Establishment and validation of a loop-mediated isothermal amplification (LAMP) assay targeting the ttrRSBCA locus for rapid detection of Salmonella spp. in food

Kreitlow, Antonia; Becker, André; Schotte, Ulrich; Malorny, Burkhard; Plötz, Madeleine; Abdulmawjood, Amir

doi:10.1016/j.foodcont.2021.107973

Cited by 12 publications

(3 citation statements)

References 39 publications

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“…Even though this may seem trivial, the success of any given method relying on this technique is highly dependent on the appropriate selection of reagents. For other techniques, extensive benchmarking of commercial master mixes [ 15 , 16 ], nucleic acid extraction kits [ 17 , 18 ], genetic targets [ 19 , 20 ], and amplification and detection platforms [ 13 , 21 ] has been performed, and they have even been compared to other existing techniques and methods [ 22 , 23 , 24 ].…”

Section: Introductionmentioning

confidence: 99%

Evaluating Commercial Loop-Mediated Isothermal Amplification Master Mixes for Enhanced Detection of Foodborne Pathogens

Costa-Ribeiro,

Lamas,

Garrido-Maestu

2024

Foods

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Loop-mediated isothermal amplification, LAMP, is nowadays the most popular isothermal nucleic acid amplification technique, and as such, several commercial, ready-to-use master mixes have flourished. Unfortunately, independent studies to determine their performance are limited. The current study performed an independent evaluation of the existing ready-to-use commercial LAMP master mixes WarmStart® LAMP Kit, LavaLAMP™ DNA Master Mix, Saphir Bst Turbo GreenMaster, OptiGene Fast Master Mix ISO-004, and SynLAMP Mix. To reduce bias, three different genes, namely ttr (Salmonella spp.), rfbE (E. coli O157), and hly (Listeria monocytogenes), were targeted. The comparison was based on amplification speed, performance with decreasing DNA concentrations, and the effect of five typical LAMP reaction additives (betaine, DMSO, pullulan, TMAC, and GuHCl). Significant differences were observed among the different master mixes. OptiGene provided the fastest amplification and showed less detrimental effects associated with the supplements evaluated. Out of the chemicals tested, pullulan provided the best results in terms of amplification speed. It is noteworthy that the different additives impacted the master mixes differently. Overall, the current study provides insights into the performance of commercial LAMP master mixes, which can be of value for the scientific community to better select appropriate reagents when developing new methods.

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Section: Introductionmentioning

confidence: 99%

Evaluating Commercial Loop-Mediated Isothermal Amplification Master Mixes for Enhanced Detection of Foodborne Pathogens

Costa-Ribeiro,

Lamas,

Garrido-Maestu

2024

Foods

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“…For the subsequent detection of LAMP products, various methods are available, which are often based on a visible color change or a generated turbidity 10 . In addition to low-cost options for performing this method, which can be of great benefit in limited laboratory environments, there are also portable real-time fluorometers or turbidimeters available on the market 11 , 12 . These offer additional features like real-time monitoring of the LAMP reaction or melting curve generation for evaluating the specificity of a LAMP product.…”

Section: Introductionmentioning

confidence: 99%

Identification of the novel potential pathogen Trueperella pecoris with interspecies significance by LAMP diagnostics

Kreitlow,

Ningrum,

Lämmler

et al. 2023

Sci Rep

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Trueperella pecoris was described as a new species of the genus Trueperella in 2021 and might be pathogenic to various animal species. However, the lack of a suitable diagnostic test system stands in the way of epidemiological surveys to clarify possible causalities. In this study, a Loop-mediated Isothermal Amplification (LAMP) assay was developed and validated that was highly specific for T. pecoris. The assay provided an analytical sensitivity of 0.5 pg/25 µL and showed 100% inclusivity and exclusivity for 11 target and 33 non-target strains, respectively. Three different DNA extraction methods were evaluated to select the most LAMP-compatible method for cell disruption in pure and complex samples. Using an on-site applicable single-buffer DNA extraction with additional heating, the cell-based detection limit was 2.3 CFU/reaction. Finally, the LAMP assay was validated by means of artificially contaminated porcine lung tissue samples in which minimal microbial loads between 6.54 and 8.37 × 103 CFU per swab sample were detectable. The LAMP assay established in this study represents a suitable diagnostic procedure for identifying T. pecoris in clinical specimens and will help to collect epidemiological data on the pathogenicity of this species.

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“…DNA amplification can be performed using a heating block and monitored with the naked eye, e.g., by turbidity or fluorescence observation ( Mori and Notomi, 2009 ; Mori et al, 2013 ). Additionally, it was shown that the LAMP technique is robust against potential reaction inhibitors and copes with simplified DNA extraction procedures ( Kaneko et al, 2007 ; Kreitlow et al, 2021 ). These characteristics make the LAMP method suitable for low-cost and rapid detection of pathogens with options for on-site application or implementation under restricted laboratory conditions often found in developing countries.…”

Section: Introductionmentioning

confidence: 99%

Combined Loop-Mediated Isothermal Amplification Assays for Rapid Detection and One-Step Differentiation of Campylobacter jejuni and Campylobacter coli in Meat Products

et al. 2021

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A loop-mediated isothermal amplification (LAMP) assay system was established, allowing rplD gene-based simultaneous detection of Campylobacter jejuni and Campylobacter coli in enriched meat products. Additionally, one-step differentiation of target species on agar plates was enabled by cdtC gene- and gyrA gene-based duplex LAMP. Both the rplD and cdtC–gyrA LAMP assays amplified the target sequences in all 62 C. jejuni and 27 C. coli strains used for determining inclusivity and revealed 100% exclusivity toward 85 tested non-target species. Throughout the entire experiments, C. jejuni and C. coli strains were 100% distinguishable by melting curves of cdtC and gyrA LAMP products. After 24-h enrichment, the rplD LAMP assay reliably detected initial inoculation levels of 10–100 CFU/g in artificially contaminated minced meat. Investigation of naturally contaminated meat samples revealed a diagnostic accuracy of 95% toward real-time PCR and 94.1% toward the standard culture method applying the 24-h incubation period. Diagnostic sensitivity and specificity, and positive and negative predictive values were 89.8, 100, 100, and 91.2%, respectively, when measured against real-time PCR, and 89.6, 98.1, 97.7, and 91.2%, respectively, when measured against the standard culture method. After 48-h enrichment, the detection limit of the rplD LAMP assay improved to initial inoculation levels of 1–10 CFU/g in artificially contaminated minced meat. Applying the 48-h incubation period on naturally contaminated meat samples resulted in 100% concordant results between rplD LAMP, real-time PCR, and the standard culture method. The established LAMP assay system was proved to be suitable for rapid meat sample screening. Furthermore, it constitutes a promising tool for investigating other Campylobacter sources and could therefore make a valuable contribution to protect consumers from foodborne illness.

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Establishment and validation of a loop-mediated isothermal amplification (LAMP) assay targeting the ttrRSBCA locus for rapid detection of Salmonella spp. in food

Cited by 12 publications

References 39 publications

Evaluating Commercial Loop-Mediated Isothermal Amplification Master Mixes for Enhanced Detection of Foodborne Pathogens

Evaluating Commercial Loop-Mediated Isothermal Amplification Master Mixes for Enhanced Detection of Foodborne Pathogens

Identification of the novel potential pathogen Trueperella pecoris with interspecies significance by LAMP diagnostics

Combined Loop-Mediated Isothermal Amplification Assays for Rapid Detection and One-Step Differentiation of Campylobacter jejuni and Campylobacter coli in Meat Products

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