Abstract
PCR multiplexing has proven to be challenging, and thus has provided limited means for pathogen genotyping. We developed a new approach for analysis of PCR amplicons based on restriction endonuclease digestion. The first stage of the restriction enzyme assay is hybridization of a target DNA to immobilized complementary oligonucleotide probes that carry a molecular marker, horseradish peroxidase (HRP). At the second stage, a target-specific restriction enzyme is added, cleaving the target-probe duplex at the corresponding restriction site and releasing the HRP marker into solution, where it is quantified colorimetrically. The assay was tested for detection of the methicillin-resistant Staphylococcus aureus (MRSA) pathogen, using the mecA gene as a target. Calibration curves indicated that the limit of detection for both target oligonucleotide and PCR amplicon was approximately 1 nM. Sequences of target oligonucleotides were altered to demonstrate that (i) any mutation of the restriction site reduced the signal to zero; (ii) double and triple point mutations of sequences flanking the restriction site reduced restriction to 50–80% of the positive control; and (iii) a minimum of a 16-bp target-probe dsDNA hybrid was required for significant cleavage. Further experiments showed that the assay could detect the mecA amplicon from an unpurified PCR mixture with detection limits similar to those with standard fluorescence-based qPCR. Furthermore, addition of a large excess of heterologous genomic DNA did not affect amplicon detection. Specificity of the assay is very high because it involves two biorecognition steps. The proposed assay is low-cost and can be completed in less than 1 hour. Thus, we have demonstrated an efficient new approach for pathogen detection and amplicon genotyping in conjunction with various end-point and qPCR applications. The restriction enzyme assay may also be used for parallel analysis of multiple different amplicons from the same unpurified mixture in broad-range PCR applications.
Highlights
PCR-based nucleic acid detection techniques have become the standard methodology in clinical and research microbiology and molecular diagnostics of infectious diseases
We developed a novel amplicon genotyping technique, and have tested the approach for detection of an important pathogen: methicillin-resistant Staphylococcus aureus (MRSA)
We describe a new approach for amplicon detection and genotyping based on specific enzymatic digestion of a target DNA
Summary
PCR-based nucleic acid detection techniques have become the standard methodology in clinical and research microbiology and molecular diagnostics of infectious diseases (for reviews see [1], [2], [3]). Direct DNA sequencing can be used for amplicon characterization. It is still a relatively expensive and time-consuming approach. Many conventional methods provide for simultaneous detection of multiple organisms of interest together with pathogen characterization and genotyping [6]. An attractive approach involves the use of ‘universal’ primers (designed from conserved sequences) to generate a mixed population of amplicons (‘broad-ranged PCR’) [7]. This approach, requires the development of additional, low-cost and rapid techniques to analyze the resulting mixture of PCR products simultaneously [2]
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