Rapid Detection of Salmonella Enterica Serovar Enteritidis from Eggs and Chicken Meat by Real‐Time Recombinase Polymerase Amplification in Comparison with the Two‐Step Real‐Time PCR

Abstract

Salmonella is the most common cause of foodborne outbreaks throughout the world and Salmonella enterica serovar Enteritidis is one of the major causes of salmonellosis. We describe a rapid, sensitive and inexpensive method for the detection of S.Enteritidis from eggs and chicken meat using the real-time recombinase polymerase amplification (Real-time RPA) and compared this assay with the two-step real-time polymerase chain reaction (PCR) (Real-time PCR). The detection sensitivity of the RPA and PCR assays was identical in pure culture, however, the reaction time was as short as 10 min compared with 40 min for the PCR assay. In food application, the detection sensitivity of the RPA assay was 10 cfu/g from eggs and 100 cfu/g from chicken samples without additional DNA purification steps before the amplification -therefore, the sensitivity of the RPA assay was 100 times greater from eggs and 10 times greater from chicken than the PCR assay. PRACTICAL APPLICATIONSNumerous epidemiological investigations attributed the source of sporadic Salmonella outbreaks as poultry and poultry-by-products including eggs. This is the first study in which a real-time RPA was developed to detect the Salmonella Enteritidis specifically. The whole procedure is fairly simple and does not require specific equipment, making it potentially applicable in the monitoring and detection of S. Enteritidis in eggs and poultry products.

Development of a multiplex real-time recombinase polymerase amplification (RPA) assay for rapid quantitative detection of Campylobacter coli and jejuni from eggs and chicken products

“…RPA operates on an enzyme-derived-pri mer binding process; the recombinase and crowding agents form a nucleoprotein complex wit h primers and recognize their complementary target sequence. The primers are then extended by Sau polymerase and the amplification is accomplished by the cyclic repetition of the proce ss at the isothermal condition (37 -42 °C) (Piepenburg, Williams, Stemple, & Armes, 2006;Euler et al, 2013;Kim & Lee, 2016). While the real-time PCR method required a 4-h runtim e for detecting Campylobacter spp.…”

“…The RPA assay has been applied to detect various viruses and a few pathogens (Boyle, et al, 2013;Aebischer, Wernike, Hoffmann, & Beer, 2014;Zaghloul & El-Shahat, 2014;Glais & Jacquot, 2015;Teoh et al, 2015;Xia et al, 2015), but there were few attempts to detect the pathogenic bacteria from foods using an RPA assay (Santiago-Felipe, Tortajada-Genaro, Morais, Puchades, & Maquieira, 2015;Kim & Lee, 2016). In addition, to the best of our knowledge, there are no published reports in which the Campylobacter spp.…”

“…have been detected in poultry products using the multiplex RPA assay. In a previous study, we successfully established the real-time RPA method for detecting and quantifying Salmonella Enteritidis (Kim & Lee, 2016). In this study, we have developed a rapid and specific multiplex real-time RPA assay to simultaneously detect C. coli and C. jejuni.…”

Recombinase Polymerase Amplification (RPA) Combined with Lateral Flow Immunoassay for Rapid Detection of Salmonella in Food

“…Combined with the cognition of recombinant technology, the researchers have paid more attention to developing point-of-care and rapid technology. As a new technology of nucleic acid isothermal amplification, recombinase polymerase amplification (RPA) has shown greater advantages in detection for its great sensitivity, low-cost, and high speed, and detection without complex instruments in resource-poor laboratories and the outdoors, which are typically lacking traditional molecular detection methods [13]. This technique mainly locates homologous sequences in double-stranded DNA by combining the recombinase with a protein-DNA complex, which is formed by binding of the primer.…”

Antimicrobial Resistance of Common Zoonotic Bacteria in the Food Chain: An Emerging Threat

“…Even though Salmonella was significantly less frequently detected in eggs and their products, they remain the most important source of outbreaks [14,20], most probably due to a large worldwide consumption coupled with low concentrations of Salmonella that cannot be detected [7]. Rapid methods with improved sensitivity are thus needed to address this shortfall, e.g., real-time recombinase polymerase amplification [21] or sequence-based methods.…”