A Colorimetric LAMP Assay for Salmonella spp. Detection: Towards a DNA Extraction-Free Approach for Pathogen Screening

Abstract

As of today, bacteriological identification and the molecular approach PCR are considered the gold standards for Salmonella spp. detection. However, these methods are time-consuming and costly due to the requirements for enrichment and nucleic acid extraction. In this study, we evaluated the reliability of a developed colorimetric loop-mediated isothermal amplification (cLAMP) assay targeting the hilA gene, using Phenol Red as an amplification indicator. Given that Phenol Red is pH-dependent, and to develop an extraction-free test, we evaluated chicken meat pretreatment and thermal treatment. First, we assessed the reliability of this test using a pure culture of Salmonella spp. and then in 50 chicken samples pretreated with optimal NaOH concentrations under standardized conditions. Samples representing extreme pH values were artificially contaminated and subjected to DNA extraction and a heat-treatment protocol. Serial dilutions of these products served as templates for LAMP reactions. The assay sensitivity was estimated to be around 3.9 CFU/µL of pure bacterial culture. In contrast, in biological samples, we detected up to 10 CFU/µL using DNA extraction, while heat treatment successfully amplified the initial solution and even some dilutions up to 10³ CFU/µL. In conclusion, our cLAMP assay demonstrated good sensitivity and provided clear evidence of its potential for in-field use without relying on prior enrichment steps and DNA extraction.

Keywords: LAMP colorimetric; Salmonella; extraction free; foodborne disease; pretreatment of sample; rapid detection.

Figure 1

Outline of the experimental design.

Figure 2

The protocol for pretreatment of chicken samples.

Figure 3

Box plot displaying the distribution of pretreated chicken samples’ pH.

Figure 4

(A) Lamp sensitivity test using hilA dilution of pure bacterial culture; (B) confirmation of amplification of the sensitivity cLAMP reactions. L: ladder 100 bp, 1: void, 2: negative control, 3: 0.39 CFU/µL, 4: 3.9 CFU/µL, 5: 3.9 × 10 CFU/µL, 6: 3.9 × 10² CFU/µL, 7: 3.9 × 10³ CFU/µL, and 8: 3.9 × 10⁴ CFU/µL, 9–11: Void.

Figure 5

Amplification plot of extracted DNA from serial dilution of Salmonella solutions.

Figure 6

Specificity results of the cLAMP method for Salmonella detection. 1: S. Enteritidis, 2: S. Typhimurium, 3: S. Kentucky, 4: S. Gallinarum Pullorum, 5–6: positive controls, 7–8: negative controls, 9: E. coli, 10: Proteus mirabillis, 11: Klebsiella spp., 12: Listeria monocytogenes, 13: Pasteurella spp., 14: Bordetella spp., 15: Staphylococcus spp., and 16: Clostridium perfringens.

Figure 7

Detection of Salmonella in artificially contaminated chicken samples in cLAMP with extracted DNA as template. The number of CFU per µL in each reaction tube, NC : Negative Control, A1: 10⁵ CFU/µL, A2: 10⁴ CFU/µL, A3: 10³ CFU/µL, A4: 10² CFU/µL, A5: 10 CFU/µL, A6: 1 CFU/µL; B1: 10⁵ CFU/µL, B2: 10⁴ CFU/µL, B3: 10³ CFU/µL, B4: 10² CFU/µL, B5: 10 CFU/µL, and B6: 1 CFU/µL.

Figure 8

Detection of Salmonella in artificially contaminated chicken samples in cLAMP with heat-treatment product as template. The number of CFU per µL in each reaction tube, NC: Negative Control, A’1: 10⁵ CFU/µL, A’2: 10⁴ CFU/µL, A’3: 10³ CFU/µL, A’4: 10² CFU/µL, A’5: 10 CFU/µL, A’6: 1 CFU/µL; B’1: 10⁵ CFU/µL, B’2: 10⁴ CFU/µL, B’3: 10³ CFU/µL, B’4: 10² CFU/µL, B’5: 10 CFU/µL, and B’6: 1 CFU/µL.

Figure 9

Amplification confirmation by gel electrophoresis for Sample A. L: ladder 100 bp. 2–8: reactions with extracted DNA as template; 10–15 reactions with heat-treatment products as templates; 1: void, 2: NC, 3: A6, 4: A5, 5: A4, 6: A3, 7: A2, 8: A1, 9: void, 10: A’6, 11: A’5, 12: A’4, 13: A’3, 14: A’2, and 15: A’1.

Figure 10

Amplification confirmation by gel electrophoresis for Sample B. L: ladder 100 bp. 2–8: reactions with extracted DNA as template; 10–15 reactions with heat-treatment products as templates; 1: void, 2: NC, 3: B6, 4: B5, 5: B4, 6: B3, 7: B2, 8: B1, 9: void, 10: B’6, 11: B’5, 12: B’4, 13: B’3, 14: B’2, and 15: B’1.