doi:10.5455/vetworld.2013.554-557 Sensitive detection of novel Indian isolate of BTV 21 using ns1 gene based real-time PCR assay

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.


Introduction
of diagnostic and research.The potential of this format to provide sensitive, specific and rapid detection and Bluetongue (BT) is an economically important, quantification of viral RNA has made it an infectious, non-contagious, insect transmitted viral indispensable tool for state-of-the-art diagnostics of disease of domestic and wild ruminants [1].The virus important animal viral pathogens [8].Integration of is prone to frequent mutations and genetic rethese assays into automated liquid handling platforms assortment leading to emergence of several serotypes for nucleic acid extraction increases the rate and [2].So far 24 serotypes have been reported from all standardization of sample throughput and decreases over the world [3].However, two more serotypes i.e.
the potential for cross-contamination.BTV 25 from Switzerland [4] and BTV 26 from By real-time RT-PCR the highly conserved Kuwait [5] have been, reported recently.In India 21 genome segment of ns1 can be targeted to detect all the different BTV serotypes have been reported from 24 BTV serotypes, as well as geographic variants different parts based upon serum neutralization and within the individual serotype.The real-time RT-PCR virus isolation [6].However, BTV serotype 21 has been based assays are capable of rapid screening of field reported from West Bengal state of India recently [7].
samples of BTV [9].A new primer-probe energy To detect effectively BT disease, nucleic acid transfer (PriProET) based real-time RT-PCR assay was based methods have been developed and evaluated for developed recently for early and rapid detection of all group specific detection.Although conventional 24 BTV serotypes along with other emerging strains of reverse transcriptase polymerase chain reaction (RT-BTV [10] The cDNA was amplified ns1 gene for detection of novel Indian BTV-21 targeting the non-structural (ns1) gene specific primer serotype which could be the first report in India to the pairs in a 25 µl using the following conditions: 25 pM best of our knowledge.

Materials and Methods
µM dNTP, 6% DMSO, 1.5 mM MgCl2 and 2.5 U of Taq polymerase.The initial denaturation step was done Bluetongue virus isolate: The novel Indian isolate of at 95ºC for 3 min for all primers, three step cyclic BTV-21 (KMNO7) originated from Andhra Pradesh, denaturation at 94ºC for 30 seconds, annealing India was grown in Baby hamster kidney-21 (BHK-21) temperature of 58ºC extension step was at 72ºC for one cell line and used for raising virus stock for RNA min and final extension at 72ºC for 8 min.The ns1 gene isolation.
based primer of real-time PCR was also subjected to RT-PCR to confirm the specificity of the primer to ns1 Extraction of viral RNA and cDNA preparation: The gene.dsRNA from cell culture grown viruses was extracted by standard procedure used in our laboratory [13].The The real-time RT-PCR: The cDNA was used for real extracted RNA was confirmed as BTV by RNA-PAGE time PCR using ns1 gene specific primers and probe in analysis.The viral RNA was further confirmed as BTV 20µM concentration which yielded better Ct value on by amplification of ns1 gene by RT-PCR.Reverse standardization.A 25 µl reaction was set having transcription was carried out in a 20 µl reaction mixture following components: Universal Master Mix (13.0 using the following components: 1.2 µg/µl viral µl), forward and reverse primers (8pM), TaqMan probe dsRNA, 1.5 µl DMSO, 30 pmol random primers.The (8pM), cDNA (2.0 µl).The above reaction mixture was mixture was heated at 99ºC for 5 min in thermal cycler transferred to the 96 well optical plate and covered with (Biorad i-cycler, USA), snap chilled on ice and then the optical cover.The reaction was carried out in ABI 7500 following reagents were added: 1 µl of 200units/µl Mo-Std.version (1.4) using the thermal condition for 40 MuLV-RT (Promega, USA), 1X RT buffer, 0.5 µl 100 cycles: initial denaturation step at 95ºC for 10 min, mM dNTPs.After allowing the primers to anneal at three step of cyclic denaturation at 94ºC for 15 seconds, 25ºC for 10 min, reverse transcription was carried out annealing temperature of 54ºC for 15 seconds, at 37ºC for 60 min in thermal cycler.The reverse extension step was carried out at 61ºC for 1 min and transcriptase was heat inactivated at 90ºC for 10 min.
final extension at 61ºC for 5 min.

Results and Discussion
and RT-PCR: The group specific ns1 gene based primer was used to confirm the sample as BTV [14].The same The real-time RT-PCR is eco-friendly and less primer was used with serially 10 fold diluted sample to health hazardous as well as economical as it avoids the use of agarose gel electrophoresis (having harmful determine the detection limit of ns1 gene based RTethidium bromide), so decreasing the risks of PCR.For real-time PCR reaction the ns1 gene specific environmental contamination.The assay is suitable for primers pair forward (606-631: 5'-GTTGAGA GACA large scale sample testing and detection of BTV from AATTAACACATGTCC-3') and reverse (705-723:5'various biological samples such as blood, semen etc. as AATGCTTCG CAAAAT CAT CC AT-3') along with well as adapted in various cell lines such as BHK-21, fluorogenic TaqMan-MGB probe corresponding to Thus, the real-time PCR had lower limit (7.0 fg) The RNA-PAGE analysis revealed classical 10 of detection compared to conventional RT-PCR (70.0 segments with 3:3:3:1 migration pattern which is fg).By comparing the results of both assays it was characteristic to BTV as reported earlier [16].RNA-revealed that the real-time RT-PCR has been found to PAGE has been used not only to identify and detect about 10 times more sensitive as compared to the BTV in samples but also to analyze genomic diversity conventional RT-PCR.With a view to develop highly within and between serotypes and re-assortments in sensitive assays for the detection of BTV, it is mixed infection [16,17].BTV-21 yielded an expected necessary to determine the threshold sensitivity of RTintense band of 123 bp without any non-specific PCR.The genome segment 10 based RT-PCR detection amplification using group specific primers targeting limit for BTV was reported as little as 100 fg to1 pg of 3 4 ns1 gene (designed for real time PCR) by RT-PCR BTV RNA, which is equivalent to 5 x 10 to 5 x 10 viral (Figure -1).The novel isolate KMNO7 (BTV-21) was particles [19].subjected to RT-PCR amplification of ns1 gene with Earlier, different gene specific RT-PCR were serially 10 fold diluted viral RNA with initial RNA developed for diagnosis of BTV from various concentration of 0.7 ng/µl and the expected 366 bp biological samples.The ns1 gene based nested RTband was observed up to the 4th dilution corresponding PCR was one of them and its detection limit was 1 3 plaque-forming unit (pfu) of BTV in Culicoides to 70.0 fg of RNA (equivalent to 3.3x10 target copies midges [20].For retrospective diagnosis of BTV in of ns1 gene) (Figure -2).The earlier workers reported frozen semen and fixed tissue samples the vp3 gene that the primers complimentarily targeting the 5' specific RT-PCR was developed with the threshold terminus of segment 5 encoding ns1 gene might be detection limit with 0.75 to 7.5 pfu of BTV on fresh suitable for development of group specific diagnosis of tissues [21].Later on, the more specific and sensitive BTV by RT-PCR [18].The group specific ns1 gene of real-time PCR technique was developed for BTV genomic segment 5 was targeted for the conventional diagnosis.The detection limit for real-time RT-PCR to RT-PCR and real-time RT-PCR as the ns1 gene is detect bluetongue virus in blood samples by TaqMan highly conserved across the all serotypes.
probe and ns1 gene specific primer was found to be The isolate KMNO7 (BTV-21) with measured 0.005 to 0.05 TCID /ml [22].Similarly the detection concentration of RNA of 0.7ng/µl was also serially 10 50 fold diluted and reverse transcribed to produce cDNA.limit of 0.1 TCID50/ml was reported using TaqMan The corresponding cDNA was used for real-time RTprobe and ns1 gene specific primer in BHK-21 cell PCR.The first dilution of RNA showed Ct value at 26 culture adapted BTV [23].The segment 10 based realcycle corresponding to 70pg/µl.As dilution level time RT-PCR assays based on the primer-probe energy increased the Ct value also increased for the transfer (PriProET) can detect 10-100 target copies of corresponding dilution.The minimum amount of RNA viral RNA of all the 24 serotypes of BTV [10].Another measured by the assay was up to level of fifth dilution real-time PCR assay showed the detection limit of less In future extensive evaluation of group specific target copies of ns1 gene) which was at the 38 cycle.