Highly sensitive and rapid identification of coxsackievirus A16 based on reverse transcription multiple cross displacement amplification combined with nanoparticle-based lateral flow biosensor assay

Jinzhi Cheng; Yu Wang; Yuhong Zhou; Jingrun Lu; Xiaomin Tang

doi:10.3389/fmicb.2023.1121930

Highly sensitive and rapid identification of coxsackievirus A16 based on reverse transcription multiple cross displacement amplification combined with nanoparticle-based lateral flow biosensor assay

Front Microbiol. 2023 Mar 8:14:1121930. doi: 10.3389/fmicb.2023.1121930. eCollection 2023.

Authors

Jinzhi Cheng¹, Yu Wang², Yuhong Zhou³, Jingrun Lu², Xiaomin Tang⁴

Affiliations

¹ School of Basic Medical Sciences, Guizhou Medical University, Guiyang, China.
² Department of Clinical Laboratory, The First People's Hospital of Guiyang, Guiyang, Guizhou, China.
³ School of Public Health, The Key Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education, Guizhou Medical University, Guiyang, China.
⁴ Laboratory of Bacterial Infectious Disease of Experimental Center, Guizhou Provincial Centre for Disease Control and Prevention, Guiyang, Guizhou, China.

Abstract

Introduction: One of the main pathogens responsible for human hand, foot, and mouth disease (HFMD), coxsackievirus A16, has put young children's health at danger, especially in countries in the Asia-Pacific region. Early quick identification is essential for the avoidance and control of the disorder since there are no vaccinations or antiviral medications available to prevent and manage CVA16 infection.

Methods: Here, we describe the creation of an easy, speedy, and accurate CVA16 infection detection approach using lateral flow biosensors (LFB) and reverse transcriptionmultiple cross displacement amplification (RT-MCDA). A group of 10 primers was developed for the RT-MCDA system in order to amplify the genes in an isothermal amplification device while targeting the highly conserved region of the CVA16 VP1 gene. Then, without requiring any extra tools, RT-MCDA amplification reaction products might well be detected by visual detection reagent (VDR) and LFB.

Results: The outcomes showed that 64°C within 40 min was the ideal reaction setting for the CVA16-MCDA test. Target sequences with <40 copies might be found using the CVA16-MCDA. There was no cross-reaction among CVA16 strains and other strains. The findings demonstrated that the CVA16-MCDA test could promptly and successfully identify all of the CVA16-positive (46/220) samples identified by the traditional real-time quantitative polymerase chain reaction (qRT-PCR) assays for 220 clinical anal swab samples. The whole process, such as the processing of the sample (15 min), the MCDA reaction (40 min), and the documenting of the results (2 min), could be finished in 1 h.

Conclusion: The CVA16-MCDA-LFB assay, which targeted the VP1 gene, was an efficient, simple, and highly specific examination that might be used extensively in rural regions' basic healthcare institutions and point-of-care settings.

Keywords: MCDA-LFB; coxsackievirus A16; hand, foot, and mouth disease; lateral flow biosensor; limit of detection; reverse transcription-multiple cross displacement amplification.