Suitability of PCR Fingerprinting, Infrequent-Restriction-Site PCR, and Pulsed-Field Gel Electrophoresis, Combined with Computerized Gel Analysis, in Library Typing of Salmonella enterica Serovar Enteritidis

Abstract

Strains of Salmonella enterica (n ‫؍‬ 212) of different serovars and phage types were used to establish a library typing computerized system for serovar Enteritidis on the basis of PCR fingerprinting, infrequent-restrictionsite PCR (IRS-PCR), or pulsed-field gel electrophoresis (PFGE). The rate of PCR fingerprinting interassay and intercenter reproducibility was low and was only increased when DNA samples were extracted at the same time and amplified with the same reaction mixtures. Reproducibility of IRS-PCR technique reached 100%, but discrimination was low (D ‫؍‬ 0.52). The PFGE procedure showed an intercenter reproducibility value of 93.3%. The high reproducibility of PFGE combined with the previously determined high discrimination directed its use for library typing. The use of PFGE with enzymes XbaI, BlnI, and SpeI for library typing of serovar Enteritidis was assessed with GelCompar 4.0 software. Three computer libraries of PFGE DNA profiles were constructed, and their ability to recognize new DNA profiles was analyzed. The results obtained pointed out that the combination of PFGE with computerized analysis could be suitable in long-term epidemiological comparison and surveillance of Salmonella serovar Enteritidis, specially if the prevalence of genetic events that could be responsible for changes in PFGE profiles in this serovar was low.

Comparison of Multilocus Sequence Typing, Pulsed-Field Gel Electrophoresis, and Antimicrobial Susceptibility Typing for Characterization of Salmonella enterica Serotype Newport Isolates

“…Pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility typing (AST) are two commonly used methods for studying microbial epidemiology and trends in the antibiotic resistance of bacteria. PFGE is currently used by the CDC PulseNet surveillance program and is generally accepted as the "gold standard" for molecular typing of Salmonella (7,18,34,41,57). Due to its limitations, many studies have compared PFGE to other genetic typing methods in attempts to identify more powerful tools for epidemiological investigations and evolutionary analyses.…”

Use of Pulsed-Field Gel Electrophoresis of Conserved XbaI Fragments for Identification of Swine Salmonella Serotypes

“…Consequently, PFGE has become a very important tool in epidemiology (14). Garaizar et al (9) and Ridley et al (14) used PFGE and computerized gel analysis to identify strains within Salmonella enterica serovar Enteritidis. They concluded that with standardization, PFGE protocols and results were reproducible among different laboratories.…”

RAPD-PCR as a Molecular Discriminative Technique for Human Pathogenic Bacteria - A Review

“…Serotyping, based on the Kauffmann-White scheme, remains the standard for classification of Salmonella isolates in outbreak investigations but now has been supplemented by a range of molecular genotyping methods [1,2]. In recent years, molecular-based techniques, such as PulsedField Gel Electrophoresis (PFGE), Restriction Fragment Length Polymorphism (RFLP), Random Amplified Polymorphic DNA (RAPD), Automatic Ribo-typing, rep-PCR, Multi-Locus Sequence Typing (MLST) have been shown to be useful methods for discrimination among isolates of pathogens [3,4]. Molecular-typing methods including rep-PCR, Multi-Locus Sequence Typing (MLST) have been shown to be useful methods for discrimination among isolates of pathogens [3,4].…”

“…In recent years, molecular-based techniques, such as PulsedField Gel Electrophoresis (PFGE), Restriction Fragment Length Polymorphism (RFLP), Random Amplified Polymorphic DNA (RAPD), Automatic Ribo-typing, rep-PCR, Multi-Locus Sequence Typing (MLST) have been shown to be useful methods for discrimination among isolates of pathogens [3,4]. Molecular-typing methods including rep-PCR, Multi-Locus Sequence Typing (MLST) have been shown to be useful methods for discrimination among isolates of pathogens [3,4]. Recent advances in molecular techniques have generated several typing methods based on PCR for genetic assessment of genetic relatedness of bacterial or fungal strains.…”