A one-step multiplex real-time RT-PCR assay for rapid and simultaneous detection of human norovirus genogroup I, II and IV

Wang, Heng-hui; Gao, Lei; Ji, Ji-mei; Ge, Zhi-jie; Zhu, Xinqiang; He, Peiyan; Chen, Zhong-wen

doi:10.1016/j.jviromet.2013.02.004

Cited by 18 publications

(10 citation statements)

References 23 publications

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“…The detection limit of the assays using Noprobe3 and Noprobe4 was similar to previous established assays at a range of 10100 copies per reaction (16,17). With such high sensitivity, our assays cannot only be applied for clinical samples in which high copy numbers of viruses are present but also for detection of environmental and food samples where the virus is present at low copy numbers.…”

Section: Resultssupporting

confidence: 80%

Development of Single-Tube Nested Real-Time PCR Assays with Long Internally Quenched Probes for Detection of Norovirus Genogroup II

et al. 2016

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The high sequence variation of RNA viruses necessitates use of degenerate primers and probes or multiple primers and probes in molecular diagnostic assays. We showed previously that PCR amplification in two rounds, first with long target-specific primers and then with short generic primers, followed by detection using long probes, can tolerate sequence variation. Here we demonstrate that long primers and probes of up to 56 nucleotides can also be applied in real-time PCR for the detection of norovirus genogroup II with improved sensitivity. Probe design (method of incorporating quenchers, use of Zen internal quencher or traditional quenchers) greatly affects the sensitivity of the real-time PCR assays.

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Section: Resultssupporting

confidence: 80%

Development of Single-Tube Nested Real-Time PCR Assays with Long Internally Quenched Probes for Detection of Norovirus Genogroup II

et al. 2016

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“…This is the first report to describe a Luminex-based laboratory-developed assay designed specifically in order to include MERS-CoV as one of targets. Several commercial Luminex-based kits for respiratory viruses are available now, such as xTAG ™ respiratory virus panel (RVP) and xTAG ™ RVP Fast (Luminex Corp.), MultiCode ® -PLx (EraGen Biosciences), ResPlex II Kit (QIAGEN) [26,32,33], however , they either involve too complicated steps, or include too many Over the past ten years, 'in-house' real-time PCR/ RT-PCR assays have been used by our lab for the diagnosis and surveillance of viral respiratory tract illnesses and showed the good characteristics of rapid and simplicity due to the advantage of simultaneous amplification and analysis, without post-amplification manipulation [34,35]. However, the primers and probes provided by provincial CDC and state CDC were designed specifically for individual target, so these 'in-house' assays for lab diagnosis were mostly monoplex.…”

Section: Discussionmentioning

confidence: 99%

“…However, the primers and probes provided by provincial CDC and state CDC were designed specifically for individual target, so these 'in-house' assays for lab diagnosis were mostly monoplex. Although multiplex real-time RT-PCR was also used in work, the limited multiplexing and throughput capacity became its obvious shortcomings due to the limited fluorescent channels in a real-time instrument and the adverse interactions between primers and probes in one reaction [20,35]. So, a multiplex real-time RT-PCR assay for more targets has to be divided into two or more panels, such as our developed real-time RT-PCR assay with two panels for six respiratory viruses in this study.…”

Section: Discussionmentioning

confidence: 99%

A multiplex liquid-chip assay based on Luminex xMAP technology for simultaneous detection of six common respiratory viruses

Luo¹,

Chen

Wang³

et al. 2017

Oncotarget

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We utilized one-step multiplex reverse transcription-PCR (RT-PCR) and Luminex xMAP technology to develop a respiratory multiplex liquid-chip assay (rMLA) for simultaneous detection of 6 common respiratory viruses, including influenza virus type A (FluA) and type B (FluB), para-influenza virus type 3 (PIV-3), respiratory syncytial virus (RSV), human metapneumovirus (MPV) and a threatening virus to China, Middle East Respiratory Syndrome coronavirus (MERS-CoV). Performance of rMLA was evaluated by comparing with real-time RT-PCR. Detection data from clinical specimens showed that the rMLA had diagnostic sensitivities of 97.10% for FluA, 94.59% for FluB, 98.68% for PIV-3, 94.87% for RSV and 95.92% for MPV (No Data for MERS-CoV due to the lack of positive specimens). Data of analytical sensitivities showed that the detection limits of the rMLA assay were 5–25 viral RNA copies per μl for FluA, FluB, PIV-3 and MERS-CoV, approximate to the real-time RT-PCR assay; while the values were 8 and 22copies/μl for MPV and RSV, lower than the real-time RT-PCR(78 and 114 copies/μl respectively). The results indicated that the rMLA is a sensitive, specific detection tool and comparable to real-time RT-PCR, especially suitable for high-throughput detection of respiratory specimens.

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“…A recently described duplex assay had no loss of sensitivity compared to the sensitivities of GI and GII monoplex assays, and when applied to surface water and groundwater samples, this assay was more efficient that conventional RT-PCR (60). Only one other study targeting all three human NoV genogroups in a multiplex assay based on GI and GII primers/probes and newly designed primers and probe for GIV has been described previously (18,61). As noted by these authors, the sensitivity of the multiplex realtime assay that was developed was lower than the sensitivities of the corresponding monoplex assays due to interactions of primers and probes, confirmed by the failure of NoV detection in three of seven food or environmental samples.…”

Section: Discussionmentioning

confidence: 99%

Environmental Detection of Genogroup I, II, and IV Noroviruses by Using a Generic Real-Time Reverse Transcription-PCR Assay

Miura

Parnaudeau

Grodzki

et al. 2013

Appl Environ Microbiol

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Norovirus is the most common agent implicated in food-borne outbreaks and is frequently detected in environmental samples. These viruses are highly diverse, and three genogroups (genogroup I [GI], GII, and GIV) infect humans. Being noncultivable viruses, real-time reverse transcription-PCR (RT-PCR) is the only sensitive method available for their detection in food or environmental samples. Selection of consensus sequences for the design of sensitive assays has been challenging due to sequence diversity and has led to the development of specific real-time RT-PCR assays for each genogroup. Thus, sample screening can require several replicates for amplification of each genogroup (without considering positive and negative controls or standard curves). This study reports the development of a generic assay that detects all three human norovirus genogroups on a qualitative basis using a one-step real-time RT-PCR assay. The generic assay achieved good specificity and sensitivity for all three genogroups, detected separately or in combination. At variance with multiplex assays, the choice of the same fluorescent dye for all three probes specific to each genogroup allows the levels of fluorescence to be added and may increase assay sensitivity when multiple strains from different genogroups are present. When it was applied to sewage sample extracts, this generic assay successfully detected norovirus in all samples found to be positive by the genogroup-specific RT-PCRs. The generic assay also identified all norovirus-positive samples among 157 archived nucleic acid shellfish extracts, including samples contaminated by all three genogroups.

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A one-step multiplex real-time RT-PCR assay for rapid and simultaneous detection of human norovirus genogroup I, II and IV

Cited by 18 publications

References 23 publications

Development of Single-Tube Nested Real-Time PCR Assays with Long Internally Quenched Probes for Detection of Norovirus Genogroup II

Development of Single-Tube Nested Real-Time PCR Assays with Long Internally Quenched Probes for Detection of Norovirus Genogroup II

A multiplex liquid-chip assay based on Luminex xMAP technology for simultaneous detection of six common respiratory viruses

Environmental Detection of Genogroup I, II, and IV Noroviruses by Using a Generic Real-Time Reverse Transcription-PCR Assay

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