Detection of murine norovirus by reverse transcription loop-mediated isothermal amplification

Hanaki, Ken-Ichi; Ike, Fumio; Kajita, Ayako; Yasuno, Wataru; Yanagiba, Misato; Goto, Mayu; Sakai, Kouji; Ami, Yasushi; Kyuwa, Shigeru

doi:10.1016/j.jviromet.2014.03.025

Cited by 11 publications

(11 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, these diagnostic methods do have some drawbacks, such as labour‐intensive and time consuming. The loop‐mediated isothermal amplification (LAMP) assay, which required shorter run time compared with PCR/qPCR, was reported for the detection of PDCoV (Hanaki et al., ). In comparison to LAMP assay, the recombinase polymerase amplification (RPA) assay, which required fewer primers (two primers) and shorter run time (20–30 min), has been widely reported in pathogen detection (Lillis et al., ; Ma, Zeng, Huang, et al., ; Ma, Cong, et al., ; Yang et al., ; Yang, Qin, Sun, et al., ).…”

Section: Introductionmentioning

confidence: 99%

Point‐of‐care diagnostic assay for rapid detection of porcine deltacoronavirus using the recombinase polymerase amplification method

Zeng

Huang

et al. 2019

Transbound Emerg Dis

View full text Add to dashboard Cite

Summary Porcine deltacoronavirus (PDCoV) has emerged and spread throughout the porcine industry in many countries over the last 6 years. PDCoV caused watery diarrhoea, vomiting and dehydration in newborn piglets. A sensitive diagnostic method would be beneficial to the prevention and control of PDCoV infection. Recombinase polymerase amplification (RPA) is an isothermal amplification method which has been widely used for virus detection. A probe‐based reverse transcription RPA (RT‐RPA) assay was developed for real‐time detection of PDCoV. The amplification can be finished in 20 min and fluorescence monitoring was performed by a portable device. The lowest detection limit of the PDCoV RT‐RPA assay was 100 copies of RNA molecules per reaction; moreover, the RT‐RPA assay had no cross‐reaction with other common swine viruses. The clinical performance of the RT‐RPA assay was evaluated using 108 clinical samples (54 intestine specimens and 54 faecal swab specimens). The coincidence rate of the detection results for clinical samples between RT‐RPA and RT‐qPCR was 97.2%. In summary, the real‐time RT‐RPA assay offers a promising alternative to RT‐qPCR for point‐of‐care detection of PDCoV.

show abstract

Section: Introductionmentioning

confidence: 99%

Point‐of‐care diagnostic assay for rapid detection of porcine deltacoronavirus using the recombinase polymerase amplification method

Zeng

Huang

et al. 2019

Transbound Emerg Dis

View full text Add to dashboard Cite

show abstract

“…To monitor and detect MNV rapidly and specifically, several molecular methods such as reverse transcription polymerase chain reaction (RT-PCR) assays and RT-quantitative PCR (RT-qPCR) assays have been developed [8,[13][14][15]. Considering that the RT-PCR and RT-qPCR methods are time-consuming, the loop-mediated isothermal amplification (LAMP) method for detection of MNV was developed [16]. However four to six primers were required in the LAMP assay [16].…”

Section: Introductionmentioning

confidence: 99%

Development and evaluation of a broadly reactive reverse transcription recombinase polymerase amplification assay for rapid detection of murine norovirus

Zeng

Feng

et al. 2018

BMC Vet Res

View full text Add to dashboard Cite

BackgroundMurine norovirus (MNV) is recognized as the most prevalent viral pathogen in captive mouse colonies. The rapid detection assay for MNV would be a useful tool for monitoring and preventing MNV infection. A recombinase polymerase amplification (RPA) assay was established in this study to provide a solution for rapid and sensitive detection of MNV.ResultsThe detection limit of the RT-RPA assay for the detection of MNV was 1 × 102 copies of RNA molecules per reaction. The assay was specific since there was no cross-reaction with other common murine viruses. In addition, the broad reactivity of the RT-RPA assay was validated using the synthesized template carrying seven point mutations among several MNV strains. The MNV RT-RPA assay could detect as few as 1 × 102 copies of the mutant per reaction, suggesting the assay could be broadly reactive against a large diversity of MNV strains. Forty eight clinical samples including 16 gastric tissue specimens, 16 cecal tissue specimens and 16 fecal specimens were tested for the validation of the new developed RT-RPA assay. The detection results of RT-RPA and RT-qPCR for clinical samples were very similar, except that a gastric tissue sample which was positive by RT-qPCR, with a RNA titer of 27 copies, was negative by RT-RPA.ConclusionsA broadly reactive RT-RPA assay was successfully established for MNV detection.

show abstract

“…Accordingly, the SYBR Green I assay has the potential to detect various MNV with sensitivity, reproducibility, and sufficient quantitative capacity. In contrast, when the TaqMan assay was performed with 1000 copies of the 7 MNV plasmids, threshold cycles for the detection of Apo960 were delayed by 6 Ct compared with those for the detection of other 6 MNV [43]. The forward primer has a mismatch at position −5 of the 3′ end to the Apo960 genome sequence.…”

Section: Discussionmentioning

confidence: 99%

A Broadly Reactive One-Step SYBR Green I Real-Time RT-PCR Assay for Rapid Detection of Murine Norovirus

et al. 2014

Self Cite

View full text Add to dashboard Cite

A one-step SYBR Green I real-time RT-PCR assay was developed for the detection and quantification of a broad range of murine noroviruses (MNVs). The primer design was based on the multiple sequence alignments of 101 sequences of the open reading frame (ORF)1−ORF2 junction of MNV. The broad reactivity and quantitative capacity of the assay were validated using 7 MNV plasmids. The assay was completed within 1 h, and the reliable detection limit was 10 copies of MNV plasmid or 0.063 median tissue culture infective doses per milliliter of RAW264 cell culture-propagated viruses. The diagnostic performance of the assay was evaluated using 158 mouse fecal samples, 91 of which were confirmed to be positive. The melting curve analysis demonstrated the diversity of MNV in the samples. This is the first report of a broadly reactive one-step SYBR Green I real-time RT-PCR assay for detecting of MNVs. The rapid and sensitive performance of this assay makes it a powerful tool for diagnostic applications.

show abstract

Detection of murine norovirus by reverse transcription loop-mediated isothermal amplification

Cited by 11 publications

References 32 publications

Point‐of‐care diagnostic assay for rapid detection of porcine deltacoronavirus using the recombinase polymerase amplification method

Point‐of‐care diagnostic assay for rapid detection of porcine deltacoronavirus using the recombinase polymerase amplification method

Development and evaluation of a broadly reactive reverse transcription recombinase polymerase amplification assay for rapid detection of murine norovirus

A Broadly Reactive One-Step SYBR Green I Real-Time RT-PCR Assay for Rapid Detection of Murine Norovirus

Contact Info

Product

Resources

About