title: Sensitive and specific quantitative detection of rotavirus A by one-step real-time reverse transcription-PCR assay without antecedent double-stranded-RNA denaturation.
abstract: A real-time quantitative reverse transcription-PCR (qRT-PCR) assay using the recombinant thermostable Thermus thermophilus (rTth) enzyme was developed to detect and quantify rotavirus A (RVA). By using rTth 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.
authors.affiliation: Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
authors.forename: Slavica
authors.initials: S
authors.lastname: Mijatovic-Rustempasic
authors.forename: Ka Ian
authors.initials: KI
authors.lastname: Tam
authors.forename: Tara K
authors.initials: TK
authors.lastname: Kerin
authors.forename: Jamie M
authors.initials: JM
authors.lastname: Lewis
authors.forename: Rashi
authors.initials: R
authors.lastname: Gautam
authors.forename: Osbourne
authors.initials: O
authors.lastname: Quaye
authors.forename: Jon R
authors.initials: JR
authors.lastname: Gentsch
authors.forename: Michael D
authors.initials: MD
authors.lastname: Bowen
mesh_terms: D005243:Feces / Q000821:virology*; D006801:Humans; D009691:Nucleic Acid Denaturation; D012330:RNA, Double-Stranded / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012401:Rotavirus / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods*
chemical_list: D012330:RNA, Double-Stranded