Article history: Received: 25-01-2018, Accepted: 13-03-2018, Published online: 10-04-2018
Corresponding author: Kalyani Putty
E-mail: email@example.comCitation: Lakshmi IK, Putty K, Raut SS, Patil SR, Rao PP, Bhagyalakshmi B, Jyothi YK, Susmitha B, Reddy YV, Kasulanati S, Jyothi JS and Reddy YN (2018) Standardization and application of real time polymerase chain reaction for rapid detection of bluetongue virus, Veterinary World, 11(4): 452-458.
Aim: The present study was designed to standardize real-time polymerase chain reaction (PCR) for detecting the bluetongue virus from blood samples of sheep collected during outbreaks of bluetongue disease in the year 2014 in Andhra Pradesh and Telangana states of India.
Materials and Methods: A 10-fold serial dilution of Plasmid PUC59 with bluetongue virus (BTV) NS3 insert was used to plot the standard curve. BHK-21 and KC cells were used for in vitro propagation of virus BTV-9 at a TCID50/ml of 105 ml and RNA was isolated by the Trizol method. Both reverse transcription -PCR and real-time PCR using TaqMan probe were carried out with RNA extracted from virus-spiked culture medium and blood to compare the sensitivity by means of finding out the limit of detection (LoD). The results were verified by inoculating the detected and undetected dilutions onto cell cultures with further cytological (cytopathic effect) and molecular confirmation (by BTV-NS1 group-specific PCR). The standardized technique was then applied to field samples (blood) for detecting BTV.
Results: The slope of the standard curve obtained was -3.23, and the efficiency was 103%. The LoD with RT-PCR was 8.269Ex103 number of copies of plasmid, whereas it was 13 with real-time PCR for plasmid dilutions. Similarly, LoD was determined for virus-spiked culture medium, and blood with both the types of PCR and the values were 103 TCID 50/ml and 104 TCID 50/ml with RT-PCR and 10° TCID 50/ml and 102 TCID 50/ml with real-time PCR, respectively. The standardized technique was applied to blood samples collected from BTV suspected animals; 10 among 20 samples were found positive with Cq values ranging from 27 to 39. The Cq value exhibiting samples were further processed in cell cultures and were confirmed to be BT positive. Likewise, Cq undetected samples on processing in cell cultures turned out to be BTV negative.
Conclusion: Real-time PCR was found to be a very sensitive as well as reliable method to detect BTV present in different types of samples, including blood samples collected from BTV-infected sheep, compared to RT-PCR. The LoD of BTV is likely influenced by sample type, possibly by the interference by the other components present in the sample.
Keywords: bluetongue virus, limit of detection, real-time polymerase chain reaction.
1. Borden, E.C., Shope, R.E. and Murphy, F.A. (1971) Physicochemical and morphological relationships of some arthropod-borne viruses to bluetongue virus-a new taxonomic group. Physicochemical and serological studies. J. Gen. Virol. 3: 261-271. [Crossref]
2. OIE. (2012) Bluetongue: Aetiology Epidemiology, Diagnosis, Prevention and Control References. Organization International des Epizootics, Paris.
3. Batten, C., Darpel, K., Henstock, M., Fay, P., Veronesi, E., Gubbins, S., Graves, S., Frost, L. and Oura, C. (2014) Evidence for transmission of bluetongue virus serotype 26 through direct contact. PLoS One, 9: e96049. [Crossref]
4. Maan, S., Maan, N.S., Belaganahalli, M.N., Potgieter, A.C., Kumar, V., Batra, K., Wright, I.M., Kirkland, P.D. and Mertens, P.P. (2016) Development and evaluation of real-time RT-PCR assays for detection and typing of bluetongue virus. PLoS One, 11: e0163014. [Crossref]
5. Elbers, A.R.W., Backx, A., Mintiens, K., Gerbier, G., Staubach, C., Hendrickx, G. and van der Spek, A. (2008) Field observations during the bluetongue serotype 8 epidemic in 2006. II. Morbidity and mortality rates, case fatality and clinical recovery in sheep and cattle in the Netherlands. Prevent. Vet. Med. 87: 31-40. [Crossref] [PubMed]
7. Gaudreault, N.N., Jasperson, D.C., Dubovi, E.J., Johnson, D.J., Ostlund, E.N., Wilson, W.C. (2015) Whole genome sequence analysis of circulating Bluetongue virus serotype 11 strains from the United States including two domestic canine isolates. J Vet Diagn Invest. 27: 442-448. [Crossref] [PubMed]
8. Maclachlan, N.J. and Mayo, C.E. (2013) Potential strategies for control of bluetongue, a globally emerging, Culicoides-transmitted viral disease of ruminant livestock and wildlife. Antiviral Res. 99: 79-90. [Crossref] [PubMed]
9. Shaw, A.E., Ratinier, M., Nunes, S.F., Nomikou, K., Caporale, M., Golder, M., Allan, K., Hamers, C., Hudelet, P., Zientara, S., Breard, E., Mertens, P. and Palmarini, M. (2013) Reassortment between two serologically unrelated bluetongue virus strains is flexible and can involve any genome segment. J Virol. 87: 543-557. [Crossref] [PubMed] [PMC]
10. Schulz, C., Breard, E., Sailleau, C., Jenckel, M., Viarouge, C., Vitour, D., Palmarini, M., Gallois, M., Hoper, D., Hoffmann, B., Beer, M. and Zientara, S. (2016) Bluetongue virus serotype 27: detection and characterization of two novel variants in Corsica, France. J. Gen. Virol. 97: 2073-2083. [Crossref]
11. Chand, K., Biswas, S.K., Pandey, A.B., Muthuchelvan, D. and Mondal, B. (2015) Bluetongue in India: A review. Adv. Anim. Vet. Sci. 3: 605-612. [Crossref]
12. Gard, G.P. and Kirkland, P.D. (1993). Bluetongue virology and serology. In: Corner, L.A., Bogust, T.Y., editors. Australian Standard Diagnostic Techniques for Animal Diseases. CSIRO for the Standing Committee on Agriculture and Resource Management, East Melbourne, Australia.
14. Wilson, W.C., Ma, H.C., Venter, E.H., van Djik, A.A., Seal, B.S. and Mecham, J.O. (2000) Phylogenetic relationships of bluetongue viruses based on gene S7. Virus Res. 67: 141-151. [Crossref]
15. Anthony, S., Jones, H., Darpel, K.E., Elliott, H., Maan, S., Samuel, A., Mellor, P.S. and Mertens, P.P.C. (2007) A duplex RT-PCR assay for detection of genome segment 7 (VP7 gene) from 24 BTV serotypes. J Virol Methods. 141: 188-197. [Crossref] [PubMed]
16. Shaw, A.E., Monaghan, P., Alpar, H.O., Anthony, S., Darpel, K.E., Batten, C.A., Guercio, A., Alimena, G., Vitale, M., Bankowska, K. and Carpenter, S. (2007) Development and initial evaluation of a real-time RT-PCR assay to detect bluetongue virus genome segment 1. J Virol Methods. 145: 115-126. [Crossref] [PubMed]
17. Toussaint, J.F., Sailleau, C., Breard, E., Zientara, S. and De Clercq, K. (2007) Bluetongue virus detection by two real-time RT-qPCRs targeting two different genomic segments. J Virol Methods.140: 115-123. [Crossref] [PubMed]
18. OIE. (2000) Manual of Standards for Diagnostic Tests and Vaccines. Organization International des Epizootics, Paris.
19. Maan, N.S., Maan, S., Belaganahalli, M., Pullinger, G., Montes, A.J.A., Gasparini, M.R., Guimera, M., Nomikou, K. and Mertens, P.P. (2015) A quantitative real-time reverse transcription PCR (qRT-PCR) assay to detect genome segment 9 of all 26 bluetongue virus serotypes. J Virol Methods. 213: 118-126. [Crossref] [PubMed]
20. Orru, G., Ferrando, M.L., Meloni, M., Liciardi, M., Savini, G. and De Santis, P. (2006) Rapid detection and quantitation of Bluetongue virus (BTV) using a Molecular Beacon fluorescent probe assay. J Virol Methods. 137: 34-42. [Crossref] [PubMed]
21. Parsonson, I.M., Della-Porta, A.J., McPhee, D.A., Cybinski, D.H., Squire, K.R.E. and Uren, M.F. (1987) Experimental infection of bulls and cows with bluetongue virus serotype 20. Aust Vet J. 64: 10-13. [Crossref] [PubMed]
22. Green, M.R., Sambrook, J. (2012) Molecular Cloning: A Laboratory Manual. 4th ed. Cold Spring Harbor Laboratory Press, New York.
23. OIE Terrestrial Manual (2012) 7th edition, Volumes 1 and 2 ISBN 978-92-9044-878-5.
24. Stanislawek, W.L., Lunt, R.A., Blacksell, S.D., Newberry, K.M., Hooper, P. and White, J.R. (1996) Detection by ELISA of bluetongue antigen directly in the blood of experimentally infected sheep. Vet. Microbiol. 52: 1-12. [Crossref]
25. Hawkes, R.A., Kirkland, P.D., Sanders, D.A., Zhang, F., Li, Z., Davis, R.J. and Zhang, N. (2000) Laboratory and field studies of an antigen capture ELISA for bluetongue virus. J. Virol. Methods 85: 137-149. [Crossref]
26. Lunt, R.A., White, J.R. and Blacksell, S.D. (1988) Evaluation of a monoclonal antibody blocking ELISA for the detection of group-specific antibodies to bluetongue virus in experimental and field sera. J. Gen. Virol. 69: 2729-2740. [Crossref]27. Celma, C.C. and Roy, P. (2009) A viral nonstructural protein regulates bluetongue virus trafficking and release. J Virol. 83: 6806-6816. [Crossref] [PubMed] [PMC]