Evaluation of a PCR Melting Profile Technique for Bacterial Strain Differentiation

Abstract

In search of an effective DNA typing technique for hospital epidemiology use, the performance and convenience of a PCR melting profile (PCR MP) technique based on using low denaturation temperatures during ligation-mediated PCR (LM PCR) of bacterial DNA was tested. A number of Escherichia coli isolates from patients of the Clinical Hospital in Gdańsk, Poland, were examined. We found that the PCR MP technique is a rapid method that offers good discriminatory power and excellent reproducibility and may be applied for epidemiological studies. The usefulness of the PCR MP for molecular typing was compared with the pulsedfield gel electrophoresis method, which is currently considered the gold standard for epidemiological studies of isolates recovered from patients and the environment. Clustering of PCR MP fingerprinting data matched pulsed-field gel electrophoresis data. The features of the PCR MP technique are discussed in comparison with conventional methods. Data presented here demonstrate the complexity of the epidemiological situation concerning E. coli that may occur in a hospital.Over the past 20 years, a significant number of DNA-based techniques have been introduced into the field of bacterial characterization and taxonomy. These genomic fingerprinting methods were developed to detect DNA sequence polymorphisms by using general principles, such as restriction endonuclease analysis, molecular hybridization, and PCR amplification. DNA fingerprinting involves the display of a set of DNA fragments from a specific DNA sample. A variety of DNA fingerprinting techniques are presently available (1-7, 11-13), most of which use PCR for detection of fragments. The choice of fingerprinting technique depends on the type of application. Ideally, a fingerprinting technique should require no prior investments in terms of sequence analysis, primer synthesis, or characterization of DNA probes. A number of fingerprinting methods which meet these requirements have been developed. The fingerprints are obtained by visualizing many parts of the genome. Differences in these fingerprints between individuals are interpreted as genetic distances. Obviously, the differences should reflect variations in DNA rather than artifacts due to a nonrobust method. Furthermore, the method should provide the appropriate level of discriminatory power, and it should be relatively rapid and cheap, especially in large-scale population genetic studies. Macrorestriction analysis of genomic DNA followed by pulsed-field gel electrophoresis (REA-PFGE) has become the "gold standard" for molecular typing (10). In recent years, some alternative techniques have been successfully applied for the typing of bacteria below the species level. These include amplification-based methods, such as amplified fragment length polymorphisms (11), random amplification of polymorphic DNA (3, 12, 13), and amplification of DNA fragments surrounding rare restriction sites (5, 6, 8). These techniques are now applied more and more because they involve less time, comparably low cos...

High-Resolution Melting-Curve Analysis of Ligation-Mediated Real-Time PCR for Rapid Evaluation of an Epidemiological Outbreak of Extended-Spectrum-Beta-Lactamase-Producing Escherichia coli

“…The LM/HRM method was developed from a previously described strategy applying conventional PCR and gel electrophoresis analysis of DNA products (6)(7)(8)16). This method could essentially be described as a PCR-based PFGE method where the resolution may be altered based on the denaturation temperature.…”

“…To allow analysis by HRM, we modified the original protocol (8) by lowering the T D from 87.2°C to 84°C. During the optimization of the T D , it was clear that the resolution of HRM patterns is highly dependent on the denaturation temperature of the PCR (data not shown).…”

“…During the optimization of the T D , it was clear that the resolution of HRM patterns is highly dependent on the denaturation temperature of the PCR (data not shown). We are currently evaluating the LM/HRM method for other species, and for each group of bacteria or fungi to be analyzed by the method, an individual T D needs to be defined, as previously shown (6)(7)(8)(9)16). The reason why the original temperature for E. coli did not provide enough resolution by HRM in the present report is probably due to the synthesis of too many amplicons, which were visualized by the gel electrophoresis pattern of the DNA products (Fig.…”

“…Previously, Masny and Płucienniczak (12) described a novel method based on ligation-mediated PCR (LM/PCR) using low denaturation temperatures (T D s), resulting in specific melting-profile DNA product patterns for bacterial and fungal isolates. The method is based on genomic cleavage by restriction enzymes and the amplification of several fragments by ligation-mediated PCR followed by an analysis of the DNA fragment patterns by gel electrophoresis (8). The method has performed successfully for Enterobacteriaceae, Candida spp., Staphylococcus aureus, and Enterococcus spp., with a discriminatory power that is comparable to that of PFGE (6)(7)(8)16).…”

Evaluation of High-Resolution Melting Curve Analysis of Ligation-Mediated Real-Time PCR, a Rapid Method for Epidemiological Typing of ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter Species) Pathogens

“…In this study, we further modified ligation-mediated PCR (LM PCR) with polyacrylamide gel analysis (12,13) for use in a singletube real-time PCR platform with high-resolution melt analysis (LMqPCR HRMA) for rapid epidemiological typing of the multiresistant ESKAPE pathogens. Following optimization, the method was evaluated blindly using a selection of ESKAPE pathogens that were previously defined by PFGE analysis as being identical, closely related, or unrelated (see the supplemental material).…”