Establishment and application of a rapid assay for GII.4/GII.17 NoV detection based on the combination of CRISPR/Cas13a and isothermal amplification
IntroductionNorovirus (NoV) is one of the most important agents responsible for viral acute gastroenteritis, among which GII.4 NoV is the predominant strain worldwide, and GII.17 NoV surpassed GII.4 in some epidemic seasons. Rapid and accurate gene recognition is essential for a timely response to N...
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Frontiers Media S.A.
2024-02-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2024.1334387/full |
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| author | Jia-Heng Li Duona Jing Yu Wang Jiayi Xu Junxuan Yu Huisha Du Qing Chen Shixing Tang Xu-Fu Zhang Xu-Fu Zhang Ying-Chun Dai |
| author_facet | Jia-Heng Li Duona Jing Yu Wang Jiayi Xu Junxuan Yu Huisha Du Qing Chen Shixing Tang Xu-Fu Zhang Xu-Fu Zhang Ying-Chun Dai |
| author_sort | Jia-Heng Li |
| collection | DOAJ |
| description | IntroductionNorovirus (NoV) is one of the most important agents responsible for viral acute gastroenteritis, among which GII.4 NoV is the predominant strain worldwide, and GII.17 NoV surpassed GII.4 in some epidemic seasons. Rapid and accurate gene recognition is essential for a timely response to NoV outbreaks.MethodsIn the present study, the highly conserved regions of GII.4 and GII.17 NoVs were identified in the junction of open reading frame (ORF) 1 and ORF2 and then amplified by isothermal recombinase-aided amplification (RAA), followed by the cleavage of CRISPR-Cas13a with screened CRISPR RNAs (crRNAs) and RAA primers. The entire detection procedure could be completed within 40 min using a thermostat, and the results could be read out by the naked eye under a portable blue light transilluminator.DiscussionThe assay showed a high sensitivity of 97.96% and a high specificity of 100.0%. It offered a low limit of detection (LOD) of 2.5×100 copies/reaction and a coincidence rate of 96.75% in 71 clinical fecal samples. Overall, rapid and inexpensive detection of GII.4/GII.17 NoVs was established, which makes it possible to be used in areas with limited resources, particularly in low-income countries. Furthermore, it will contribute to assessing transmission risks and implementing control measures for GII.4/GII.17 NoVs, making healthcare more accessible worldwide. |
| first_indexed | 2024-03-08T04:52:02Z |
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| issn | 1664-302X |
| language | English |
| last_indexed | 2024-04-24T15:21:45Z |
| publishDate | 2024-02-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-cfc67576f0374697a6686abe5d707a5c2024-04-02T07:48:48ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2024-02-011510.3389/fmicb.2024.13343871334387Establishment and application of a rapid assay for GII.4/GII.17 NoV detection based on the combination of CRISPR/Cas13a and isothermal amplificationJia-Heng Li0Duona Jing1Yu Wang2Jiayi Xu3Junxuan Yu4Huisha Du5Qing Chen6Shixing Tang7Xu-Fu Zhang8Xu-Fu Zhang9Ying-Chun Dai10Guangdong Provincial Key Laboratory of Tropical Disease Research, Department of Epidemiology, School of Public Health, Southern Medical University, Guangzhou, ChinaGuangdong Provincial Key Laboratory of Tropical Disease Research, Department of Epidemiology, School of Public Health, Southern Medical University, Guangzhou, ChinaGuangdong Provincial Key Laboratory of Tropical Disease Research, Department of Epidemiology, School of Public Health, Southern Medical University, Guangzhou, ChinaGuangdong Provincial Key Laboratory of Tropical Disease Research, Department of Epidemiology, School of Public Health, Southern Medical University, Guangzhou, ChinaGuangdong Provincial Key Laboratory of Tropical Disease Research, Department of Epidemiology, School of Public Health, Southern Medical University, Guangzhou, ChinaGuangdong Provincial Key Laboratory of Tropical Disease Research, Department of Epidemiology, School of Public Health, Southern Medical University, Guangzhou, ChinaGuangdong Provincial Key Laboratory of Tropical Disease Research, Department of Epidemiology, School of Public Health, Southern Medical University, Guangzhou, ChinaGuangdong Provincial Key Laboratory of Tropical Disease Research, Department of Epidemiology, School of Public Health, Southern Medical University, Guangzhou, ChinaThe Fifth Affiliated Hospital, Southern Medical University, Guangzhou, ChinaSchool of Traditional Chinese Medicine, Southern Medical University, Guangzhou, ChinaGuangdong Provincial Key Laboratory of Tropical Disease Research, Department of Epidemiology, School of Public Health, Southern Medical University, Guangzhou, ChinaIntroductionNorovirus (NoV) is one of the most important agents responsible for viral acute gastroenteritis, among which GII.4 NoV is the predominant strain worldwide, and GII.17 NoV surpassed GII.4 in some epidemic seasons. Rapid and accurate gene recognition is essential for a timely response to NoV outbreaks.MethodsIn the present study, the highly conserved regions of GII.4 and GII.17 NoVs were identified in the junction of open reading frame (ORF) 1 and ORF2 and then amplified by isothermal recombinase-aided amplification (RAA), followed by the cleavage of CRISPR-Cas13a with screened CRISPR RNAs (crRNAs) and RAA primers. The entire detection procedure could be completed within 40 min using a thermostat, and the results could be read out by the naked eye under a portable blue light transilluminator.DiscussionThe assay showed a high sensitivity of 97.96% and a high specificity of 100.0%. It offered a low limit of detection (LOD) of 2.5×100 copies/reaction and a coincidence rate of 96.75% in 71 clinical fecal samples. Overall, rapid and inexpensive detection of GII.4/GII.17 NoVs was established, which makes it possible to be used in areas with limited resources, particularly in low-income countries. Furthermore, it will contribute to assessing transmission risks and implementing control measures for GII.4/GII.17 NoVs, making healthcare more accessible worldwide.https://www.frontiersin.org/articles/10.3389/fmicb.2024.1334387/fullnorovirusgenotype GII.4/GII.17CRISPR/Cas13aRAAdetection |
| spellingShingle | Jia-Heng Li Duona Jing Yu Wang Jiayi Xu Junxuan Yu Huisha Du Qing Chen Shixing Tang Xu-Fu Zhang Xu-Fu Zhang Ying-Chun Dai Establishment and application of a rapid assay for GII.4/GII.17 NoV detection based on the combination of CRISPR/Cas13a and isothermal amplification Frontiers in Microbiology norovirus genotype GII.4/GII.17 CRISPR/Cas13a RAA detection |
| title | Establishment and application of a rapid assay for GII.4/GII.17 NoV detection based on the combination of CRISPR/Cas13a and isothermal amplification |
| title_full | Establishment and application of a rapid assay for GII.4/GII.17 NoV detection based on the combination of CRISPR/Cas13a and isothermal amplification |
| title_fullStr | Establishment and application of a rapid assay for GII.4/GII.17 NoV detection based on the combination of CRISPR/Cas13a and isothermal amplification |
| title_full_unstemmed | Establishment and application of a rapid assay for GII.4/GII.17 NoV detection based on the combination of CRISPR/Cas13a and isothermal amplification |
| title_short | Establishment and application of a rapid assay for GII.4/GII.17 NoV detection based on the combination of CRISPR/Cas13a and isothermal amplification |
| title_sort | establishment and application of a rapid assay for gii 4 gii 17 nov detection based on the combination of crispr cas13a and isothermal amplification |
| topic | norovirus genotype GII.4/GII.17 CRISPR/Cas13a RAA detection |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2024.1334387/full |
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