Loop-mediated isothermal amplification assays for the detection of antimicrobial resistance elements in Vibrio cholera.

Abstract

Background: The bacterium Vibrio cholerae causes diarrheal illness and can acquire genetic material leading to multiple drug resistance (MDR). Rapid detection of resistance-conferring mobile genetic elements helps avoid the prescription of ineffective antibiotics for specific strains. Colorimetric loop-mediated isothermal amplification (LAMP) assays provide a rapid and cost-effective means for detection at point-of-care since they do not require specialized equipment, require limited expertise to perform, and can take less than 30 min to perform in resource limited regions. LAMP output is a color change that can be viewed by eye, but it can be difficult to design primer sets, determine target specificity, and interpret subjective color changes.

Methods: We developed an algorithm for the in silico design and evaluation of LAMP assays within the open-source PCR Signature Erosion Tool (PSET) and a computer vision application for the quantitative analysis of colorimetric outputs. First, Primer3 calculates LAMP primer sequence candidates with settings based on GC-content optimization. Next, PSET aligns the primer sequences of each assay against large sequence databases to calculate sufficient sequence similarity, coverage, and primer arrangement to the intended taxa, ultimately generating a confusion matrix. Finally, we tested assay candidates in the laboratory against synthetic constructs.

Results: As an example, we generated new LAMP assays targeting drug resistance in V. cholerae and evaluated existing ones from the literature based on in silico target specificity and in vitro testing. Improvements in the design and testing of LAMP assays, with heightened target specificity and a simple analysis platform, increase utility for in-field applications. Overall, 9 of the 16 tested LAMP assays had positive signal through visual and computer vision-based detection methods developed here. Here we show LAMP assays tested on synthetic AMR gene targets for aph(6), varG, floR, qnrVC5, and almG, which allow for resistance to aminoglycosides, penicillins, carbapenems, phenicols, fluoroquinolones, and polymyxins respectively.