Rapid Identification of Biothreat and Other Clinically Relevant Bacterial Species by Use of Universal PCR Coupled with High-Resolution Melting Analysis

Abstract

A rapid assay for eubacterial species identification is described using high-resolution melt analysis to characterize PCR products. Unique melt profiles generated from multiple hypervariable regions of the 16S rRNA gene for 100 clinically relevant bacterial pathogens, including category A and B biothreat agents and their surrogates, allowed highly specific species identification.Rapid and accurate diagnostic tools are critical for infectious disease surveillance and early diagnosis of disease (8,12). A simple platform which could deliver broad-based screening and specific pathogen identification would be invaluable for the timely recognition of emerging and biothreat (BT) outbreaks, as well as commonly encountered clinical infections (2,7,9,11,12).We previously reported a probe-based PCR assay, which utilizes conserved and variable 16S rRNA gene sequences for initial broad-based eubacterial detection and subsequent identification of specific bacterial agents (11). The assay demonstrated high analytical sensitivity but was limited by an inability to differentiate closely related pathogens due to decreased specificity of the TaqMan probe chemistry and high sequence homology within selected hypervariable regions of the 16S rRNA gene. Probe-based amplicon characterization accordingly limits testing to a finite number of anticipated pathogens. Alternative strategies for amplicon analysis, such as sequencing and mass spectrometry, allow broader-scale product characterization but are costly, time-consuming, and lacking in throughput (1, 6). High-resolution melt analysis (HRMA) offers a simple, low-cost, closed-tube approach to amplicon analysis with the capacity for single-nucleotide discrimination and easy integration with PCR analysis (10). We report a unique strategy for the rapid, highly specific identification of BTrelated and non-BT-related bacterial pathogens which couples eubacterial PCR with HRMA.

Identification of non-tuberculous mycobacteria by real-time PCR coupled with a high-resolution melting system

“…between members of the M. fortuitum group) ( Table 1). Similar findings have been reported in the literature (Yang et al, 2009). These unsatisfactory observations may have resulted from: (i) multiple different species in the M. fortuitum group, as discriminated by 16S rRNA gene sequencing, (ii) nucleotide polymorphisms of the 16S rRNA gene and ITS region within the species (e.g.…”

“…Previous studies have shown that HRMA is capable of distinguishing different sequence types with the same G+C content (Slinger et al, 2007) or discriminating single-nucleotide differences between closely related species (Yang et al, 2009). Despite its theoretically high discrimination power, we found that amplicons of different NTM species could generate similar melting curves (e.g.…”

“…The difference plots were generated by subtracting these curves from the reference curve. Compared with the primary melting curve, the difference plot minimizes the effect of interfering factors and can easily be differentiated by visual inspection in a clinical laboratory (Won et al, 2010;Yang et al, 2009).…”

“…Previous studies have combined HRMA with real-time PCR assays for rapid detection and identification of clinically important bacteria (Ajitkumar et al, 2012;Chen et al, 2011;Cheng et al, 2006, Ricchi et al, 2011Choi et al, 2010;Douarre et al, 2012;Pang et al, 2011;Pietzka et al, 2009;Tortoli, 2009;Won et al, 2010;Yang et al, 2009). The literature suggests that a combined real-time PCR-HRMA assay is a novel and rapid technique suitable for the genotyping of drug-resistant isolates of M. tuberculosis (Chen et al, 2011;Pietzka et al, 2009;Ramirez et al, 2010).…”

The Potential of High Resolution Melting Analysis (Hrma) to Streamline, Facilitate and Enrich Routine Diagnostics in Medical Microbiology

“…However, a well-proved broader coverage is desirable for routine use. So far, the best achievement towards an optimum system was made by Yang et al 25 , who covered 100 clinically important bacterial species, though, reproducibility and specificity of this assay in routine use still has to be evaluated.…”