Development of a PCR assay for the identification of Salmonella enterica serovar Brandenburg

Abstract

Currently, Salmonella enterica serovar Brandenburg is identified serologically on the basis of two surface antigens, somatic (O) polysaccharide and flagellar (H) proteins. This procedure is time-consuming and requires expensive typing reagents. To overcome these problems, a PCR method was developed and validated for the identification of S. Brandenburg. Portions of the invA, rfbJ(B), fliC and fljB genes were targeted for amplification using four pairs of oligonucleotide primers. To validate the assay, genomic DNA from an array of 72 Salmonella strains representing 28 serotypes and 5 non-Salmonella strains from 4 different genera was subjected to PCR. The four targeted genes were correctly amplified only from S. Brandenburg. These results indicate that this PCR assay is a simple, rapid, reliable and reproducible method for the identification of S. Brandenburg that will aid in surveillance, prevention and control of this pathogen. INTRODUCTIONSince 1996, Salmonella enterica serovar Brandenburg has been a major cause of ovine abortions and mortality, leading to significant economic impact to farmers in New Zealand (Clark, 2000;Clark et al., 2004;Roe, 1999;Smart, 2000). Sheep that have recovered from clinical disease may become carriers and excrete organisms in faeces. S. Brandenburg has also been isolated from humans and other animals, including horses, cattle, goats, deer, pigs and dogs. In New Zealand, human cases occurred through contact with infected animals and not through consumption of animal products (Clark et al., 2004). Therefore, rapid detection of S. Brandenburg would aid in controlling the spread of this pathogen to both animals and humans.The number of Salmonella serotypes reported by 2002 was 2541 (CDC, 2007). The somatic O-antigen, together with phase-1 (H1) and phase-2 (H2) flagellar antigens, forms the basis for Salmonella serotyping. Each Salmonella serotype has a unique combination of O, H1 and H2 antigens. Laboratory methods for the identification of S. Brandenburg include serotyping, which is performed by the identification of O, H1 and H2 antigens according to the Kauffmann-White scheme (Popoff & Le Minor, 2001). This procedure is time-consuming, laborious and costly. Rapid and inexpensive PCR assays for the detection of Salmonella at the genus level have been developed (Rahn et al., 1992), but the number of assays to determine the Salmonella serotype is limited and includes those to detect serotypes Enteriditis (Lampel et al., 1996), Gallinarum (Shah et al., 2005), Typhi (Farrell et al., 2005;Kumar et al., 2006) and Typhimurium (Leon-Velarde et al., 2004). To date, there is no serotype-specific PCR assay for the detection of S. Brandenburg.Somatic O-antigens are polymers of the O-subunit, and the rfb gene clusters are responsible for much of its variation. Variability of O-antigens among serogroups is due to differences in the type and arrangement of sugars in the Osubunit (Fitzgerald et al., 2003; Gillespie et al., 2003). The filament of flagella is a polymer of flagellin monomers that are ...

Detection of invA gene of Salmonella from milkfish (Chanos chanos) at Sidoarjo wet fish market, Indonesia, using polymerase chain reaction technique

“…Typhimurium gene as previously reported by Darwin and Miller [23]. This is also consistent with research conducted by Pererat and Murray [24] that the results of PCR on various serotypes of Salmonella showed positive results, while PCR results on non- Salmonella strains such as E. coli , Klebsiella, Proteus , and Shigella were negative for the invA gene.…”

“…This gene is located in pathogenicity island I or referred to as Salmonella Pathogenicity Island (SPI); the DNA region is related to the pathogenicity of Salmonella enterica and is owned by all serotypes [26]. SPI serves to add complex virulence by bacteria to the infected host [24].…”

Development of a novel multiplex PCR assay for the identification of Salmonella enterica Typhimurium and Enteritidis

“…Polymerase chain reaction (PCR) methods have shown promise in microbial diagnostics over the past decades because of their rapidity and higher accuracy compared to traditional antiserabased serotyping (Kardos, Farkas, Antal, Nogrady, & Kiss, 2007;Kim et al, 2009;Octavia & Lan, 2010;Perera & Murray, 2008;Trafny, Kozlowska, & Szpakowska, 2006). The detection specificity of PCR methods depends on the specific binding of the primer set to the target sequences in the organisms.…”

One-Step Triplex High-Resolution Melting Analysis for Rapid Identification and Simultaneous Subtyping of Frequently Isolated Salmonella Serovars

“…PFGE and MLST are still expensive and time-consuming methods and are therefore of limited value for routine subtyping (14,24). Most PCR-based subtyping procedures allow the detection of only a single serotype (17,20), and even in multiplex-PCR-based approaches, the number of identifiable serotypes is low and requires a multistep protocol (18). Advances in whole-genome sequencing will further facilitate the identification of suitable markers and therefore improve molecular subtyping procedures (3).…”