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Standardization and Application of Real-Time Polymerase Chain Reaction for Rapid Detection of Bluetongue Virus


Affiliations
1 Department of Bacteriology and Mycology, Veterinary Biological and Research Institute, Labbipeta, Vijayawada - 520 010, Andhra Pradesh, India
2 Department of Veterinary Microbiology and Biotechnology, College of Veterinary Science, PVNRT Veterinary University, Hyderabad - 500 030, Telangana, India
3 Ella Foundation, Genome Valley, Turkapally, Shameerpet Mandal, Hyderabad - 500 078, Telangana, India
4 Department of Virology, Veterinary Biological and Research Institute, Labbipeta, Vijayawada - 520 010, Andhra Pradesh, India
 

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 8.269E×103 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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  • Standardization and Application of Real-Time Polymerase Chain Reaction for Rapid Detection of Bluetongue Virus

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Authors

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

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 8.269E×103 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.

References