Page 139 - GPD-4-1

Gene & Protein in Disease Rotavirus diversity in Uttar Pradesh

2.2. Extraction of RV double-stranded RNA (ds-RNA) Lithuania), 0.2 mM dNTPs, 0.6 pmol of forward primer,
from stool samples 0.6 pmol of reverse primer, 5.0 μL of first-strand cDNA,
and NFW (Amresco, USA). The cycling parameters
All plasticware and glassware were decontaminated using
0.05% (v/v) diethyl pyrocarbonate to remove RNase/ included an initial denaturation at 95°C for 5 min, followed
DNase activity before the extraction of RV dsRNA from by 35 cycles of denaturation at 95°C for 30 s, annealing
stool samples. The RNA extraction was performed at temperatures ranging from 48°C to 55°C for 30 s, and
utilizing either TRIzol LS reagent (Invitrogen, USA) or extension at 72°C for 1 to 2 min, concluding with a final
RNAsure Virus kit (Nucleopore, Genetix Biotech Asia extension at 72°C for 10 min. The gene-specific primer
Ltd., India), adhering to the manufacturers’ instructions. pairs used in the present study are detailed in Table 1, with
their respective annealing temperatures and product sizes.
The extracted dsRNA was kept in 20 μL of RNA storage
solution (Riboreserve, Amresco, USA) and stored at −20°C To increase the genetic diversity among RV strains, two or
for future use. A positive control stool sample containing more primer pairs were used to amplify each gene segment.
rotaviruses maintained by the Division of Veterinary Public The amplified PCR products were subsequently resolved
Health at IVRI, Bareilly, Uttar Pradesh, was incorporated through electrophoresis on a 1.5% (w/v) agarose gel in
Tris-borate buffer. The DNA bands were visualized with
at every stage of the experiments, from dsRNA extraction
to RV detection through ribonucleic acid-polyacrylamide an ultraviolet transilluminator and the image was captured
gel electrophoresis (RNA-PAGE) and reverse transcription using a gel documentation system (Vilber, Germany).
polymerase chain reaction (RT-PCR). To purify the amplicons, the QIAquick Gel Extraction
Kit (Qiagen, Germany) was employed according to the
2.3. Detection of RV by RNA-PAGE manufacturer’s instructions. The eluted products were
stored at -20°C for future use and nucleotide sequencing.
The dsRNA extracted from each sample was analyzed Furthermore, samples whose VP7 gene failed to be
using RNA-PAGE (comprising a 7.5% resolving gel and a amplified using 9Con1/9Con2 primers were subsequently
5.0% stacking gel), followed by silver staining. 10,11
amplified with primer pairs Vp7-F59/Vp7-R995 and TNG
2.4. Detection of RV by RT-PCR C1- F/TNG C2-R.
A two-step RT-PCR assay was optimized using positive 2.5. RVA genotyping
control samples by fine-tuning the concentrations of The G genotype of RV was determined through nucleotide
various reagents and cycling conditions. To enhance sequencing of the VP7 gene amplified products resulting
coverage of the 3’ end, reverse transcription utilizing the from all three primer pairs. The nucleotide sequencing
random primer “FR26RV-N” paired with the poly T-tagged was conducted by Eurofins Genomics India Pvt. Ltd.
primer “FR40RV-T” was also optimized, employing the in Bangalore, India. The DNA sequences obtained
M-MuLV reverse transcriptase (RT) enzyme. To start were analyzed using the Basic Local Alignment Search
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the RT-PCR, the dsRNA denaturation and elimination of Tool (BLAST) available in the National Center for
secondary structures were performed in a final volume Biotechnology Information (NCBI) (https://www.ncbi.
of 14 μL reaction mix, comprising 3.0 μL of FR26RV-N nlm.nih.gov/) and classified using RotaC (http://rotac.
(10 pmol), 1.0 μL of FR40RV-T (10 pmol), 1.5 μL of regatools.be), a freely available web-based tool that
dimethyl sulfoxide, 3.5 μL of nuclease-free water (NFW), can be used for fast RV genotype differentiation of all
and 5.0 μL of dsRNA. The reaction mix was spun and 11 group A RV gene segments according to the new
incubated at 95°C for 5 min in a thermocycler (Eppendorf, guidelines proposed by the RV Classification Working
Germany), followed by snap chilling on ice for 1 – 5 min. Group. To identify the P genotypes, a partial length of
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Next, the reaction mix was added with 11 μL of other the VP4 gene segment was amplified using the previously
reagents, including 2.5 μL of ×5 RT buffer, 2.0 μL of 10 mM mentioned protocol, but specific primers (con3F/con2R
dithiothreitol, 0.5 μL of 10 mM dNTPs, 0.25 μL of RNase and Wang F20/Wang 1185R) and different annealing
inhibitor (40 U/μL), 0.25 μL of M-MuLV RT (20 U/μL), conditions were applied (Table 1). Similarly, for I and
and 5.5 μL of NFW. First-strand cDNA synthesis was E genotyping, full-length VP6 (1340 bp), and NSP4
performed at 37°C for 60 min, followed by 65°C for 10 min. genes (722 bp) were amplified accordingly (Table 1). 3,14,15
The resulting first-strand cDNA was either used for PCR All amplified products were subjected to agarose gel
immediately or stored at −20°C for later use. electrophoresis, visualization, and image acquisition as
The PCR assay was set up in a total reaction volume mentioned in Section 2.4. The identification of other
of 25 μL consisting of 1× PCR buffer with MgCl , 1.25 U genotypes was carried out similarly to the method used
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of Dream Taq DNA polymerase (Thermo Scientific, for G genotypes.

Volume 4 Issue 1 (2025) 3 doi: 10.36922/gpd.6237