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 <i>Salmonella</i> culture as reference methods for detecting <i>Salmonella</i> contamination in animal products and animal production environmental samples.Methods: The SalInvA01 primer, derived from the <i>InvA</i> 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: <i>Salmonella</i> 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, <i>Salmonella</i> 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, <i>Salmonella</i> 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 <i>Salmonella</i> detection, which used <i>InvA</i> gene, was highly specific, sensitive, and convenient for identifying <i>Salmonella</i>. Furthermore, this method could be used for <i>Salmonella</i> monitoring and primary screening in animal products and animal production environmental samples.