Characterization of Salmonella Serovars by PCR-Single-Strand Conformation Polymorphism Analysis

Abstract

PCR-restriction fragment length polymorphism (PCR-RFLP) and PCR-single-strand conformation polymorphism (PCR-SSCP) analyses were carried out on the 1.6-kb groEL gene from 41 strains of 10 different Salmonella serovars. Three HaeIII RFLP profiles were recognized, but no discrimination between the serovars could be achieved by this technique. However, PCR-SSCP analysis of the groEL genes of various Salmonella serovars produced 14 SSCP profiles, indicating the potential of this technique to differentiate different Salmonella serovars (interserovar differentiation). Moreover, PCR-SSCP could differentiate strains within a subset of serovars (intraserovar discrimination), as three SSCP profiles were produced for the 11 Salmonella enterica serovar Enteritidis strains, and two SSCP profiles were generated for the 7 S. enterica serovar Infantis and five S. enterica serovar Newport strains. PCR-SSCP has the potential to complement classical typing methods such as serotyping and phage typing for the typing of Salmonella serovars due to its rapidity, simplicity, and typeability.Salmonellae are the etiologic agents of different diseases collectively referred to as salmonellosis. Human salmonellosis can be divided into four syndromes: enteric fever (typhoid-like disease), gastroenteritis (food poisoning), bacteremia with or without gastroenteritis, and the asymptomatic carrier state.On a global scale, the incidence of typhoid fever is decreasing, while that of nontyphoidal salmonellosis is increasing, although both remain major health problems. The World Health Organization has estimated that annually there are close to 17 million cases of typhoid fever, with nearly 600,000 deaths, and 1.3 billion cases of acute gastroenteritis or diarrhea due to nontyphoidal salmonellosis, with 3 million deaths (8,23,26).To curb both typhoidal and nontyphoidal salmonellae, laboratory-based surveillance of human and animal infections is a necessary first step of any prevention strategy. Phenotypic methods play an important role in the identification to the genus level. Serotyping is used for primary typing of strains, while phage typing and antibiogram are used for subdivision of serotypes (33). However, serotyping of Salmonella is laborious due to the large number of recognized serotypes, i.e., over 2, 400 (1, 27).In addition, a number of molecular typing methods have also been used to try to improve the identification of salmonellae and also to differentiate strains below the level of serotypes. These DNA-related techniques include ribotyping (3), pulsedfield gel electrophoresis (PFGE) (18,20,32), IS200 fingerprinting (4), PCR-ribotyping (12), ribosomal DNA intergenic spacer amplification and heteroduplex analysis (9), amplified fragment length polymorphism (1, 21), automated 5Ј nuclease PCR assay (7), and random amplified polymorphic DNA analysis (30).In recent times, various molecular techniques that detect base sequence changes in bacteria have been used as tools in epidemiological typing. One of the most widely used techniques for the i...

PCR-RFLP Analysis of a fliC Gene Fragment in Avian Salmonella Isolates

“…This shows that this method with the mentioned enzyme does not have the potential be applied for identification of our isolates. Nair et al using PCR-RFLP on the groEL gene amplicon of Salmonella digested with HaeIII, found a low discriminatory capacity, as only three different profiles were obtained, and different serotypes of Salmonella shared the same restriction profile (17). In our study we had eight different serotypes with four restriction profiles, which showed that our study is in agreement with their study.…”

Multiplex PCR-Based Method for Identification of Common Clinical Serotypes ofSalmonella entericasubsp.enterica

“…These include ribotyping (12), ribosomal DNA intergenic spacer amplification (22), random amplification of DNA polymorphism (35), IS200 analysis (13,38), real-time PCR (21), PCR-single-strand conformation polymorphism analysis (27), amplified fragment length polymorphism (37), sequence analysis (26), multiplex PCR (2), and DNA microarrays (8,31). Other laboratories have taken protein-based approaches to type Salmonella enterica by methods including protein arrays (7) and mass spectrometry (40).…”

Molecular Diagnosis of Enteric Fever: Progress and Perspectives

“…The widely researched targets for S. Typhi PCR-based assays include the S. Typhi flagellin gene fliC-d (Song et al, 1993;Hague et al, 1999Hague et al, , 2001Kumar et al, 2002;Prakash et al, 2005;Ambati et al, 2007;Hatta & Smits 2007;Nandagopal et al, 2010;Nath et al, 2010), the viaB region encoding the Vi antigen of S. Typhi (Hashimoto et al, 1995), the Salmonella invasion gene invA (Cocolin et al, 1998), hilA gene encoding a transcription factor of S. Typhi (Sánchez-Jiménez & Cardona-Castro, 2004), Vi polysaccharide export ATP-binding protein vexC gene (Farrell et al, 2005), ST5 gene (Aziah et al, 2007), an iron-regulated gene iroB (Bäumler et al, 1997), 5S-23S spacer region (Zhu et al, 1996), and a heat shock protein groEL gene (Nair et al, 2002).…”

“…Cocolin et al developed a PCR-microtitre plate hybridization technique for detection of S. Typhi invA by PCR, and demonstrated enhanced sensitivity and faster availability of results in comparison to a standard agarose gel electrophoresis approach (Cocolin et al, 1998). Other PCR assays were also researched on different gene targets in order to find a rapid and sensitive detection of S. Typhi in clinical specimens (Zhu et al, 1996;Bäumler et al, 1997;Nair et al, 2002;Sánchez-Jiménez & Cardona-Castro, 2004;Farrell et al, 2005;Nizami et al, 2006;Aziah et al, 2007 ).…”