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 BHK21 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 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.
Highlights
Bluetongue (BT) is an economically important, infectious, non-contagious, insect transmitted viral disease of domestic and wild ruminants [1]
The ns1 gene based group specific PCR showed a single 366 bp amplicon in agarose gel electrophoresis confirmed the sample as bluetongue virus (BTV)
Bluetongue virus isolate: The novel Indian isolate of BTV-21 (KMNO7) originated from Andhra Pradesh, India was grown in Baby hamster kidney-21 (BHK-21) cell line and used for raising virus stock for RNA isolation
Summary
Bluetongue (BT) is an economically important, infectious, non-contagious, insect transmitted viral disease of domestic and wild ruminants [1]. The virus is prone to frequent mutations and genetic reassortment leading to emergence of several serotypes [2]. Serotypes have been reported from all over the world [3]. Two more serotypes i.e. BTV from Switzerland [4] and BTV from Kuwait [5] have been, reported recently. In India 21 different BTV serotypes have been reported from different parts based upon serum neutralization and virus isolation [6]. BTV serotype 21 has been reported from West Bengal state of India recently [7]
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