Development of a lateral flow dipstick test for the detection of 4 strains of Salmonella spp. in animal products and animal production environmental samples based on loop-mediated isothermal amplification

Abstract

Objective: This study aimed to develop loop-mediated isothermal amplification (LAMP) combined with lateral flow dipstick (LFD) and compare it with LAMP-AGE, polymerase chain reaction (PCR), and standard Salmonella culture as reference methods for detecting Salmonella contamination in animal products and animal production environmental samples.

Methods: The SalInvA01 primer, derived from the InvA gene and designed as a new probe for LFD detection, was used in developing this study. Adjusting for optimal conditions by temperature, time, and reagent concentration includes evaluating the specificity and limit of detection. The sampling of 120 animal product samples and 350 animal production environmental samples was determined by LAMP-LFD, comparing LAMP-AGE, PCR, and the culture method.

Results: Salmonella was amplified using optimal conditions for the LAMP reaction and a DNA probe for LFD at 63°C for 60 minutes. The specificity test revealed no cross-reactivity with other microorganisms. The limit of detection of LAMP-LFD in pure culture was 3×102 CFU/mL (6 CFU/reaction) and 9.01 pg/μL in genomic DNA. The limit of detection of the LAMP-LFD using artificially inoculated in minced chicken samples with 5 hours of pre-enrichment was 3.4×104 CFU/mL (680 CFU/reaction). For 120 animal product samples, Salmonella was detected by the culture method, LAMP-LFD, LAMP-AGE, and PCR in 10/120 (8.3%). In three hundred fifty animal production environmental samples, Salmonella was detected in 91/350 (26%) by the culture method, equivalent to the detection rates of LAMP-LFD and LAMP-AGE, while PCR achieved 86/350 (24.6%). When comparing sensitivity, specificity, positive predictive value, and accuracy, LAMP-LFD showed the best results at 100%, 95.7%, 86.3%, and 96.6%, respectively. For Kappa index of LAMP-LFD, indicated nearly perfect agreement with culture method.

Conclusion: The LAMP-LFD Salmonella detection, which used InvA gene, was highly specific, sensitive, and convenient for identifying Salmonella. Furthermore, this method could be used for Salmonella monitoring and primary screening in animal products and animal production environmental samples.

Keywords: Animal Products; Detection; InvA Gene; Lateral Flow Dipstick (LFD); Loopmediated Isothermal Amplification; Salmonella spp.

Figure 1

Optimization of SalInvA01-DIG LAMP reaction temperature. The LAMP-LFD assay were optimized temperature and assessed based on 2% agarose gel electrophoresis (A) and lateral flow dipstick (B) negative control without DNA template (NC), control line (C), and test line (T). LAMP-LFD, loop-mediated isothermal amplification combined with lateral flow dipstick.

Figure 2

The specificity test of SalInvA01-DIG primer sets using LAMP-LFD, Lane NC represents negative control (without DNA template), control line (C) and test line (T). LAMP-LFD, loop-mediated isothermal amplification combined with lateral flow dipstick.

Figure 3

Limit of detection (LOD) for detection of Salmonella spp. in pure culture by using LAMP-AGE (A) and LAMP-LFD (B). Lane M represents 100 bp DNA ladder marker, Lane NC represents negative control (without DNA template). LAMP-LFD, loop-mediated isothermal amplification combined with lateral flow dipstick.

Figure 4

Limit of detection (LOD) for detection of purified DNA of Salmonella by using LAMP-AGE (A) and LAMP-LFD (B). Lane M represents 100 bp DNA ladder marker, Lane NC represents negative control (without DNA template). Lane M represents 100 bp DNA ladder marker, Lane NC represents negative control (without DNA template). LAMP-LFD, loop-mediated isothermal amplification combined with lateral flow dipstick.

Figure 5

Limit of detection (LOD) for detection of Salmonella in artificial contamination chicken without pre-enrichment by using LAMP-AGE (A) and LAMP-LFD (B). Lane M represents 100 bp DNA ladder marker, Lane NC represents negative control (without DNA template). LAMP-LFD, loop-mediated isothermal amplification combined with lateral flow dipstick.

Figure 6

Limit of detection (LOD) for detection of Salmonella in artificial contamination chicken with pre-enrichment by using LAMP-AGE (A) and LAMP-LFD (B). Lane M represents 100 bp DNA ladder marker, Lane NC represents negative control (without DNA template). LAMP-LFD, loop-mediated isothermal amplification combined with lateral flow dipstick.

Figure 7

The detection results of Salmonella in animal products using LAMP-AGE. (A) pork, (B) minced pork, (C) chicken meat, (D) minced chicken, (E) beef, (F) chicken ball, (G) pork ball, (H) beef ball, (I–G) chicken drumstick and (K–L) chicken fillet. Lane M represents 100 bp DNA ladder marker, Lane PC represents positive control Lane NC represents negative control (without DNA template).

Figure 8

The detection results of Salmonella in animal products using LAMP-LFD. (A) pork, (B) minced pork, (C) chicken meat, (D) minced chicken, (E) beef, (F) chicken ball, (G) pork ball, (H) beef ball, (I–G) chicken drumstick and (K–L) chicken fillet. PC represents positive control NC represents negative control (without DNA template). LAMP-LFD, loop-mediated isothermal amplification combined with lateral flow dipstick.

Figure 9

The detection results of Salmonella in animal products using PCR assay. (A) pork, (B) minced pork, (C) chicken meat, (D) minced chicken, (E) beef, (F) chicken ball, (G) pork ball, (H) beef ball, (I–G) chicken drumstick and (K–L) chicken fillet. Lane M represents 100 bp DNA ladder marker, Lane PC represents positive control Lane NC represents negative control (without DNA template). LAMP-LFD, loop-mediated isothermal amplification combined with lateral flow dipstick.