Detection assay of polymyxin resistance coding mcr-1 gene based on CRISPR/Cas13a system

Abstract

Introduction: Polymyxins are reserved as an ultimate defense against multidrug-resistant bacteria. The emergence of the polymyxin resistance gene mcr-1 poses a potential risk for the treatment of severe infections caused by Gram-negative bacteria. Timely detection and monitoring the mcr-1 gene are essential for guiding anti-infective therapy and controlling the spread of polymyxin resistance. Quantitative real-time PCR (qPCR) is one of the common methods for detecting resistance genes. However, qPCR has equipment dependency, and is not feasible in primary healthcare settings. Currently, there remains a lack of a highly sensitive and portable method for detecting the mcr-1 gene.

Methods: We established and optimized detection assays of the mcr-1 gene based on CRISPR/Cas13a system and lateral flow strips. The detection method was preliminarily evaluated using clinical isolates from Escherichia coli, compared with qPCR.

Results: The method for detecting the mcr-1 gene based on the CRISPR/Cas13a system and lateral flow strips was established, with a detection limit of 100 copies/mL. This method demonstrated high analytical specificity, with no cross-reactivity detected in non-mcr-1 and non-resistant strains. Among 36 clinical isolates, the method identified 31 strains as positive for the mcr-1 gene, and had a 100% concordance rate with the results of qPCR.

Conclusions: We established a detection method for the polymyxin resistance mcr-1 gene based on the CRISPR/Cas13a system. This method enables visual readouts without instruments, making it potentially applicable to primary healthcare settings and field surveillance.

Keywords: CRISPR/Cas13a; antibiotic resistance; lateral flow strip; mcr-1; polymyxin.

Figure 1

The schematic of CRISPR-ERASE assay for detecting the mcr-1 gene. Plasmid DNA was extracted from Escherichia coli, amplified the mcr-1 gene fragment by recombinase-assisted amplification (RAA) and transcribed into an RNA sequence. When CRISPR/Cas13a system recognizes the RNA target and degrades reporter RNA. The cleaved reporter RNA can be captured on a lateral flow strip and the results can be read with the naked eye using ERASE strips.

Figure 2

Design and screening of RAA primers and crRNAs for the detection of the mcr-1 gene. (A) The schematic diagram of the design of RAA primers and crRNA. (B) The dynamic fluorescence curves within 40 min of 3 different crRNAs for CRISPR-ERASE assay (n = 3). (C) The fluorescence values of different crRNAs designed for the mcr-1 gene were compared at the 40 min after detection reaction. ****p < 0.0001. The data were expressed as mean ± SEM (n = 3). (D) The fluorescence values produced at 40 min of CRSIPR fluorescence detection reaction with crRNA-1 and different RAA amplification primer combinations of mcr-1 gene. The data were expressed as mean ± SEM (n = 3).

Figure 3

Analytical sensitivity and specificity evaluation of the CRISPR-ERASE assay for the detection of the mcr-1 gene. (A) Comparison of fluorescence values produced by CRISPR fluorescence detection of mcr-1 plasmid. TH means the threshold using the mean fluorescence value of the negative control group plus three times the standard deviation. ***p<0.001; ****p<0.0001. The data were expressed as mean ± SD (n = 3). (B) The results of qPCR at different mcr-1 plasmid concentrations (10³ ~10^-1 copies/μL) (n = 3). (C) The results of CRISPR-ERASE at different mcr-1 plasmid concentrations (10³ ~10^-1 copies/μL). Test band disappeared and control band was visible meaning that the test result is positive. Repeat each result three times. NC represents Negative Control. (D) CRISPR-ERASE assay was used to detect the mcr-1 gene of different strains. PC represents Positive Control; Escherichia coli (+) represents mcr-1-positive Escherichia coli; S.aureus represents Staphylococcus aureus; H. pylori represents Helicobacter pylori; K. pneumoniae represents Klebsiella pneumoniae (n = 3).

Figure 4

Preliminary validation of CRISPR-ERASE detection system to detect mcr-1 gene in isolated Escherichia coli samples. (A) The results of antimicrobial susceptibility testing for NO.31-33 isolated samples. (B) The MIC results of antimicrobial susceptibility testing for 36 isolated samples. (C) The results of CRISPR-ERASE detection assay for 36 isolated samples. PC represents Positive Control.NC represents Negative Control. (D) The results of qPCR for 36 isolated samples.