Standardization and application of real-time polymerase chain reaction for rapid detection of bluetongue virus

Research (Published online: 10-04-2018)

7. Standardization and application of real-time polymerase chain reaction for rapid detection of bluetongue virus

I. Karthika Lakshmi, Kalyani Putty, Satya Samparna Raut, Sunil R. Patil, P. P. Rao, B. Bhagyalakshmi, Y. Krishna Jyothi, B. Susmitha, Y. Vishnuvardhan Reddy, Sowmya Kasulanati, J. Shiva Jyothi and Y. N. Reddy

Veterinary World, 11(4): 452-458

I. Karthika Lakshmi: Department of Bacteriology and Mycology, Veterinary Biological and Research Institute, Labbipeta, Vijayawada - 520 010, Andhra Pradesh, India.

Kalyani Putty: Department of Veterinary Microbiology and Biotechnology, College of Veterinary Science, PVNRT Veterinary University, Hyderabad - 500 030, Telangana, India.

Satya Samparna Raut: Department of Veterinary Microbiology and Biotechnology, College of Veterinary Science, PVNRT Veterinary University, Hyderabad - 500 030, Telangana, India.

Sunil R. Patil: Department of Veterinary Microbiology and Biotechnology, College of Veterinary Science, PVNRT Veterinary University, Hyderabad - 500 030, Telangana, India.

P. P. Rao: Ella Foundation, Genome Valley, Turkapally, Shameerpet Mandal, Hyderabad - 500 078, Telangana, India.

B. Bhagyalakshmi: Department of Veterinary Microbiology and Biotechnology, College of Veterinary Science, PVNRT Veterinary University, Hyderabad - 500 030, Telangana, India.

Y. Krishna Jyothi: Department of Virology, Veterinary Biological and Research Institute, Labbipeta, Vijayawada - 520 010, Andhra Pradesh, India.

B. Susmitha: Ella Foundation, Genome Valley, Turkapally, Shameerpet Mandal, Hyderabad - 500 078, Telangana, India.

Y. Vishnuvardhan Reddy: Ella Foundation, Genome Valley, Turkapally, Shameerpet Mandal, Hyderabad - 500 078, Telangana, India.

Sowmya Kasulanati: Department of Veterinary Microbiology and Biotechnology, College of Veterinary Science, PVNRT Veterinary University, Hyderabad - 500 030, Telangana, India.

J. Shiva Jyothi: Department of Veterinary Microbiology and Biotechnology, College of Veterinary Science, PVNRT Veterinary University, Hyderabad - 500 030, Telangana, India.

Y. N. Reddy: Ella Foundation, Genome Valley, Turkapally, Shameerpet Mandal, Hyderabad - 500 078, Telangana, India.

doi: 10.14202/vetworld.2018.452-458

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Article history: Received: 25-01-2018, Accepted: 13-03-2018, Published online: 10-04-2018

Corresponding author: Kalyani Putty

E-mail: kalyaniputty@gmail.com

Citation: 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.

Abstract

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.

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