Development and Evaluation of Rapid and Accurate One-tube RPA-CRISPR-Cas12b-based Detection of Mcr-1 and Tet(X4)

Overview

Journal Appl Microbiol Biotechnol

Specialties Biomedical Engineering
Biotechnology
Microbiology

Date 2024 May 27

PMID 38801527

Authors

Yu Wang

Huan Chen

Qingyun Pan

Jing Wang

Xinan Jiao

Yunzeng Zhang

Affiliations

Soon will be listed here.

Abstract

The emergence and quick spread of the plasmid-mediated tigecycline resistance gene tet(X4) and colistin resistance gene mcr-1 have posed a great threat to public health and raised global concerns. It is imperative to develop rapid and accurate detection systems for the onsite surveillance of mcr-1 and tet(X4). In this study, we developed one-tube recombinase polymerase amplification (RPA) and CRISPR-Cas12b integrated mcr-1 and tet(X4) detection systems. We identified mcr-1- and tet(X4)-conserved and -specific protospacers through a comprehensive BLAST search based on the NCBI nt database and used them for assembling the detection systems. Our developed one-tube RPA-CRISPR-Cas12b-based detection systems enabled the specific detection of mcr-1 and tet(X4) with a sensitivity of 6.25 and 9 copies within a detection time of ~ 55 and ~ 40 min, respectively. The detection results using pork and associated environmental samples collected from retail markets demonstrated that our developed mcr-1 and tet(X4) detection systems could successfully monitor mcr-1 and tet(X4), respectively. Notably, mcr-1- and tet(X4)-positive strains were isolated from the positive samples, as revealed using the developed detection systems. Whole-genome sequencing of representative strains identified an mcr-1-carrying IncI2 plasmid and a tet(X4)-carrying IncFII plasmid, which are known as important vectors for mcr-1 and tet(X4) transmission, respectively. Taken together, our developed one-tube RPA-CRISPR-Cas12b-based mcr-1 and tet(X4) detection systems show promising potential for the onsite detection of mcr-1 and tet(X4). KEY POINTS: • One-tube RPA-CRISPR-Cas12b-based mcr-1 and tet(X4) detection systems were developed based on identified novel protospacers. • Both detection systems exhibited high sensitivity and specification with a sample-to-answer time of less than 1 h. • The detection systems show promising potential for onsite detection of mcr-1 and tet(X4).

Citing Articles

Validation of Recombinase Polymerase Amplification with In-House Lateral Flow Assay for Gene Detection of Colistin Resistant Isolates.

Ullah N, Suchanta N, Pimpitak U, Santanirand P, Am-In N, Chaichanawongsaroj N Antibiotics (Basel). 2024; 13(10).

PMID: 39452250 PMC: 11505259. DOI: 10.3390/antibiotics13100984.

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