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Sensitive and specific quantitative detection of rotavirus A by one-step real-time reverse transcription-PCR assay without antecedent double-stranded-RNA denaturation
- Source :
- Journal of clinical microbiology. 51(9)
- Publication Year :
- 2013
-
Abstract
- A real-time quantitative reverse transcription-PCR (qRT-PCR) assay using the recombinant thermostable Thermus thermophilus (r Tth ) enzyme was developed to detect and quantify rotavirus A (RVA). By using r Tth polymerase, significant improvement was achieved over the existing real-time RT-PCR assays, which require denaturation of the RVA double-stranded RNA (dsRNA) prior to assay setup. Using a dsRNA transcript for segment 7, which encodes the assay target NSP3 gene, the limit of detection for the improved assay was calculated to be approximately 1 genome copy per reaction. The NSP3 qRT-PCR assay was validated using a panel of 1,906 stool samples, 23 reference RVA strains, and 14 nontarget enteric virus samples. The assay detected a diverse number of RVA genotypes and did not detect other enteric viruses, demonstrating analytical sensitivity and specificity for RVA in testing stool samples. A XenoRNA internal process control was introduced and detected in a multiplexed qRT-PCR format. Because it does not require an antecedent dsRNA denaturation step, this assay reduces the possibility of sample cross-contamination and requires less hands-on time than other published qRT-PCR protocols for RVA detection.
- Subjects :
- Microbiology (medical)
Rotavirus
viruses
Biology
medicine.disease_cause
Nucleic Acid Denaturation
Real-Time Polymerase Chain Reaction
Sensitivity and Specificity
law.invention
Feces
law
Virology
medicine
Humans
Gene
Polymerase
RNA, Double-Stranded
Reverse Transcriptase Polymerase Chain Reaction
RNA
Thermus thermophilus
Viral Load
biology.organism_classification
Molecular biology
Reverse transcription polymerase chain reaction
RNA silencing
Recombinant DNA
biology.protein
Subjects
Details
- ISSN :
- 1098660X
- Volume :
- 51
- Issue :
- 9
- Database :
- OpenAIRE
- Journal :
- Journal of clinical microbiology
- Accession number :
- edsair.doi.dedup.....c34ffed2657ac7caab958e5b17ed798b