Rapid and highly sensitive detection of Escherichia coli O157:H7 in food with loop‐mediated isothermal amplification coupled to a new bioluminescent assay

Abstract

Testing for bioluminescent pyrophosphate is a convenient method of DNA detection without complex equipments, but it is insufficiently sensitive and offers no particular time advantage over other rapid detection methods. The shortcomings of the traditional bioluminescent pyrophosphate method have been addressed by using 2-deoxyadenosine-5-(α-thio)-triphosphate (dATPαS) instead of dATP for LAMP, thus reducing the high background signal and generating a constant background value. In this study, LAMP coupled to a novel bioluminescent pyrophosphate assay was developed to detect E. coli O157:H7. The new method has a limit of detection of <10 copies/μL or 5 CFU/mL; its sensitivity is higher than that of the conventional LAMP assay. Moreover, a food-borne pathogen can be detected when a single DNA template is included in the LAMP assay, making it 100 times more sensitive than the traditional LAMP method. Three hundred food samples were tested with this assay and the accuracy of detection was verified with a culture method and MALDI Biotyper. The assay only took 90-120 min and detected <10 copies of the pathogen. This method had the advantages of rapidity, sensitivity, and simplicity, so it is very competitive for the rapid and highly sensitive detection of food-borne pathogens.

A Novel Approach to the Bioluminescent Detection of the SARS-CoV-2 ORF1ab Gene by Coupling Isothermal RNA Reverse Transcription Amplification with a Digital PCR Approach

“…Relative to the conventional reagent mixture, the optimized reagent mixture had the same amplification effect in RT-LAMP (Figure 2A), consistent with our previous findings [33] that dATPαS can eliminate false positive signal and better facilitate amplification than dATP in LAMP attributable to dATP (Figure 2B). Although this bioluminescent signalbased method could be used to detect SARS-CoV-2, it could not achieve real-time and accurate quantification.…”

Rapid Detection of Escherichia coli O157:H7 by Loop-Mediated Isothermal Amplification Coupled with a Lateral Flow Assay Targeting the z3276 Genetic Marker

“…Thus, the new method of z3276-LAMP-LF in our study effectively detects lower concentrations of E. coli O157:H7. Third, the performance of many current methods for the detection of E. coli O157:H7 has been evaluated in artificially contaminated samples (Ravan et al 2016;Zhang et al 2013;Li and Chen 2012) as well as practical samples collected from slaughterhouses and farms (Holland et al 2000;Fei et al 2020;Goma et al 2019). In this study, we evaluated the performance of the z3276-LAMP-LF assay using artificially contaminated ground beef and bovine feces.…”

“…It involves the amplification of six regions of the target with four pairs of primers under isothermal conditions (generally 60-65℃) in less than an hour (Notomi et al 2000). LAMP assays have been previously developed for the detection of E. coli O157:H7 (Rani et al 2021;Fei et al 2020;Zhang et al 2013), but these assays were not sufficiently specific to reliably detect this serotype. In response, Ravan and colleagues (Ravan et al 2016) developed an LAMP method that targeted the genetic marker z3276 for the detection of E. coli O157:H7, which had a detection limit of 10 CFU/reaction in pure bacterial culture.…”

A carbon-black-embedded poly(dimethylsiloxane)-paper hybrid device for energy-efficient nucleic-acid amplification in point-of-care testing

“…Primers used in this study were the same as those in a previous study conducted by Fei et al 35 A DNA sample (1 mL) was added to…”