Integrating loop-mediated isothermal amplification with lateral flow assay to achieve a highly sensitive method for detecting Streptococcus suis Genome in raw pork

Abstract

Streptococcus suis (S.suis), a zoonotic foodborne pathogen prevalent in Southeast Asia, poses a substantial threat to human and animal health because of its ability to cause severe and life-threatening illnesses. To address this challenge, a rapid and highly sensitive detection platform for S. suis in raw pork was developed by integrating loop-mediated isothermal amplification (LAMP) and a lateral flow assay (LFA), S. suis LAMP-LFA. LAMP reactions targeting the S. suis glutamate dehydrogenase (gdh) gene were optimized for specific detection of S. suis within 45 min at an isothermal temperature of 65 °C. The assay exhibited marked sensitivity, with a detection limit of 100 fg for genomic DNA extracted from S. suis cultures. Notably, this method showed no cross-reactivity with other bacterial contaminants commonly found in raw pork. The resulting LAMP amplicons were effectively detected using LFA, with a test limit of 10¹ CFU per 25 g of raw pork. S. suis LAMP-LFA proved to be highly specific and reliable, with no false-positives detected in spiked pork samples or pork samples containing other bacterial contaminants. Due to its high sensitivity, specificity, and rapid turnaround time, the proposed technique has immense potential as a field-deployable screening test for S. suis detection in raw pork, contributing to enhanced food safety and public health protection.

Keywords: Glutamate dehydrogenase (gdh) gene; Lateral flow assays; Loop-mediated isothermal amplification; Raw pork; Streptococcus suis.

Fig. 1

The schematic diagram of S. suis LAMP-LFA. LAMP, loop-mediated isothermal amplification; LFA, lateral flow assay; FITC, fluorescein isothiocyanate; CGC, colloidal gold conjugate; Ig, immunoglobulin.

Fig. 2

The optimum temperature of LAMP reaction. Four different temperatures including 55 (A), 60 (B), 65 (C) and 70 (D) °C at 1 h of incubation time. Two amount of DNA template: 40 ng and 40 pg. M: 1 kb marker, Neg: sterile distilled water (negative control). LAMP, loop-mediated isothermal amplification. The full and non-adjusted images are provided in Supplementary Figs. 1–4.

Fig. 3

The optimum incubation time of LAMP reaction. Four different reaction times including 30 (A), 45 (B), 60 (C) and 70 (D) min at 65 °C. Sample amount: 40 ng and 40 pg. M: 1 kb marker. Neg: sterile distilled water (negative control). LAMP, loop-mediated isothermal amplification. The full and non-adjusted images are provided in Supplementary Figs. 5–7.

Fig. 4

The specificity of LAMP reaction. 40 ng of DNA from different bacterial strains including S. suis serotype 1 and 2, E. coli, E. faecalis, S. aureus, and S. enteritidis. M: 1 kb marker. Neg: sterile distilled water (negative control). LAMP, loop-mediated isothermal amplification. The full and non-adjusted images are provided in Supplementary Fig. 8.

Fig. 5

The sensitivity of the LAMP reaction. S. suis serotype 1 DNA were amplified by LAMP reaction at 65 °C for 45 min (A) compared with the standard PCR (B). The gel electrophoresis images of LAMP-amplified S. suis serotype 2 DNA samples (C) compared with the standard PCR (D). M: 1 kb marker. Neg: sterile distilled water (negative control). LAMP, loop-mediated isothermal amplification. The full and non-adjusted images are provided in Supplementary Figs. 9–12.

Fig. 6

The binding activity of anti-FITC-CGC and specificity. Dot blot reaction for testing the binding activity (A) and specificity with the LAMP product of other bacterial strains (B). LAMP, loop-mediated isothermal amplification.

Fig. 7

The sensitivity and specificity of S. suis LAMP-LFA. The positive reaction of S. suis LAMP-LFA for the detection of S. suis serotype 1 (A) and serotype 2 (B). The specificity of S. suis LAMP-LFA with other bacterial strains (C). LAMP, loop-mediated isothermal amplification; LFA, lateral flow assay.

Fig. 8

Detection of S. suis serotype 1 in spiked raw pork samples. The gel electrophoresis images of S. suis serotype 1 amplified by the LAMP method (A), S. suis LAMP-LFA (B), and conventional PCR reaction (C). Samples: raw pork samples spiked with S. suis from 10° to 10⁸ CFU/25 g (before amplification), M: marker, Neg: raw pork without S. suis. LAMP, loop-mediated isothermal amplification; LFA, lateral flow assay. The full and non-adjusted images are provided in Supplementary Figs. 13–14.

Fig. 9

Detection of S. suis serotype 2 in spiked raw pork samples. The gel electrophoresis images of S. suis serotype 2 amplified by LAMP method (A), S. suis LAMP-LFA (B), and conventional PCR reaction (C). Samples: raw pork samples spiked with S. suis from 10° to 10⁸ CFU/25 g (pre-amplification), M: marker, Neg: raw pork without S. suis. LAMP, loop-mediated isothermal amplification; LFA, lateral flow assay. The full and non-adjusted images are provided in Supplementary Figs. 15–16.

Fig. 10

Detection of S. suis in actual raw pork samples. The gel electrophoresis image of LAMP products of S. suis serotype 1 and 2, and pork samples (A) and S. suis LAMP-LFA (B). M: DNA marker. LAMP, loop-mediated isothermal amplification; LFA, lateral flow assay. The full and non-adjusted images are provided in Supplementary Fig. 17.