificity of the qRT-PCR was confirmed by the negative control and other six viruses. The findings demonstrated that TaqMan qRT-PCR could only detect BCoV. This verified the qRT-PCR had an excellent specificity. It is obvious that this TaqMan qRT-PCR assay can detect only BCoV with stronger sensitivity and reproducibility than other real-time PCR methods. The sensitivity test indicated the minimum detection limit of the TaqMan qRT-PCR was 4.72 × 101copies/μL, or 47.2 copies/μL. Sensitivity of the TaqMan qRT-PCR assay was increased by 100-fold as compared to universal PCR with a good inter-assay and intra-assay reproducibility. Thereby, based on the high sensitivity of the assay of this qRT-PCR assay it may be a cost-effective method to diagnose BCoV infections and indentify the etiologic agents of diarrhea syndrome in the dairy farms.


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References


Alfieri A.A., Alfieri A.F. & Takiuchi E. 2016. Detection of Bovine Coronavirus by Conventional Reverse Transcription Polymerase Chain Reaction. Animal Coronaviruses. 3: 101-113.

Amer H.M. & Almajhdi F.N. 2011. Development of a SYBR Green I based real-time RT-PCR assay for detection and quantification of bovine coronavirus. Mecular and Cellular Probes. 25: 101-107.

Beaudeau F., Ohlson A. & Emanuelson U. 2010. Associations between bovine coronavirus and bovine respiratory syncytial virus infections and animal performance in Swedish dairy herds. Journal of Dairy Science. 93: 1523-1533.

Boileau M.J. & Kapil S. 2010. Bovine coronavirus associated syndromes. Veterinary clinics of North America Food Animal Practice. 26: 123-146.

Chaharaein B., Omar A.R., Aini I., Yusoff K. & Hassan S.S. 2009. Detection of H5, H7 and H9 subtypes of avian influenza viruses by multiplex reverse transcription polymerase chain reaction. Microbiological Research. 164: 174-179.

Decaro N., Martella V., Ricci D., Elia G., Desario C. & Campolo M. 2005. Genotypespecific fluorogenic RT PCR assays for the detection and quantitation of canine coronavirus type I and type II RNA in faecal samples of dogs. Journal of Virology Methods. 130: 72-78.

Gomez D.E., Arroyo L.G., Poljak Z., Viel L. & Weese J.S. 2017. Detection of Bovine Coronavirus in Healthy and Diarrheic Dairy Calves. Journal of Veterinary Internal Medicine. 31: 1884-1891.

Gunn L., Collins P.J., O'Connell M.J. & O'Shea H. 2015. Phylogenetic investigation of enteric bovine coronavirus in Ireland reveals partitioning between European and global strains. Irish Veterinary Journal. 68: 31-35.

Hasoksuz M., Hoet A.E., Loerch S.C., Wittum T.E., Nielsen P.R. & Saif L.J. 2002. Detection of respiratory and enteric shedding of bovine coronaviruses in cattle in an Ohio feedlot. Journal of Veterinary Diagnostic Investigation. 14: 308-313.

He Q., Guo Z., Zhang B., Yue H. & Tang C. 2019. First detection of bovine coronavirus in Yak (Bos grunniens) and a bovine coronavirus genome with a recombinant HE gene. Journal of General Virology. 100: 793-803.

Kanno T., Ishihara R., Hatama S. & Uchida I. 2018. A long-term animal experiment indicating persistent infection of bovine coronavirus in cattle. Journal of Veterinary Medical Science. 80: 1134-1137.

Keha A., Xue L., Yan S. & Yue H. 2019. Prevalence of a novel bovine coronavirus strain with a recombinant hemagglutinin/esterase gene in dairy calves in China. Transbound Emerging Disease. Doi: 10.1111/tbed.13228.

Kim J.H., Jang J.H., Yoon S.W., Noh J.Y., Ahn M.J. & Kim Y. 2018. Detection of bovine coronavirus in nasal swab of non-captive wild water deer, Korea. Transbound Emerging Disease. 65: 627-631.

La Rocca S.A. 2009. A short target real-time RT-PCR assay for detection of pestiviruses infecting cattle. Journal of Virology Methods. 161: 122-127.

Mebus C.A., Stair E.L., Rhodes M.B. & Twiehaus M.J. 1973. Neonatal calf diarrhea: propagation, attenuation, and characteristics of a coronavirus-like agent. American Journal of Veterinary Research. 34: 145-150.

Mohamed F.F., Mansour S.M., El-Araby I.E., Mor S.K. & Goyal S.M. 2017. Mecular detection of enteric viruses from diarrheic calves in Egypt. Archives of Virology. 162: 129-137.

Santos N., Honma S., Timenetsky Mdo C., Linhares A.C., Ushijima H. & Armah G.E. 2008. Development of a microtiter plate hybridization based PCR-enzyme-linked immunosorbent assay for identification of clinically relevant human group A rotavirus G and P genotypes. Journal of Clinical Microbiology. 46: 462-429.

Schoeman D. & Fielding B.C. 2019. Coronavirus envelope protein: current knowledge. Virology Journal. 16: 69-72.

Shin J., Tark D., Le V.P., Choe S., Cha R.M. & Park G.N. 2019. Genetic characterization of bovine coronavirus in Vietnam. Virus Genes. 55: 415-420.

Snodgrass D.R., Terzolo H.R., Sherwood D., Campbell I., Menzies J.D. & Synge B.A. 1986. Aetiology of diarrhoea in young calves. Veterinary Record. 119: 31-34.

Symes S.J., Allen J.L., Mansell P.D., Woodward K.L., Bailey K.E. & Gilkerson J.R. 2018. First detection of bovine noroviruses and detection of bovine coronavirus in Australian dairy cattle. Australian Veterinary Journal. 96: 203-208.

Traven M., Bjornerot L. & Larsson B. 1999. Nationwide survey of antibodies to bovine coronavirus in bulk milk from Swedish dairy herds. Veterinary Record. 144: 527-529.

Wolff C., Emanuelson U., Ohlson A., Alenius S. & Fall N. 2015. Bovine respiratory syncytial virus and bovine coronavirus in Swedish organic and conventional dairy herds. Acta Veterinaria Scandinavica. 57: 2-6.

Yan L.F., Lanny W., Pace B.B., Floyd D.W. & Zhang S. 2016. Failed detection of Bovine viral diarrhea virus 2 subgenotype a (BVDV-2a) by direct fluorescent antibody test on tissue samples due to reduced reactivity of field isolates to raw anti-BVDV antibody. Journal of Veterinary Diagnostic Investigation. 28: 150-157.

Zhang Y., Liu H. & Wang X.D. 2015. A novel real-time RT-PCR with TaqM an-MGB probes and its application in detecting BVDV infections in dairy farms. Journal of Integrative Agriculture. 14: 1637-1643.

Zhao J.J., Cheng D., Li N., Sun Y., Shi Z. & Zhu QH. 2008. Evaluation of a multiplex real-time RT-PCR for quantitative and differential detection of wild-type viruses and C-strain vaccine of Classical swine fever virus. Veterinary Microbiology. 126: 1-10.




DOI: https://doi.org/10.22456/1679-9216.97156

Copyright (c) 2019 Jin-Jing Geng, Zhuan-di Gong, Qyong-yi Li, Xiao-yun Shen, Suo-cheng Wei

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