Establishment and application of a rapid assay for GII.4/GII.17 NoV detection based on the combination of CRISPR/Cas13a and isothermal amplification

Jia-Heng Li; Duona Jing; Yu Wang; Jiayi Xu; Junxuan Yu; Huisha Du; Qing Chen; Shixing Tang; Xu-Fu Zhang; Ying-Chun Dai

doi:10.3389/fmicb.2024.1334387

Establishment and application of a rapid assay for GII.4/GII.17 NoV detection based on the combination of CRISPR/Cas13a and isothermal amplification

Front Microbiol. 2024 Feb 8:15:1334387. doi: 10.3389/fmicb.2024.1334387. eCollection 2024.

Authors

Jia-Heng Li¹, Duona Jing¹, Yu Wang¹, Jiayi Xu¹, Junxuan Yu¹, Huisha Du¹, Qing Chen¹, Shixing Tang¹, Xu-Fu Zhang^{2

3}, Ying-Chun Dai¹

Affiliations

¹ Guangdong Provincial Key Laboratory of Tropical Disease Research, Department of Epidemiology, School of Public Health, Southern Medical University, Guangzhou, China.
² The Fifth Affiliated Hospital, Southern Medical University, Guangzhou, China.
³ School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, China.

Abstract

Introduction: Norovirus (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.

Methods: In 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.

Discussion: The 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.

Keywords: CRISPR/Cas13a; RAA; detection; genotype GII.4/GII.17; norovirus.

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. The study was supported by the Key-Area Research and Development Program of Guangdong Province (2022B1111020002), the National Natural Science Foundation of China (grant numbers 31771007 and 81773975), and the Natural Science Foundation of Guangdong Province, China (grant number 2019A1515010951).