Use of internal standard RNA molecules for the RT-PCR amplification of the faeces-borne RNA viruses

Kim, Kisoon; Park, Jeongkoo; Chung, Youngjin; Cheon, Doo‐Sung; Lee, Il-Beom; Lee, Seokgyu; Yoon, Jaedeuk; Cho, Haewol; Song, Chang Yong; Lee, Kwang-Ho

doi:10.1016/s0166-0934(02)00016-2

Cited by 7 publications

(2 citation statements)

References 19 publications

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“…Therefore, to avoid such false negative results, the internal RNA control set up previously [ 21 ] was used in this real-time SYBR Green RT-PCR assay. It was synthesized in vitro from a foreign DNA template, in order to decrease interference with norovirus amplicons [ 33 , 34 ]. It has the advantages of representing no risk for human or animal health because it does not contain any infectious material, and being stable compared to live control viruses that can evolve and change during their replication.…”

Section: Discussionmentioning

confidence: 99%

A SYBR Green RT-PCR assay in single tube to detect human and bovine noroviruses and control for inhibition

Scipioni

Mauroy

Saegerman

et al. 2008

Virol J

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Background: Noroviruses are single-stranded RNA viruses belonging to the family Caliciviridae. They are a major cause of epidemic and sporadic gastroenteritis in humans and clinical signs and lesions of gastroenteritis were reported in bovines. Due to their genetic proximity, potential zoonotic transmission or animal reservoir can be hypothesized for noroviruses. RT-PCR has become the "gold standard" for the detection of noroviruses in faecal and environmental samples. With such samples, the control for inhibition of the reaction during amplification and detection is crucial to avoid false negative results, which might otherwise not be detected. The aim of the reported method is to detect, with a SYBR Green technology, a broad range of noroviruses with a control for inhibition.

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Section: Discussionmentioning

confidence: 99%

A SYBR Green RT-PCR assay in single tube to detect human and bovine noroviruses and control for inhibition

Scipioni

Mauroy

Saegerman

et al. 2008

Virol J

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“…Additionally, we used these RT-PCR assays to control assays for other viral pathogens, e.g., norovirus genogroups I and II, hepatitis A virus, and human immunodeficiency virus (data not shown). The incorporation of an internal control into each RT-PCR tube is important to identify inhibitors and to eliminate falsenegative results, even when problematic specimens such as stool or bronchial lavage fluids are used (9,10,22). Commercially produced diagnostic kits are available for relatively few pathogens.…”

Section: Discussionmentioning

confidence: 99%

Use of Bacteriophage MS2 as an Internal Control in Viral Reverse Transcription-PCR Assays

2005

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Diagnostic systems based on reverse transcription (RT)-PCR are widely used for the detection of viral genomes in different human specimens. The application of internal controls (IC) to monitor each step of nucleic acid amplification is necessary to prevent false-negative results due to inhibition or human error. In this study, we designed various real-time RT-PCRs utilizing the coliphage MS2 replicase gene, which differ in detection format, amplicon size, and efficiency of amplification. These noncompetitive IC assays, using TaqMan, hybridization probe, or duplex scorpion probe techniques, were tested on the LightCycler and Rotorgene systems. In our approach, clinical specimens were spiked with the control virus to monitor the efficiency of extraction, reverse transcription, and amplification steps. The MS2 RT-PCR assays were applied for internal control when using a second target hepatitis C virus RNA in duplex PCR in blood donor screening. The 95% detection limit was calculated by probit analysis to 44.9 copies per PCR (range, 38.4 to 73.4). As demonstrated routinely, application of MS2 IC assays exhibits low variability and can be applied in various RT-PCR assays. MS2 phage lysates were obtained under standard laboratory conditions. The quantification of phage and template RNA was performed by plating assays to determine PFU or via real-time RT-PCR. High stability of the MS2 phage preparations stored at ؊20°C, 4°C, and room temperature was demonstrated.

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Development of homologous viral internal controls for use in RT-PCR assays of waterborne enteric viruses

Parshionikar

Cashdollar

Fout

2004

Journal of Virological Methods

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Use of internal standard RNA molecules for the RT-PCR amplification of the faeces-borne RNA viruses

Cited by 7 publications

References 19 publications

A SYBR Green RT-PCR assay in single tube to detect human and bovine noroviruses and control for inhibition

A SYBR Green RT-PCR assay in single tube to detect human and bovine noroviruses and control for inhibition

Use of Bacteriophage MS2 as an Internal Control in Viral Reverse Transcription-PCR Assays

Development of homologous viral internal controls for use in RT-PCR assays of waterborne enteric viruses

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