Assessment of the quantitative real-time polymerase chain reaction using a cDNA standard for human group A rotavirus

Min, Bok Soon; Noh, Yoon Ju; Shin, Jin Ho; Lee, Ji Hyun; Min, Kyung Il; Ryu, Seung Rel; Kim, Byoung Guk; Park, Mi Kyung; Choi, Seung Eun; Yang, Eun Hee; Park, Sue Nie; Hur, Sook Jin; Ahn, Byung Yoon

doi:10.1016/j.jviromet.2006.06.028

Cited by 35 publications

(20 citation statements)

References 20 publications

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“…Traditional methods such as cell culture for enteric virus detection remained cumbersome, costly and time-consuming (Jean et al 2002). Recently, more molecular approaches had been used to detect rotavirus, astrovirus, and norovirus from sewage water samples (Baggi et al 2001;Jean et al 2002;Meleg et al 2006;Min et al 2006;He et al 2008;Katayama et al 2008). …”

Section: Introductionmentioning

confidence: 99%

One-year monthly survey of rotavirus, astrovirus and norovirus in three sewage treatment plants in Beijing, China and associated health risk assessment

Zhang

et al. 2011

Water Science and Technology

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Section: Introductionmentioning

confidence: 99%

One-year monthly survey of rotavirus, astrovirus and norovirus in three sewage treatment plants in Beijing, China and associated health risk assessment

Zhang

et al. 2011

Water Science and Technology

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“…A few RT-real-time quantitative PCR (qPCR) assays, which are based mainly on Sybr green chemistry, have been published (14,17,23). Only two rotavirus-targeted TaqMan RT-qPCR approaches have been developed, which were tested uniquely on human strains and proved to detect only type G1 and types G1, G2, and G4, respectively (15,19).…”

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confidence: 99%

Sensitive Detection of Multiple Rotavirus Genotypes with a Single Reverse Transcription-Real-Time Quantitative PCR Assay

et al. 2008

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“…Most previously published RNA detection assays for RVA required a denaturation step prior to RNA addition to the RT or RT-PCR mixture (13,14,17,28,29,(31)(32)(33)35). Typically, RVA dsRNA and oligonucleotides are mixed in a reaction vessel and heated for 5 min at 95 to 97°C followed by 1 min incubation on ice.…”

Section: Discussionmentioning

confidence: 99%

“…Previously, the standard curves for quantification of RVA were generated using a variety of standards-native viral RNA, plasmids, cDNA, and ssRNA transcripts. Using the various standards, reported LODs of RVA qRT-PCR assays ranged from an estimated 44 copies of genomic RNA per reaction (14) to approximately 1 cDNA target copy per reaction (29). Ward et al (36) determined the LOD of 4 published qRT-PCR assays (13,(16)(17)(18) to be 2.5 copies per reaction using plasmid standards.…”

Section: Discussionmentioning

confidence: 99%

“…qRT-PCR assays offer several advantages over traditional RT-PCR, including increased sensitivity, higher throughput, and faster turnaround time as well as quantification of viral loads. Several qRT-PCR assays have been developed for detection of RVA targeting VP2 (28),VP4 (29,30), VP6 (17,19,(31)(32)(33), VP7 (18,30), NSP3 (13)(14)(15)(16)34), and NSP4 (35) genes. Due to the genetic diversity of VP4 and VP6 gene segments, the NSP3 gene, encoded by genomic segment 7, has been shown to be the best target for detection of a wide variety of RVA genotypes (13-16, 34, 36).…”

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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

Mijatovic-Rustempasic

Tam

Kerin

et al. 2013

J Clin Microbiol

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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. Group A rotaviruses (RVA) are estimated to cause 453,000 deaths among infants and young children each year (1). The genome of RVA, members of the Reoviridae family, consists of 11 double-stranded RNA (dsRNA) segments which encode six viral structural proteins (VP1 to VP4, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6) (2). Surrounding the dsRNA are three concentric protein layers, namely, a central core (VP2), a middle protein layer (VP6), and an outer capsid composed of VP7 and VP4 proteins, while the viral RNA-dependent RNA polymerase (VP1) and the RNA capping enzyme (VP3) are packaged within the core shell (3). Traditionally, viral classification has been based on the serological characteristics and sequence diversity of the outer capsid proteins, VP7 (glycosylated, G-type) and VP4 (protease sensitive, P-type) (4). Although 27 G genotypes and 35 P genotypes have been identified to date (5) Numerous techniques have been developed to detect RVA in stool samples. These techniques include, but are not limited to, virus isolation in cell culture, electron microscopy (EM), enzyme immunoassays (EIA), coupled reverse transcription and PCR amplification (RT-PCR), and real-time quantitative RT-PCRs (qRTPCRs) (2, 7-19). The molecular techniques are more rapid than the cell culture-based techniques and are more sensitive than EM or EIA. The threshold for detection of RVA by EM is approximately 10 7 viral particles/ml of stool (20). EIAs are 10 to 100 times more sensitive than EM assays, but the sensitivity and specificity of EIAs are more variable (21-23). A number of studies using RT-PCR assays targeting the RVA VP4, VP6, and VP7 gene segments have shown increases of 15% to 27% in the rate of RVA detection in comparison with . RT-PCR assays employ either one-step or two-step reverse transcription followed by an ...

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Assessment of the quantitative real-time polymerase chain reaction using a cDNA standard for human group A rotavirus

Cited by 35 publications

References 20 publications

One-year monthly survey of rotavirus, astrovirus and norovirus in three sewage treatment plants in Beijing, China and associated health risk assessment

One-year monthly survey of rotavirus, astrovirus and norovirus in three sewage treatment plants in Beijing, China and associated health risk assessment

Sensitive Detection of Multiple Rotavirus Genotypes with a Single Reverse Transcription-Real-Time Quantitative PCR Assay

Sensitive and Specific Quantitative Detection of Rotavirus A by One-Step Real-Time Reverse Transcription-PCR Assay without Antecedent Double-Stranded-RNA Denaturation

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