Assessment of loop-mediated isothermal amplification assays for Escherichia coli detection

INTRODUCTION: As of lately, loop-mediated isothermal amplification (LAMP) has become a powerful alternative to the polymerase chain reaction (PCR) in the molecular diagnostic world, especially for pathogen detection in clinical and food samples. LAMP is a powerful and new method of nucleic acid amplification, with the ability to detect DNA at a very low level. In comparison to other molecular methods such as PCR and Real-Time PCR, LAMP eliminates the need for sophisticated thermal cyclers. High efficiency of DNA amplification by LAMP significantly shortens the whole amplification process.

OBJECTIVES: This study aims to assess the efficacy of two different LAMP kits, WarmStart (Real-Time LAMP) and Colorimetric LAMP in detection of Escherichia coli. We tested and optimized the kits for the detection of E. coli ATCC® 25922 directly from bacterial suspension and indirectly using bacterial genomic DNA (gDNA). LAMP results were then compared with the results of E. coli detection using standard PCR methodology.

METHOD / DESIGN: For use in LAMP detection of E. coli directly from the bacterial suspension, we prepared eight bacterial dilutions in concentrations ranging from 1.5x108 - 1.5x101 CFU/mL. All bacterial dilutions were then used for LAMP detection using WarmStart Colorimetric LAMP 2X Master Mix (New England BioLabs) for colorimetric LAMP and WarmStart LAMP Kit (New England BioLabs) for Real-Time LAMP. Real-Time LAMP reactions were done on the Genie® II, an instrument for isothermal nucleic acid amplification (Figure 1). LAMP primers (F3, B3, FIP, BIP, LF, BF) (Figure 2) for the malB gene are taken from the literature. For indirect detection we extracted genomic DNA from E. coli using GeneJET Genomic DNA Purification Kit (Thermo Scientific). Extracted gDNA with the concentration 50 ng/μl was used for preparation of ten-fold dilutions (10-1 - 10-4 ), and these dilutions were used in both Colorimetric and WarmStart LAMP assays in order to compare limits of detection with and without DNA extraction. Finally, we used E. coli genomic DNA serial dilutions for standard PCR, using F3 and B3 primers for malB gene.

RESULTS: The results showed that both LAMP methods (WarmStart and Colorimetric LAMP) enabled the detection of E. coli directly, in suspension (without prior DNA isolation) for bacterial concentration of 1.5 x 10 8 CFU/mL (Figure 3, Chart 1). Additionally, the results of Colorimetric LAMP using serial dilutions of E. coli gDNA showed detection in all tested concentrations, except the lowest (10 -4 ) (Figures 4-5). On the other hand, the WarmStart LAMP kit enabled E. coli gDNA detection in all tested gDNA dilutions (Chart 2, Figure 6). The conventional PCR methods failed to detect E. coli malB gene product for the lowest DNA concentration (Figure 7.). Additionally, DNA bends on agarose gels are more intensive in the LAMP products compared to those obtained with PCR products.