An ultrasensitive, rapid and portable method for screening oseltamivir-resistant virus based on CRISPR/Cas12a combined with immunochromatographic strips

Hao Zhang; Fayu Yang; Man Yang; Jing Liu; Mi Wang; Chenzhong Fei; Lifang Zhang; Feiqun Xue; Chuangang Zhu; Yingchun Liu; Feng Gu

doi:10.3724/abbs.2022163

An ultrasensitive, rapid and portable method for screening oseltamivir-resistant virus based on CRISPR/Cas12a combined with immunochromatographic strips

Acta Biochim Biophys Sin (Shanghai). 2022 Nov 25;54(11):1630-1636. doi: 10.3724/abbs.2022163.

Authors

Hao Zhang^{1

2}, Fayu Yang^{1

2}, Man Yang^{1

3}, Jing Liu^{1

2}, Mi Wang^{1

2}, Chenzhong Fei^{1

2}, Lifang Zhang^{1

2}, Feiqun Xue^{1

2}, Chuangang Zhu^{1

3}, Yingchun Liu^{1

2}, Feng Gu^{1

2}

Affiliations

¹ Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China.
² Key Laboratory of Veterinary Chemical Drugs and Pharmaceutics, Ministry of Agriculture and Rural Affairs, Shanghai 200241, China.
³ National Reference Laboratory for Animal Schistosomiasis, Shanghai 200241, China.

Abstract

Influenza is a significant public health challenge because of the emergence of antigenically shifted or highly virulent strains. The neuraminidase inhibitor oseltamivir is used as an antiviral drug in clinical treatment. However, its therapeutic effects can be greatly compromised by the emergence of drug-resistant mutant viruses. Thus, there is an urgent need to distinguish drug-resistant strains with a simple method. To address this, in the present study, we develop a rapid, sensitive and convenient molecular diagnosis method based on CRISPR/Cas12a technology and lateral flow detection (LFD). By targeting mutant sequences amplified by recombinase polymerase amplification (RPA) reaction, crRNA is designed to develop the CRISPR/Cas12a assay, and 2000 copies can be directly observed by the naked eye under blue light-emitting diode (LED) light. Combined with LFD, the limit of detection of RPA-CRISPR/Cas12a-LFD is about 20 copies of target sequence per reaction. Collectively, RPA-CRISPR/Cas12a-LFD method provides a novel alternative for the sensitive, specific and portable detection to diagnose oseltamivir-resistant mutant strains.

Keywords: CRISPR/Cas12a; influenza virus; lateral flow detection; sensitivity.

MeSH terms

CRISPR-Cas Systems
Nucleic Acid Amplification Techniques* / methods
Oseltamivir / pharmacology
Recombinases / metabolism
Viruses* / metabolism

Substances

Oseltamivir
Recombinases

Grants and funding

This work was supported by the grants from the National Natural Science Foundation of China (31871247 and 32071443), the Public-Interest Scientific Institution Basal Research Fund (Y2019PT10, 2019JB04, and Y2020XK18), and the Chinese Academy of Agricultural Sciences Grants (CAAS-Y2019YJ07-03).