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Development and Evaluation of a Rapid GII Norovirus Detection Method Based on CRISPR-Cas12a Cover

Development and Evaluation of a Rapid GII Norovirus Detection Method Based on CRISPR-Cas12a

Polish Journal of Microbiology
Volume 73 (2024): Issue 1 (March 2024)
By: Xinyi Hu,  Pei He,  Tong Jiang and  Jilu Shen  
Open Access
|Mar 2024

Figures & Tables

Fig. 1.

Detection principle of RPA-Cas12a-fluorescence analysis and RPA-Cas12a-immunochromatography detection method. A) RPA-Cas12a method detection process; B) principle of Immunochromatography Flow Strip. C) the schematic diagram for judging the results of immunochromatography flow strips
Detection principle of RPA-Cas12a-fluorescence analysis and RPA-Cas12a-immunochromatography detection method. A) RPA-Cas12a method detection process; B) principle of Immunochromatography Flow Strip. C) the schematic diagram for judging the results of immunochromatography flow strips

Fig. 2.

RPA amplified fragments and design results of crRNA.
RPA amplified fragments and design results of crRNA.

Fig. 3.

Verification of sensitivity and specificity of the RPA-Cas12a-fluorometric and RPA-Cas12a-immunochromatographic assays. A) Sensitivity curves of different concentrations of norovirus positive quality control products amplified by RPA; B) specificity of RPA-Cas12a-fluorometric assay; C) RPA-Cas12a-immunochromatographic results of different concentrations of Norovirus; D) specificity of RPA-Cas12a-immunochromatographic assay
Verification of sensitivity and specificity of the RPA-Cas12a-fluorometric and RPA-Cas12a-immunochromatographic assays. A) Sensitivity curves of different concentrations of norovirus positive quality control products amplified by RPA; B) specificity of RPA-Cas12a-fluorometric assay; C) RPA-Cas12a-immunochromatographic results of different concentrations of Norovirus; D) specificity of RPA-Cas12a-immunochromatographic assay

Fig. 4.

Detection of GII norovirus in clinical samples based on CRISPR-Cas12a. A, B, C) Positive results of RPA-Cas12a-fluorescence assay of 24 samples; D) positive results of RPA-Cas12a-immunochromatographic assay of 24 samples; E) negative results of RPA-Cas12a-immunochromatographic assay of 11 samples; F, G) qPCR results of 24 positive samples
Detection of GII norovirus in clinical samples based on CRISPR-Cas12a. A, B, C) Positive results of RPA-Cas12a-fluorescence assay of 24 samples; D) positive results of RPA-Cas12a-immunochromatographic assay of 24 samples; E) negative results of RPA-Cas12a-immunochromatographic assay of 11 samples; F, G) qPCR results of 24 positive samples

Comparison between RPA-cas12a method and qPCR_

RPA-Cas12aQuantitative Real-Time PCRTotal
+−
+24024
−01111
Total241135

Bacteria and viruses involved in this study_

NameSource of strains
Shigella flexneriCMCC(B)51572
Escherichia coli O157:H7NCTC12900
Vibrio parahaemolyticusATCC® 17802™
Salmonella TyphimuriumClinical isolates
Campylobacter jejuniATCC® 33291™
Yersinia enterocoliticaCMCC(B)50024
Human astrovirusClinical isolates
RotavirusATCC® VR-2274™
Enteric adenovirusATCC® VR-930™

Primers and crRNA sequences_

OligonucleotideSequence (5’–3’)
RPA forward primersCCTCTCTTCACGGACCCTCTTTCTACAGC
RPA reverse primersTTCATTCACAAAATTGGGAGCCAGATTGC
crRNAAGUGCCUGGGAGAAAGAUGUCGUUU
ssDNA reportersFAM-TTATTATT-BHQ1
FAM-TTATTATT-Biotin
DOI: https://doi.org/10.33073/pjm-2024-009 | Journal eISSN: 2544-4646 | Journal ISSN: 1733-1331
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Language: English
Page range: 89 - 97
Submitted on: Oct 28, 2023
Accepted on: Jan 22, 2024
Published on: Mar 4, 2024
Published by: Polish Society of Microbiologists
In partnership with: Paradigm Publishing Services
Publication frequency: 4 issues per year
Keywords:
GII norovirus,
gastroenteritis,
RPA,
CRISPR-Cas12a,
diagnostic
Related subjects:
Life sciences,
Microbiology and virology

© 2024 Xinyi Hu, Pei He, Tong Jiang, Jilu Shen, published by Polish Society of Microbiologists
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

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