Sensitive detection of novel Indian isolate of BTV 21 using ns1 gene based real-time PCR assay

TM Vishwaradhya, P Minakshi, Ranjan Koushlesh, Supriya A, Pawan Kumar, Gaya Prasad
2013 Veterinary World  
Aim: The study was conducted to develop ns1 gene based sensitive real-time reverse transcriptase PCR (real-time RT-PCR) assay for diagnosis of India isolates of bluetongue virus (BTV). Materials and Methods: The BTV serotype 21 isolate (KMNO7) was isolated from Andhra Pradesh and propagated in BHK-21 cell line in our laboratory. The Nucleic acid (dsRNA) of virus was extracted using Trizol method and cDNA was prepared using a standard protocol. The cDNA was allowed to ns1 gene based group
more » ... based group specific PCR to confirm the isolate as BTV. The viral RNA was diluted 10 folds and the detection limit of ns1 gene based RT-PCR was determined. Finally the 10fold diluted viral RNA was subjected to real-time RT-PCR using ns1 gene primer and Taq man probe to standardized the reaction and determine the detection limit. Results: The ns1 gene based group specific PCR showed a single 366 bp amplicon in agarose gel electrophoresis confirmed the sample as BTV. The ns1 gene RT-PCR using 10 fold diluted viral RNA showed the detection limit of 70.0 fg (equivalent to 3 3.3x10 target copies of ns1 gene) per reaction in 1% agarose gel electrophoresis. The ns1 gene based real time RT-PCR was 2 successfully standardized and the detection limit was found to be 7.0 fg (equivalent to 3.3x10 target copies of ns1 gene) per reaction. Conclusion: The ns1 gene based real-time RT-PCR was successfully standardized and it was found to be 10 times more sensitive than conventional RT-PCR.
doi:10.5455/vetworld.2013.554-557 fatcat:j4llxy5ksjattmkkx6tm7hauie