Naked-eye detection with loop-mediated isothermal amplification for P. carotovorum subsp. carotovorum in agricultural products.

Pectobacterium carotovorum causing soft-rot disease requires on-site detection before the distribution of agricultural products. Loop-mediated isothermal amplification (LAMP), which is resistant to food inhibitors, is known for its high detection sensitivity for pathogens and when coupled with lateral flow immunoassay (LFA) enables visualizations. For detection of soft-rot disease, we developed a LAMP-LFA system targeting 16S ribosomal RNA, a partial sequence gene of P. carotovorum subsp. carotovorum. The LAMP-LFA was performed at 60 °C for 50 min followed by hybridization of digoxygenin-labeled LAMP amplicon and biotinylated probe. Detection sensitivity was 3.22 × 101 CFU/mL in pure culture, which specifically detected the target. In Chinese cabbage and potato, the target was detected up to low levels of 1.57 × 102 CFU/g and 1.29 × 102 CFU/g, respectively. This study showed potential applicability as a sensitive point-of-care system for soft-rot disease bacteria detection in agricultural products. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01315-z.

Diverse methods of reducing and confirming false-positive results of loop-mediated isothermal amplification assays: A review.

Loop-mediated isothermal amplification (LAMP), a rapid and sensitive isothermal nucleic acid amplification method, is a promising alternative to other molecular amplification techniques due to its superior specificity and sensitivity. However, due to primer dimerization, LAMP results in nonspecific and nontemplate amplification. And during the amplification confirmation process, there is carry-over contamination. These factors can result in false-positive results that overestimate the amount of DNA, preventing accurate detection. This review outlined several techniques for reducing false-positive LAMP results before amplification and confirming false-positive results after amplification. Before the amplification step, DNA polymerase activity can be decreased with organic additives such as dimethyl sulfoxide, betaine, and pullulan to prevent nonspecific amplification. The enzyme uracil-DNA-glycosylase (UDG) can eliminate false-positive results caused by carry-over contamination, and the hot-start effect with gold nanoparticles can reduce nonspecific amplification. When confirming false-positive results using clustered regularly interspaced short palindromic repeats, guide RNA accurately detects LAMP amplification, allowing differentiation from nonspecific amplification. By confirming amplification, the colorimetric change in the deoxyribozyme (DNAzyme) formed by the reaction of the G-quadruplex sequence of the LAMP amplicon and hemin can distinguish false-positive results. Lateral flow immunoassay can distinguish false-positive results by accurately recognizing hybridized probes to LAMP amplicons.

A review of mechanism analysis methods in multi-species biofilm of foodborne pathogens.

Biofilms are an aggregation of microorganisms that have high resistance to antimicrobial agents. In the food industry, it has been widely studied that foodborne pathogens on both food surfaces and food-contact surfaces can form biofilms thereby threatening the safety of the food. In the natural environment, multi-species biofilms formed by more than two different microorganisms are abundant. In addition, the resistance of multi-species biofilms to antimicrobial agents is higher than that of mono-species biofilms. Therefore, studies to elucidate the mechanisms of multi-species biofilms formed by foodborne pathogens are still required in the food industry. In this review paper, we summarized the novel analytical methods studied to evaluate the mechanisms of multi-species biofilms formed by foodborne pathogens by dividing them into four categories: spatial distribution, bacterial interaction, extracellular polymeric substance production and quorum sensing analytical methods.

Thermophilic helicase-dependent amplification-based CRISPR/Cas12a system: Detection of stx2 in Escherichia coli O157:H7 by controlling primer dimers.

BACKGROUND: s: To overcome the limitation of polymerase chain reaction (PCR), isothermal amplification methods such as thermophilic helicase-dependent amplification (tHDA) have been developed. However, formation of primer dimer due to the single amplification temperature are major problems of tHDA. When cross-dimerization of forward and reverse primer occurred, false-positive results can be found on the lateral flow assay (LFA) which is one of the major detection methods widely used as a point of care diagnosis. Therefore, specific method of detecting only the target amplicon is required. RESULTS: In this study, a tHDA-based CRISPR/Cas12a system was developed to detect low levels of Escherichia coli O157:H7 in fresh salad mix without the false-positive results produced by primer dimers. For the comparison of the effect in eliminating false-positive results by CRISPR/Cas12a system, LFA was also evaluated. The tHDA-based CRISPR/Cas12a system detected as low as 101 CFU/mL E. coli O157:H7 in bacterial pure culture. In LFA false-positive results were produced due to the primer dimer, whereas the primer dimer produced by tHDA was not detected in the CRISPR/Cas12a system. These results indicated that the CRISPR/Cas12a system eliminated the formation of primer dimer. In fresh salad mix, the tHDA-based CRISPR/Cas12a system combined with the filter concentration method detected 103 CFU/g E. coli O157:H7. CONCLUSION: This study was the first to amplify stx2 of E. coli O157:H7 with tHDA as an isothermal amplification method and detected the amplicon without false-positive results by combining tHDA with CRISPR/Cas12a. Therefore, this study showed great potential for detecting low levels of E. coli O157:H7 present in fresh salad mix.

Enhanced detection of Listeria monocytogenes using tetraethylenepentamine-functionalized magnetic nanoparticles and LAMP-CRISPR/Cas12a-based biosensor.

BACKGROUND: Listeria monocytogenes is a pathogenic bacterium that can lead to severe illnesses, especially among vulnerable populations. Therefore, the development of rapid and sensitive detection methods is vital to prevent and manage foodborne diseases. In this study, we used tetraethylenepentamine (TEPA)-functionalized magnetic nanoparticles (MNPs) and a loop-mediated isothermal amplification (LAMP)-based CRISPR/Cas12a-based biosensor to concentrate and detect, respectively, L. monocytogenes. LAMP enables DNA amplification at a constant temperature, providing a highly suitable approach for point-of-care testing (POCT). The ability of CRISPR/Cas12a to cleave ssDNA reporter, coupled with TEPA-functionalized MNPs effective attachment to negatively charged bacteria, forms a promising biosensor. RESULTS: The LAMP assay was meticulously developed by selecting specific primers and designing crRNA sequences targeting a specific region within the hly gene of L. monocytogenes. We selected primer and refined the amplification conditions by systematically exploring a temperature range from 59 °C to 69 °C, ensuring the attainment of optimal performance. This process was complemented by systematic optimization of LAMP-CRISPR/Cas12a system parameters. In particular, we successfully established the optimal ssDNA reporter concentrations (0-1.2 µM) and Cas12a-mediated trans-cleavage times (0-20 min), crucial components that underpin the effectiveness of the LAMP-CRISPR/Cas12a-based biosensor. For optimizing parameters in capturing L. monocytogenes using TEPA-functionalized MNPs, capture efficiency was significantly enhanced through adjustments in TEPA-functionalized MNPs concentration, incubation times, and magnetic separation duration. Large-volume (20 mL) magnetic separation exhibited a 10-fold sensitivity improvement over conventional methods. Utilizing TEPA-functionalized MNPs, the LAMP-CRISPR/Cas12a-based biosensor achieved detection limits of 100 CFU mL-1 in pure cultures and 100 CFU g-1 in enoki mushrooms. SIGNIFICANCE: The integration of this novel technique with the LAMP-CRISPR/Cas12a-based biosensor enhances the accuracy and sensitivity of L. monocytogenes detection in foods, and it can be a promising biosensor for POCT. The 10-fold increase in sensitivity compared to conventional methods makes this approach a groundbreaking advancement in pathogenic bacteria detection for food safety and public health.

Review of multi-species biofilm formation from foodborne pathogens: multi-species biofilms and removal methodology.

Multi-species biofilms are ubiquitous worldwide and are a concern in the food industry. Multi-species biofilms have a higher resistance to antimicrobial therapies than mono-species biofilms. In addition, multi-species biofilms can cause severe foodborne diseases. To remove multi-species biofilms, controlling the formation process of extracellular polymeric substances (EPS) and quorum sensing (QS) effects is essential. EPS disruption, inhibition of QS, and disinfection have been utilized to remove multi-species biofilms. This review presents information on the formation and novel removal methods for multi-species biofilms.

Development of modified enrichment broth for short enrichment and recovery of filter-injured Salmonella Typhimurium.

The filter concentration method facilitates the rapid detection of foodborne pathogens. The filter concentration method lowered the limit of detection (LOD) of artificially inoculated cabbage with Salmonella Typhimurium; however, the procedure injured foodborne pathogens during filtering procedure. Thus, to detect injured pathogens under the detection limit, an enrichment broth promoting pathogen resuscitation and growth is required. To rapidly recover, cultivate and lower the time to result (TTR) of S. Typhimurium detection after filter concentration method, a brain heart infusion (BHI) broth-based modified enrichment broth (MEB) was developed. The MEB was developed by fitting growth curves to a modified Gompertz model; 1.00 g/L of sodium pyruvate, 0.20 g/L proline and 2.0 g/L magnesium sulphate additives were optimized as additional components to rapidly grow filter-injured S. Typhimurium. As a result, the rate of filter-injured S. Typhimurium went from 100% to 0.0% using MEB within 3.5 h. In contrast, BHI required 4 h and buffered peptone water (BPW) required more than 4 h to decrease the injury rate to 0.0%. Using MEB, BHI and BPW, filter-injured S. Typhimurium in cabbages were enriched to 4.056 ± 0.026 Log CFU/25 g, 3.571 ± 0.187 Log CFU/25 g and 3.708 ± 0.156 Log CFU/25 g, respectively. Additionally, 1-9 CFU/mL S. Typhimurium in cabbage was detected within 3.0 h, including 1 h enrichment with MEB, whereas 5.0 h was required for BHI and BPW. Thus, the MEB developed in this study showed great potential as a short enrichment broth for the rapid detection of filter-injured S. Typhimurium.