Differentiation of Staphylococcus spp. by high-resolution melting analysis

Slaný, Michal; Vaněrková, Martina; Němcová, Eva; Žaloudíková, Barbora; Růžička, Filip; Freiberger, Tomáš

doi:10.1139/w10-091

Cited by 16 publications

(16 citation statements)

References 22 publications

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“…This is of particular interest for genera such as Staphylococcus in which some species may be pathogenic and others commensal; these are often poorly resolved by single 16S genes [36], [37]. This genus contains extremely diverse species found throughout the human microbiome [38] and includes organisms of clinical interest such as S. aureus .…”

Section: Resultsmentioning

confidence: 99%

Simultaneous Quantification of Multiple Bacteria by the BactoChip Microarray Designed to Target Species-Specific Marker Genes

et al. 2013

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Bacteria are ubiquitous throughout the environment, the most abundant inhabitants of the healthy human microbiome, and causal pathogens in a variety of diseases. Their identification in disease is often an essential step in rapid diagnosis and targeted intervention, particularly in clinical settings. At present, clinical bacterial detection and discrimination is primarily culture-based, requiring both time and microbiological expertise, especially for bacteria that are not easily cultivated. Higher-throughput molecular methods based on PCR amplification or, recently, microarrays are reaching the clinic as well. However, these methods are currently restricted to a small set of microbes or based on conserved phylogenetic markers such as the 16S rRNA gene, which are difficult to resolve at the species or strain levels. Here, we designed and experimentally validated the BactoChip, an oligonucleotide microarray for bacterial detection and quantification. The chip allows the culture-independent identification of bacterial species, also determining their relative abundances in complex communities as occur in the commensal microbiota or in clinical settings. The microarray successfully distinguished among bacterial species from 21 different genera using 60-mer probes targeting a novel set of in silico identified high-resolution marker genes. The BactoChip additionally proved accurate in determining species-level relative abundances over a 100-fold dynamic range in complex bacterial communities and with a low limit of detection (0.1%). In combination with the continually increasing number of sequenced bacterial genomes, future iterations of the technology could enable to highly accurate clinically-oriented tools for rapid assessment of bacterial community composition and relative abundances.

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Section: Resultsmentioning

confidence: 99%

Simultaneous Quantification of Multiple Bacteria by the BactoChip Microarray Designed to Target Species-Specific Marker Genes

et al. 2013

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“…Although HRMA cannot replace sequencing-based genotyping methods, this new methodology can potentially complement them (Lévesque et al, 2011). Additionally, because HRMA is a non-destructive method, the HRMA products could be used directly in a sequencing analysis if necessary and the dye would not introduce any interference (Slany et al, 2010;Vossen et al, 2009). …”

Section: Discussionmentioning

confidence: 99%

A novel high-resolution melting analysis-based method for Yersinia pseudotuberculosis genotyping

Souza¹,

Falcão²

2012

Journal of Microbiological Methods

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Yersinia pseudotuberculosis is an enteric pathogen that is environmentally widespread and is known to cause human and animal infections. The development of a fast and inexpensive typing system is necessary to facilitate epidemiological studies of Y. pseudotuberculosis infections. In this study, we aimed to develop a method of Y. pseudotuberculosis genotyping based on determining differences in single-nucleotide polymorphisms (SNPs) using a high-resolution melting analysis (HRMA). Using a set of nine primer pairs, ten SNPs were screened from sequences in the 16S rRNA, glnA, gyrB and recA sequences of 12 Y. pseudotuberculosis strains that were deposited in the GenBank database. The genetic diversity of a collection of 40 clinical Y. pseudotuberculosis strains was determined using the HRMA method and the multilocus sequence typing (MLST) technique was used for comparison. Different melting profiles were found in five out of a total of nine analyzed fragments. A phylogenetic tree was constructed from the nucleotides that were identified in the nine analyzed fragments, and the tree demonstrated that Y. pseudotuberculosis strains were separated into two groups. The first cluster was composed of strains from the 1/O:1a serogroup and the second of strains from the 2/O:3 serogroup. The separation into two clusters based on distinct bio-serogroups of Y. pseudotuberculosis was consistent with the results in the MLST database. The simple and highly reproducible HRMA assay developed by us may be used as a rapid and cost-effective method to genotype Y. pseudotuberculosis strains of O:1 and O:3 serogroups and it can complement sequence-based methods facilitating epidemiological studies of this Yersinia species.

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“…HRM has been widely utilized for a variety of applications. 24–26 In the tuberculosis arena, HRM has been used for detecting rifampin, isoniazid, streptomycin, and fluoroquinolone resistant M. tuberculosis . 27–31 In this study, we describe an HRM technique to detect pncA mutations and compare these data to sequencing and phenotypic PZA susceptibilities.…”

Section: Introductionmentioning

confidence: 99%

Pyrazinamide susceptibility testing of Mycobacterium tuberculosis by high resolution melt analysis

et al. 2014

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Pyrazinamide (PZA) plays the important role in shortening the tuberculosis treatment period and in treating MDR-TB. Phenotypic PZA susceptibility methods are limited because they require specialized acidified media, which increases costs and complexity. In this study we developed a genotypic high resolution melt (HRM) analysis technique to detect pncA mutations associated with PZA resistant M. tuberculosis. Seven overlapping primer pairs were designed to cover the entire pncA gene and upstream regions. Each gene segment was individually amplified by real-time PCR followed by HRM analysis. The assay was evaluated on 98 clinical M. tuberculosis isolates (41 PZA susceptible by MGIT method, 55 PZA resistant, 2 undetermined). HRM was 94% concordant to full-length sequencing results, with most discrepancies attributable to mixed populations per HRM or transversions. Sequencing and HRM yielded 82% and 84% concordance, respectively, to phenotypic PZA susceptibilities by MGIT, with most discrepancies attributable to isolates with wild-type pncA but phenotypic PZA resistance. This HRM technique is a simple and high-throughput method for screening clinical M. tuberculosis samples for PZA resistance.

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Differentiation of Staphylococcus spp. by high-resolution melting analysis

Cited by 16 publications

References 22 publications

Simultaneous Quantification of Multiple Bacteria by the BactoChip Microarray Designed to Target Species-Specific Marker Genes

Simultaneous Quantification of Multiple Bacteria by the BactoChip Microarray Designed to Target Species-Specific Marker Genes

A novel high-resolution melting analysis-based method for Yersinia pseudotuberculosis genotyping

Pyrazinamide susceptibility testing of Mycobacterium tuberculosis by high resolution melt analysis

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