High-resolution melting for analysis of short sequence repeats in Mycobacterium avium subsp. paratuberculosis

Ricchi, M.; Barbieri, Gianluca; Cammi, G.; Garbarino, C.; Arrigoni, N.

doi:10.1111/j.1574-6968.2011.02371.x

Cited by 13 publications

(13 citation statements)

References 14 publications

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“…Only three distinct MV types were observed for all strains among which the MV 2 type was found to be specific to the bisontype group. Among these 5 loci, only MIRU-VNTR 1067 was found to be polymorphic, in agreement with other studies (30,48). Considering the results of the present and previous studies, MIRU 1 and MIRU 4 may be excluded from future studies since these loci appear monomorphic for cattle strains from different areas of the world (24,31).…”

Section: Discussionsupporting

confidence: 78%

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Genetic Structure of Mycobacterium avium subsp. paratuberculosis Population in Cattle Herds in Quebec as Revealed by Using a Combination of Multilocus Genomic Analyses

Sohal¹,

Arsenault²,

Labrecque³

et al. 2014

J Clin Microbiol

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c Mycobacterium avium subsp. paratuberculosis is the etiological agent of paratuberculosis, a granulomatous enteritis affecting a wide range of domestic and wild ruminants worldwide. A variety of molecular typing tools are used to distinguish M. avium subsp. paratuberculosis strains, contributing to a better understanding of M. avium subsp. paratuberculosis epidemiology. In the present study, PCR-based typing methods, including mycobacterial interspersed repetitive units/variable-number tandem repeats (MIRU-VNTR) and small sequence repeats (SSR) in addition to IS1311 PCR-restriction enzyme analysis (PCR-REA), were used to investigate the genetic heterogeneity of 200 M. avium subsp. paratuberculosis strains from dairy herds located in the province of Quebec, Canada. The majority of strains were of the "cattle type," or type II, although 3 strains were of the "bison type." A total of 38 genotypes, including a novel one, were identified using a combination of 17 genetic markers, which generated a Simpson's index of genetic diversity of 0.876. Additional analyses revealed no differences in genetic diversity between environmental and individual strains. Of note, a spatial and spatiotemporal cluster was evidenced regarding the distribution of one of the most common genotypes. The population had an overall homogeneous genetic structure, although a few strains stemmed out of the consensus cluster, including the bison-type strains. The genetic structure of M. avium subsp. paratuberculosis populations within most herds suggested intraherd dissemination and microevolution, although evidence of interherd contamination was also revealed. The level of genetic diversity obtained by combining MIRU-VNTR and SSR markers shows a promising avenue for molecular epidemiology investigations of M. avium subsp. paratuberculosis transmission patterns.

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Section: Discussionsupporting

confidence: 78%

“…In agreement with other studies, MLSSR typing was found to be the most discriminatory typing method (17,44,48). However, different groups have used distinct sets of SSR loci.…”

Section: Discussionsupporting

confidence: 75%

Genetic Structure of Mycobacterium avium subsp. paratuberculosis Population in Cattle Herds in Quebec as Revealed by Using a Combination of Multilocus Genomic Analyses

Sohal¹,

Arsenault²,

Labrecque³

et al. 2014

J Clin Microbiol

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“…To the best of the authors' knowledge, this is the first article describing the application of HRM analysis for SSR genotyping in fungal pathogens. Genotyping with HRM analysis is a robust and reproducible method, and may be an appropriate alternative to using a capillary sequencer when loci show a reduced number of alleles (Ricchi et al ., ). In the current work, HRM analysis was able to detect both SSR length and SNP‐based polymorphisms in SSR, SSR flanking regions and homopolymeric traits, overcoming the issue related to allelic size homoplasy.…”

Section: Discussionmentioning

confidence: 97%

HRM analysis provides insights on the reproduction mode and the population structure of Gnomoniopsis castaneae in Europe

et al. 2016

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Gnomoniopsis castaneae is an emergent nut rot agent of chestnut in southern Europe. To elucidate its population genetics, three simple sequence repeat (SSR) and two hypervariable markers were developed and assessed through high‐resolution melting (HRM) analysis on 132 isolates collected from 10 sites in Italy, France and Switzerland. High allele diversity (ranging from 0.23 to 0.40 depending on site) and number of haplotypes (49) were observed. More than 70% of the molecular variance could be accounted among isolates within sites. Multilocus analysis showed absence of linkage disequilibrium, suggesting a predominant role played by sexual reproduction and random mating. Data analyses indicated the presence of at least two putative distinct subpopulations and this was confirmed by several approaches, including analysis of shared haplotypes, multivariate and Bayesian analyses. Based on data of allelic diversity, the possibility that the pathogen could have been introduced is discussed. This work assessed the genetic variability and the sexual strategies of G. castaneae in Europe, adding useful information on the epidemiology of this fungal plant pathogen.

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“…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).…”

Section: Introductionmentioning

confidence: 99%

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

Perng

Chen

Chiueh

et al. 2012

Journal of Medical Microbiology

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Non-tuberculous mycobacteria (NTM) are increasingly important opportunistic pathogens responsible for a variety of clinical diseases. The aim of this study was to evaluate a novel technique, real-time PCR coupled with high-resolution melting analysis (real-time PCR-HRMA), for NTM identification. Two pairs of unique primers targeted to the 16S rRNA gene and the 16S-23S internal transcribed spacer region were selected for further evaluation. A total of 149 mycobacterial clinical isolates were subjected to analysis using the real-time PCR-HRMA system. Overall, 134 NTM identified by the 16S rRNA full-gene sequencing method were categorized into four major groups: Mycobacterium avium complex, Mycobacterium chelonae group, Mycobacterium gordonae and Mycobacterium fortuitum group. Of the 134 prevalent mycobacterial isolates, 101 mycobacteria (75.4 %) could be identified correctly by the real-time PCR-HRMA system. The individual sensitivities for the M. avium complex, M. chelonae group, M. gordonae and M. fortuitum groups were 90.9, 89.1, 100 and 36.8 %, respectively. The specificity of identifying these groups varied from 96.4 to 100 %. When identification failed, mostly it was attributable to various species in the M. fortuitum group. The real-time PCR-HRMA system is therefore a rapid and sensitive method for identifying prevalent NTM in a clinical laboratory.

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High-resolution melting for analysis of short sequence repeats in Mycobacterium avium subsp. paratuberculosis

Cited by 13 publications

References 14 publications

Genetic Structure of Mycobacterium avium subsp. paratuberculosis Population in Cattle Herds in Quebec as Revealed by Using a Combination of Multilocus Genomic Analyses

Genetic Structure of Mycobacterium avium subsp. paratuberculosis Population in Cattle Herds in Quebec as Revealed by Using a Combination of Multilocus Genomic Analyses

HRM analysis provides insights on the reproduction mode and the population structure of Gnomoniopsis castaneae in Europe

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

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