This site needs JavaScript to work properly. Please enable it to take advantage of the complete set of features!
Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log in
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
doi: 10.1038/srep40244.

Development of a Recombinase Polymerase Amplification Assay for Detection of Epidemic Human Noroviruses

Affiliations

Development of a Recombinase Polymerase Amplification Assay for Detection of Epidemic Human Noroviruses

Matthew D Moore et al. Sci Rep. .

Abstract

Human norovirus is a leading cause of viral gastroenteritis worldwide. Rapid detection could facilitate control, however widespread point-of-care testing is infrequently done due to the lack of robust and portable methods. Recombinase polymerase amplification (RPA) is a novel isothermal method which rapidly amplifies and detects nucleic acids using a simple device in near real-time. An RT-RPA assay targeting a recent epidemic human norovirus strain (GII.4 New Orleans) was developed and evaluated in this study. The assay successfully detected purified norovirus RNA from multiple patient outbreak isolates and had a limit of detection of 3.40 ± 0.20 log10 genomic copies (LGC), which is comparable to most other reported isothermal norovirus amplification methods. The assay also detected norovirus in directly boiled stool, and displayed better resistance to inhibitors than a commonly used RT-qPCR assay. The assay was specific, as it did not amplify genomes from 9 non-related enteric viruses and bacteria. The assay detected norovirus in some samples in as little as 6 min, and the entire detection process can be performed in less than 30 min. The reported RT-RPA method shows promise for sensitive point-of-care detection of epidemic human norovirus, and is the fastest human norovirus amplification method to date.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Time to signal detection of different RT-RPA primer pairs.
The RT-RPA assay was performed as described using the NOP1 probe. The template used was 7.0 LGC of purified GII.4 New Orleans RNA per reaction.
Figure 2
Figure 2. Sensitivity of RT-RPA assay for purified GII.4 New Orleans RNA as predicted using probit regression analyses.
Serial dilutions of purified RNA from three selected patient stool samples were used as templates in RT-RPA reactions for 8 replicates each and the number of positive samples at each dilution was used for separate probit regressions. A representative regression analysis (for sample 29) is pictured. The points mark the proportion of samples positive at each log10 genomic copy level; the solid line marks the predicted frequency of samples positive as a function of log10 genomic copy input, and the dotted line is a visual marker at the 95% level of sample positivity.

References

    1. Ahmed S. M. et al.. Global prevalence of norovirus in cases of gastroenteritis: a systematic review and meta-analysis. Lancet Infect. Dis. 3099, 1–6 (2014). - PMC - PubMed
    1. Scharff R. L. Economic burden from health losses due to foodborne illness in the United States. J. Food Prot. 75, 123–31 (2012). - PubMed
    1. Lee B. Y. et al.. Economic value of norovirus outbreak control measures in healthcare settings. Clin. Microbiol. Infect. 17, 640–6 (2011). - PMC - PubMed
    1. Lee B. Y. et al.. Economic Impact of Outbreaks of Norovirus Infection in Hospitals. Infect. Control Hosp. Epidemiol. 32, 191–193 (2011). - PMC - PubMed
    1. Butot S., Zuber S. & Baert L. Sample preparation prior to molecular amplification: Complexities and opportunities. Curr. Opin. Virol. 4, 66–70 (2014). - PubMed

Publication types

MeSH terms

Cite

AltStyle によって変換されたページ (->オリジナル) /