Synchronous Detection of BPV and BVDV with Duplex Taqman qPCR Method

Zhuan-di Gong, Xiao-yun Shen, Hao-qin Liang, Jin-jing Geng, Qion-yi Li, Ruo-fei Feng, Suo-cheng Wei


Background: Bovine parvovirus (BPV) and bovine viral diarrhea virus (BVDV) are commonly etiologies causing diarrhea in dairy herds. BPV is a member of bocaparvovirus genus with a non-enveloped capsid. BVDV, belonging to Pestivirus genus in Flaviviridae, possesses a single-stranded RNA, and is classified into BVDV-1 and BVDV-2 genotypes according to the 5’UTR sequence. 21 genetic groups of BVDV-1 and four groups of BVDV-2 have been found. Diagnosis of viral diarrhea is often relied on virus detection by isolation or detection of serum antibody. The main objective of the present study was to establish a duplex real time PCR (qPCR) based on Taqman probe to detect synchronously BPV and BVDV.

Materials, Methods & Results: TaqMan probe and primers were designed and synthesized from the sequences of conserved 5′ - untranslated regions (5′ UTR) of Haden strain of BPV and NADL strain of BVDV. The cDNAs were transcribed in vitro to make standard curves before optimizing the assay. DNA/PCR products were ligated into pMD18-T vector, and then used to transfer BL-21 competent cells to acquire the recombinant plasmids of pMD18-T-BPV and pMD18-T-BVDV. Optimum reaction conditions were comparatively selected. The sensitivity, specificity and reproducibility of TaqMan probe qRT-PCR were evaluated respectively. The results showed the concentrations of pMD18-T-BPV or pMD18-T-BVDV were 2.0 × 1010 DNA copies/μL, respectively. A duplex Taqman qPCR method was developed by optimizing the amplification conditions to simultaneously detect BPV and BVDV. The assay targets at highly conserved VP2 gene of BPV and 5′ UTR gene of BVDV. This qPCR assay was assessed for specificity and sensitivity using DNA of BPV and cDNA of BVDV. For clinical validation, 308 samples were tested from clinically diarrhea calves. The results showed that optimum annealing temperature was achieved in 43.2 ℃ fro duplex BPV and BPIV. Dynamic curves and standard curves were created following amplification of recombinant plasmids using the optimized duplex Taqman BPV and BVDV, with an amplification efficiency of 95.69%. Duplex Taqman qPCR could only detect DNA of BPV and cDNA of BVDV with a strong specificity. The detection limitation was as low as 2.0 × 102 copies/μL of pMD18-T-BPV plasmid and 2.0 × 101 copies/μL for pMD18-T-BVDV plasmid, respectively. Sensitivity of detection was 100-fold higher than conventional PCR. Duplex Taqman qPCR had excellent repeatability or stability with less than 1.2% of intra-assay and inter-assay. 35 and 47 positive feces samples were identified using duplex Taqman qPCR in comparison to 30 and 42 positives for universal PCR, respectively.

Discussion: The bovine viral diarrhea virus (BVDV) is a key pathogenic factor in bovine diarrhea. Currently, few effective measures are available for the treatment or prevention for BVDV and BPV infections in animals. The technique was proven to be repeatable and linear over a range of at least 5 magnitudes, from 101 to 105 RNA/DNA copies, thus ensuring an accurate measurement of BPV DNA and BVDV RNA loads in clinical samples. In conclusion, a duplex Taqman qPCR was established for detecting simultaneously BPV and BVDV. Taqman qPCR method was rapid and specific assay. This assay was 100-fold sensitive than conventional PCR. It will be propitious to rapidly and differentially diagnose pathogens of viral diarrhea of dairy farms. Taqman qPCR method was rapid and specific assay and had a sensitivity of 2.0 copies/μL.

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Alpay G. & Weşilag K. 2015. Serological relationships among subgroups in bovine viral diarrhea virus genotype 1 (BVDV- 1). Veterianry Microbiology. 175: 1-6.

Bauermann F.V., Flores E.F. & Ridpath F.J. 2012. Antigenic relationships between Bovine viral diarrhea virus 1 and 2 and HoBi virus: possible impacts on diagnosis and control. Journal of Veterinary Diagnostic Investigation. 24: 253-260.

Chang J., Zhang Y., Yang D., Jiang Z., Wang F. & Yu L. 2019. Potent neutralization activity against type O foot-and-mouth disease virus elicited by a conserved type O neutralizing epitope displayed on bovine parvovirus virus-like particles. Journal of General Virology. 100: 187-198.

Cibulski S.P., Teixeira T.F., Dos Santos H.F., de Sales Lima F.E., Scheffer C.M. & Varela A.P. 2016. Ungulate copiparvovirus 1 (bovine parvovirus 2): characterization of a new genotype and associated viremia in different bovine age groups. Virus Genes. 52(2): 134-137.

de Los Angeles Ribas M., Tejero Y Cordero Y., Perez D Sausy A . & Muller C.P. 2019. Identification of human parvovirus B19 among measles and rubella suspected patients from Cuba. Journal of Medical Virology. DOI: 10.1002/jmv.25444.

de Souza W.M., Dennis T., Fumagalli M.J., Araujo J., Sabino-Santos G., Fgm M., Acrani G.O.. Aot C., Romeiro M.F. & Modha S. 2018. Novel Parvoviruses from Wild and Domestic Animals in Brazil Provide New Insights into Parvovirus Distribution and Diversity. Viruses. 10 (2): 143-148.

Decaro N., Lanave G., Lucente M.S., Mari V., Varello K. & Losurdo M. 2014. Mucosal disease-like syndrome in a calf persistently infected by Hobi-like pestivirus. Journal of Clinical Microbiology. 50(12): 2946-2954.

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 Virological Methods. 130(1): 72-78.

Dubey P., Mishra N., Rajukumar K., Behera S.P., Kalaiyarasu S. & Nema R.K. 2015. Development of a RT-PCR ELISA for simultaneous detection of BVDV-1 BVDV-2 and BDV in ruminants and its evaluation on clinical samples. Journal of Virological Methods. 213(1): 50-56.

Francisco J.D. 2017. Bovine viral diarrhea virus (BVDV) genetic diversity in Spain: A review. Spanish Journal of Agricultural Research. 15: 2171-9292.

Fredericksen F., Delgado F., Cabrera C., Yanez A., Gonzalo C. & Villalba M. 2015. The effects of reference genes in qRT-PCR assays for determining the immune response of bovine cells (MDBK) infected with the Bovine Viral Diarrhea Virus 1 (BVDV-1). Gene. 569: 95-103.

Gomez-Romero N., Basurto-Alcantara F.J., Verdugo-Rodriguez A., Bauermann F.V. & Ridpath J.F. 2017. Genetic diversity of bovine viral diarrhea virus in cattle from Mexico. Journal of Veterinary Diagnostic Investigation. 29: 362-365.

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.

Ikuta N., De Oliveira Solla Sobral F., Lehmann F.K., da Silveira P.V., de Carli S. & Casanova Y.S. 2014. Taqman real-time PCR assays for rapid detection of avian pathogenic Escherichia coli isolates. Avian Diseases. 58: 628-631.

Kailasan S., Halder S., Gurda B., Bladek H., Chipman P.R. & McKenna R. 2015. Structure of an enteric pathogen bovine parvovirus. Journal of Virology. 89(3): 2603-2614.

Karikalan M., Rajukumar K., Mishra N., Kumar M., Kalaiyarasu S. & Rajesh K. 2016. Distribution pattern of bovine viral diarrhoea virus type 1 genome in lymphoid tissues of experimentally infected sheep. Veterinary Research Communications. 40: 55-61.

Kuta A. & Woźniakowski G.P. 2015. Cross-priming amplification for detection of bovine viral diarrhoea virus species 1 and 2. Journal of Applied Microbiology. 119: 632-639.

Lifang Y., 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(3): 150-157.

Liu H., Yang H., Zhao H., Li Y., Zhao H. & Li T. 2015. Isolation and Identification of Bovine Parvovirus and its replication kinetics in Different Cells. Chinese Journal of Veterinary Science. 35: 1770-1776.

Mohamed F.F., Mansour S.M., El-Araby I.E., Mor S.K. & Goyal S.M. 2017. Molecular detection of enteric viruses from diarrheic calves in Egypt. Archives of Virology. 162(2): 129-137.

Mohamed F.F., Mansour S.M.G., Orabi A., El-Araby I.E., Ng T.F.F. & Mor S.K. 2018. Detection and genetic characterization of bovine kobuvirus from calves in Egypt. Archives of Virology. 163(7): 1439-1447.

Peddireddi L., Foster K.A., Poulsen E.G., An B., Hoang Q.H. & O'Connell C. 2018. Molecular detection and characterization of transient bovine viral diarrhea virus (BVDV) infections in cattle commingled with ten BVDV persistently infected cattle. BMC Veterinary Research. 30: 413-22.

Qiu J., Soderlund-Venermo M. & Young N.S. 2017. Human Parvoviruses. Clinical Microbiology Reviews. 30: 43-49.

Sakurai A., Nomura N., Nanba R., Sinkai T., Iwaki T. & Obayashi T. 2011. Rapid typing of influenza viruses using super high-speed quantitative real-time PCR. Journal of Virological Methods. 178(1): 75-81.

Sareyy B. & İbrahim B. 2017. Development of multiplex RT-PCR for detection and differentiationof foot-and-mouth disease virus O and A serotypes in Turkey. Turkish Journal of Veterinary And Animal Sciences. 41(5): 764-769.

Walraph J., Zoche-Golob V., Weber J. & Freick M. 2018. Decline of antibody response in indirect ELISA tests during the periparturient period caused diagnostic gaps in Coxiella burnetii and BVDV serology in pluriparous cows within a Holstein dairy herd. Research in Veterinary Science. 118(2): 91-96.

Zhang S.Q., Tan B., Li P., Wang F.X., Guo L. & Yang Y. 2014. Comparison of conventional RT-PCR reverse-transcription loop-mediated isothermal amplification and SYBR green I-based real-time RT-PCR in the rapid detection of bovine viral diarrhea virus nucleotide in con-taminated commercial bovine sera batches. Journal of Virological Methods. DOI: 10.1016/j.jviromet.2014.05.020.

Zhang Y., Liu H., Wu X., Wang X., Li J. & Zhao Y. 2015. A novel real-time RT-PCR with TaqMan-MGB probes and its application in detecting BVDV infections in dairy farms. Journal of Integrative Agriculture. 14(9): 1637-1643.

Zoccola R., Mazzei M., Carrozza M.L., Ricci E., Forzan M. & Pizzurro F. 2017. A newly developed BVDV-1 RT-qPCR Taqman assay based on Italian isolates: evaluation as a diagnostic tool. Folia Microbiologica. 62(2): 279-286.


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