# Establishment and application of a dual RPA-LFD rapid detection method for Salmonella Pullorum and Salmonella Enteritidis

## TL;DR

This study developed a fast and accurate method to detect two types of Salmonella in poultry using a combination of RPA and LFD technology.

## Contribution

A dual RPA-LFD method for simultaneous detection of S. Pullorum and S. Enteritidis is established and validated.

## Key findings

- The dual RPA-LFD method detected S. Pullorum and S. Enteritidis within 20 minutes with high sensitivity.
- The method showed no cross-reactivity with other common pathogens and matched traditional BAM results.
- Detection limits were 1.56 × 102 CFU/mL for S. Pullorum and 1.38 × 102 CFU/mL for S. Enteritidis.

## Abstract

Salmonella species are known to cause a significant decline in poultry production performance and to contaminate various stages of the breeding process, with Salmonella Pullorum (S. Pullorum) and Salmonella Enteritidis (S. Enteritidis) being the predominant serotypes responsible for infection in poultry. For rapid diagnosis at an early stage, we developed a method involving dual recombinase polymerase amplification (RPA) combined with a lateral flow dipstick (LFD) in this study, and primers were designed to target the traJ gene of S. Pullorum and the Sdf Ⅰ gene of S. Enteritidis. The primers and probes were screened, and the reaction conditions were optimized. The results showed that dual RPA successfully amplified S. Pullorum and S. Enteritidis DNA within 15 min at 37°C, and when combined with LFD, the entire process (amplification and detection) was completed within 20 min. The detection limits for S. Pullorum and S. Enteritidis were 1.56 × 102 CFU/mL and 1.38 × 102 CFU/mL, respectively. The developed dual RPA-LFD method specifically targets S. Pullorum and S. Enteritidis and exhibits no cross-reactivity with other common pathogenic microorganisms. The results for the clinical samples were fully consistent with those obtained using the Bacteriological Analytical Manual (BAM) method. The results of this study demonstrated that the developed dual RPA-LFD method is simple, rapid, specific, and highly sensitive for the simultaneous visual detection of S. Pullorum and S. Enteritidis, providing a technical reference for primary veterinary laboratories and veterinary field tests.