Rapid Detection and Differentiating of the Predominant Salmonella Serovars in Chicken Farm by TaqMan Multiplex Real-Time PCR Assay

Abstract

Salmonella has been known as an important zoonotic pathogen that can cause a variety of diseases in both animals and humans. Poultry are the main reservoir for the Salmonella serovars Salmonella Pullorum (S. Pullorum), Salmonella Gallinarum (S. Gallinarum), Salmonella Enteritidis (S. Enteritidis), and Salmonella Typhimurium (S. Typhimurium). The conventional serotyping methods for differentiating Salmonella serovars are complicated, time-consuming, laborious, and expensive; therefore, rapid and accurate molecular diagnostic methods are needed for effective detection and prevention of contamination. This study developed and evaluated a TaqMan multiplex real-time PCR assay for simultaneous detection and differentiation of the S. Pullorum, S. Gallinarum, S. Enteritidis, and S. Typhimurium. In results, the optimized multiplex real-time PCR assay was highly specific and reliable for all four target genes. The analytical sensitivity corresponded to three colony-forming units (CFUs) for these four Salmonella serovars, respectively. The detection limit for the multiplex real-time PCR assay in artificially contaminated samples was 500 CFU/g without enrichment, while 10 CFU/g after pre-enrichment. Moreover, the multiplex real-time PCR was applied to the poultry clinical samples, which achieved comparable results to the traditional bacteriological examination. Taken together, these results indicated that the optimized TaqMan multiplex real-time PCR assay will be a promising tool for clinical diagnostics and epidemiologic study of Salmonella in chicken farm and poultry products.

Microbial profile of broiler carcasses processed at a university scale mobile poultry processing unit

“…The presence and absence of Salmonella on broiler carcasses after scalding, evisceration, and final chilling steps were tested using PCR for the ompC gene. This method has been used to detect Salmonella on poultry carcasses in numerous studies ( de Freitas et al, 2010 ; Vichaibun and Kanchanaphum, 2020 ; Xin et al, 2021 ). The PCR test results are shown in Table 3 .…”

A Real-Time PCR Approach for Rapid Detection of Viable Salmonella Enteritidis in Shell Eggs

“…Molecular tests to serotype Salmonella, such as whole genome sequencing and microarrays, are alternatives which circumvent the lengthy testing turnaround time and reduce uncertainty [7,[34][35][36]. WGS can enhance discriminatory data for Salmonella isolates, which can be used to accurately determine Salmonella serotypes [37,38].…”

Genome-Wide Searching Single Nucleotide-Polymorphisms (SNPs) and SNPs-Targeting a Multiplex Primer for Identification of Common Salmonella Serotypes

“…Molecular investigation and typing of Salmonella , the SNP-based typing techniques are essential for identifying the source of a foodborne outbreak. However, Salmonella serovars identification methods are based on mainly three basic mechanisms, including restriction fragment analysis of Salmonella DNA, PCR amplification of target genes, and SNP-based identification at specific loci in the whole genome sequence [ 6 , 20 , 28 ]. To date, several molecular approaches have been applied to detect Salmonellae , such as PFGE [ 39 ], phage typing [ 40 ], and multilocus sequence typing (MLST) [ 41 ], but they have some limitations.…”

“…The current research aims to identify novel sensitive, and reliable serovar-specific targets and develop an m-PCR method for Salmonella serovars to facilitate timely prevention and treatment. Several developed detection methods are conducted for Salmonella serovars Enteritidis [ 28 ], and Typhimurium [ 29 ], since they top the list of the most prevalent serotypes [ 30 , 31 , 32 ]. S .…”