Listeria monocytogenes is a major foodborne pathogen of global health concern. Herein, the rapid diagnosis of L. monocytogenes has been achieved using loop-mediated isothermal amplification (LAMP) based on the phosphatidylcholine-phospholipase C gene (plcB). Colorimetric detection was then performed through the formation of DNA concatemers and a gold nanoparticle/DNA probe complex (GNP/DNA probe). The overall detection process was accomplished within approximately 1 h with no need for complicated equipment. The limits of detection for L. monocytogenes in the forms of purified genomic DNA and pure culture were 800 fg and 2.82 CFU mL⁻¹, respectively. No cross reactions were observed from closely related bacteria species. The LAMP-GNP/DNA probe assay was applied to the detection of 200 raw chicken meat samples and compared to routine standard methods. The data revealed that the specificity, sensitivity, and accuracy were 100, 90.20, and 97.50%, respectively. The present assay was 100% in conformity with LAMP-agarose gel electrophoresis assay. Five samples that were negative by both assays appeared to have the pathogen at below the level of detection. The assay can be applied as a rapid direct screening method for L. monocytogenes.

In this study, we successfully developed a simple and rapid method for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli using gold nanoparticles and the aptamer aptasensor. We screened 25 specific DNA aptamer candidates against these pathogens using whole-cell Systematic Evolution of Ligands by EXponential enrichment. Among them, Ap6 was selected due to its low energy minimization values of −12.25 and −27.67 kcal/mol derived from MFold and RNAFold analysis, respectively. The assay presented in this study allowed the visual colorimetric detection of labeled colloidal gold nanoparticles as well as determination of UV absorbance at 625 and 525 nm under optimized conditions. The detection limit of this aptasensor was as less as 10⁵ CFU/ml. A random investigation of 50 meat samples, including ham and chicken sausages, collected from the local market revealed 96% accuracy, 96% specificity, and 100% sensitivity of the assay. The colorimetric aptasensor can accomplish one-step detection without pre-culture, DNA extraction, and amplification. Hence, it is an easy, rapid, specific, and qualitative assay that can be used as a point-of-care testing to directly detect multiplex foodborne pathogens.

Functionalized DNA sequences are promising sensing elements to combine with transducers for bio-sensing specific target microbes. As an application example, this paper demonstrates in situ detection of loop-mediated isothermal amplification products by hybridizing them with thiolated-ssDNA covalently anchored on the electrodes of a quartz crystal microbalance (QCM). Such hybridization leads to a frequency signal, which is suitable for monitoring real-time LAMP amplification based on mass-sensing: it detects interactions between the complementary nucleobases of LAMP products in solution and the thiolated-ssDNA probe sequence on the gold surface. Target DNA LAMP products cause irreversible frequency shifts on the QCM surfaces during hybridization in the kHz range, which result from both changes in mass and charge on the electrode surface. In order to confirm the LAMP assay working in the QCM sensing system at elevated temperature, the sky blue of positive LAMP products solution was achieved by using the Hydroxy Naphthol Blue (HNB) and agarose gel electrophoresis. Since on-QCM sensing of DNA hybridization leads to irreversible sensor responses, this work shows characterization by X-ray photoelectron spectroscopy (XPS) core spectra of S2p, N1s, Mg1s, P2p and C1s. XPS results confirmed that indeed both DNA and by-products of LAMP attached to the surface. Listeria monocytogenes DNA served to study in-situ detection of amplified LAMP products on DNA-functionalized surfaces.

Porcine epidemic diarrhea virus (PEDV) and porcine circovirus type 2 (PCV2) are both important global pathogenic viruses which have a significant impact on the swine industry. In this study, a duplex loop-mediated isothermal amplification (duplex LAMP) method was developed in combination with lateral flow dipstick (LFD) for simultaneous detection of PEDV and PCV2 using specific sets of primers and probes designed based on the conserved regions of a spike gene (KF272920) and an ORF gene (EF493839), respectively. The limit of detection (LOD) values of the duplex LAMP-LFD for the detection of PEDV and PCV2 were 0.1 ng/µL and 0.246 ng/µL, respectively. The LOD of duplex LAMP-LFD was 10-times more sensitive than conventional PCR and RT-PCR-agarose gel-electrophoresis (PCR-AGE and RT-PCR-AGE). No cross-reaction to each other and to other pathogenic viruses that can infect pigs were observed according to analytical specificity tests. The duplex LAMP-LFD method for the simultaneous detection of PEDV and PCV2 co-infection could be completed within approximately 1.5 h, and only a simple heating block was required for isothermal amplification. The preliminary validation using 50 swine clinical samples with positive and negative PEDV and/or PCV2 revealed that the sensitivity, specificity, and accuracy of duplex LAMP-LFD were all 100% in comparison to conventional PCR and RT-PCR. Hence, this study suggests that duplex LAMP-LFD is a promising tool for the early detection and initial screening of PEDV and PCV2, which could be beneficial for prevention, planning, and epidemiological surveys of these diseases.

Treponema pallidum subspp. pallidum is a spirochaete bacterium that causes syphilis, one of the most common sexually transmitted diseases. Syphilis progresses through four distinct stages, each characterized by specific symptoms, namely primary, secondary, latent, and late (tertiary) syphilis. Serology has been considered the primary diagnostic approach. However, it is plagued by problems such as the limited specificity of nontreponemal tests and the inadequate correlation of treponemal tests with disease activity. In this study, we focused on the development of a loop-mediated isothermal amplification assay utilizing hydroxy naphthol blue (LAMP-HNB) for the diagnosis of T. pallidum subspp. pallidum. Specifically, this study seeks to determine the analytical sensitivity (limit of detection; LOD) and analytical specificity. Four hundred clinical serum samples were analyzed for diagnostic sensitivity, specificity, and predictive value, and each technique’s 95% confidence intervals (95% CI, p < 0.05) were evaluated. The limit of detection for polymerase chain reaction with agarose gel electrophoresis (PCR-AGE), the loop-mediated isothermal amplification assay combined with agarose gel electrophoresis (LAMP-AGE), and LAMP-HNB were 116 pg/µL, 11.6 pg/µL, and 11.6 pg/ µL, respectively. Analytical specificity examinations indicated the absence of cross-reactivity with Leptospira interrogans, Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, human immunodeficiency virus (HIV), and healthy human serum in PCR-AGE, LAMP-AGE, and LAMP-HNB. The diagnostic sensitivity, diagnostic specificity, positive predictive value (PPV), and negative predictive value (NPV) for PCR-AGE were 100.00 (100.00)%, 94.50 (94.40–94.60)%, 94.79 (94.69–94.88)%, and 100.00 (100.00)%, respectively. While, for LAMP-AGE and LAMP-HNB, they were 100.00 (100.00)%, 91.00 (90.87–91.13)%, 91.74 (91.63–91.86)%, and 100.00 (100.00)%, respectively. The LAMP-HNB test is simple, rapid, highly sensitive, and highly specific, without requiring expensive equipment. In the future, the LAMP-HNB assay may develop into a single-step diagnostic process, enabling the use as point-of-care testing for the diagnosis, prevention, and management of syphilis infection.