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

Abstract

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 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 3.3x103 target copies of ns1 gene) per reaction in 1% agarose gel electrophoresis. The ns1 gene based real time RT-PCR was successfully standardized and the detection limit was found to be 7.0 fg (equivalent to 3.3x103 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.