Rapid Colorimetric Assay for Detection of Listeria monocytogenes in Food Samples Using LAMP Formation of DNA Concatemers and Gold Nanoparticle-DNA Probe Complex

Abstract

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.

Optimization of isothermal amplification method for Mycobacteriumtuberculosisdetection and visualization method for fieldwork

“…In this study, gel electrophoresis, lateral flow test strips, and Warmstart colorimetric LAMP 2X master mix kit were used. Methods based on precipitation detection and colorimetric methods using pH-sensitive dye are widely used to visualize products in the LAMP method [37]. As the amplicon is formed in the LAMP studies by 100 times more than RT-PCR and conventional PCR, the resulting precipitate and reaction can be detected by the naked eye with the changing pH [37].…”

“…Methods based on precipitation detection and colorimetric methods using pH-sensitive dye are widely used to visualize products in the LAMP method [37]. As the amplicon is formed in the LAMP studies by 100 times more than RT-PCR and conventional PCR, the resulting precipitate and reaction can be detected by the naked eye with the changing pH [37]. Due to this advantage of the method, complex devices and laboratory dependence are eliminated.…”

Detection of Streptococcus pneumoniae in Urine by Loop-Mediated Isothermal Amplification

“…13 Finally, the direct visualization of the results (amplicons) is possible based on the presence of turbidity, or by fluorescence or colorimetric detection. [13][14][15][16][17] DNA-based detection methods used for S. pneumonia commonly utilize blood pleural fluid and nasopharyngeal aspirates, induced sputum, and nonbronchoscopic bron-choalveolar lavage fluid samples. 4,18 Compared with these other types of biofluids, urine offers several advantages for children, including its convenience of sampling and its ease of use for handling and storing.…”

Combination of Loop-Mediated Isothermal Amplification and AuNP-Oligoprobe Colourimetric Assay for Pork Authentication in Processed Meat Products

“…While the LAMP method generates DNA amplicons, its combination with the AuNP colourimetry can enable a clear and sensitive detection of DNA or RNA of interest. To this end, the LAMP-gold nanoprobe methods have been applied successfully for a wide range of fields including diagnosis of diseases (Lou et al 2020;Najian et al 2016;Qiuhua et al 2014), microbial drug resistance (Ckumdee et al 2016;Veigas et al 2013), forensic sciences (Watthanapanpituck et al 2014), and pathogen detection in foods (Garrido-Maestu et al 2017;Kong et al 2018;Seetang-Nun et al 2013;Suebsing et al 2013;Wachiralurpan et al 2018).…”