Use of High-Resolution Melting and Melting Temperature-shift assays for specific detection and identification of Bacillus anthracis based on single nucleotide discrimination

Derzelle, Sylviane; Mendy, Christiane; Laroche, Séverine; Madani, Nora

doi:10.1016/j.mimet.2011.08.005

Cited by 15 publications

(10 citation statements)

References 33 publications

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“…Several genetic fingerprinting techniques representing the complete genome, such as repetitive element sequence‐based PCR (rep‐PCR) and randomly amplified polymorphic DNA (RAPD), have also been widely applied to differentiate bacterial species and/or strains (Rossi and others ; Iacumin and others ; Švec and others ; Fonseca and others ). Recently, real‐time PCR‐based intercalting dye assays have provided a new approach for the rapid differentiation of closely related species according to specific melting temperature (Tm) and high‐resolution melting analysis dependent on the detection of small differences between DNA sequences in a double‐stranded DNA molecules (Derzelle and others ; Porcellato and others ).…”

Section: Introductionmentioning

confidence: 99%

Application of Different Molecular Techniques for Characterization of Catalase‐Positive Cocci Isolated from Sucuk

Kesmen

Yarimcam

Aslan

et al. 2014

Journal of Food Science

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This study was carried out for the characterization and discrimination of the indigenous Gram positive, catalase-positive cocci (GCC) population in sucuk, a traditional Turkish dry-fermented sausage. Sucuk samples, produced by the traditional method without starter culture were collected from 8 local producers in Kayseri/Turkey and a total of 116 GCC isolates were identified by using different molecular techniques. Two different molecular fingerprinting methods; namely, randomly amplified polymorphic DNA-PCR (RAPD-PCR) and repetitive extragenic palindrome-PCR (rep-PCR), were used for the clustering of isolates and identification at species level was carried out by full length sequencing of 16S rDNA. Combining the results obtained from molecular fingerprinting and 16S rDNA sequencing showed that the dominant GCC species isolated from the sucuk samples was Staphylococcus saprophyticus followed by Staphylococcus succinus and Staphylococcus equorum belonging to the Staphylococcus genus. Real-time PCR DNA melting curve analysis and high-resolution melting (HRM) analysis targeting the V1 + V3 regions of 16S rDNA were also applied for the discrimination of isolates belonging to different species. It was observed statistically different Tm values and species-specific HRM profiles for all except 2 species (S. saprophyticus and Staphylococcus xylosus) that have high 16S rDNA sequence similarity. The combination of rep-PCR and/or PCR-RAPD with 16S rRNA gene sequencing was an efficient approach for the characterization and identification of the GCC population in spontaneously fermented sucuk. On the other hand, intercalating dye assays were found to be a simple and very promising technique for the differentiation of the GCC population at species level.

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Section: Introductionmentioning

confidence: 99%

Application of Different Molecular Techniques for Characterization of Catalase‐Positive Cocci Isolated from Sucuk

Kesmen

Yarimcam

Aslan

et al. 2014

Journal of Food Science

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“…HRM has proven to be an efficient and labor-saving way to detect sequence variations such as SNPs in humans, plants, and microorganisms (Millat et al, 2009;Derzelle et al, 2011;Li et al, 2012). In plants, such an approach has been applied to detect SSRs, which was used for genotype identification and authenticity testing (Jaakola et al, 2010;Ganopoulos et al, 2011;Distefano et al, 2012;Madesis et al, 2012).…”

Section: Discussionmentioning

confidence: 99%

Limitation of high-resolution melting curve analysis for genotyping simple sequence repeats in sheep

Yang¹,

Yuan²,

Guo³

et al. 2014

Genet. Mol. Res.

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ABSTRACT. Variation in microsatellite or simple sequence repeat (SSR) loci has, until recently, relied heavily on the use of gel-based methods that can be both time consuming and difficult to genotype. Non gel-based systems are therefore important to increase simplicity and improve turn-around time without compromising assay sensitivity and accuracy. In this report, we assessed the latest of the non-gelbased methods, high-resolution melting (HRM) curve analysis. HRM is a technique that monitors exactly the decreasing fluorescence of intercalating dye in the process of dissociation of double-stranded DNA. The measurement immediately follows polymerase chain reaction in a one-step, closed-tube method. Four SSR loci of different complexity in sheep, namely MAF209, MCM140, CB226, and SRCRSP5, were assessed using the LightScanners System with LC Greens PLUS DNA binding dye. In order to improve the accuracy of genotyping, we applied internal oligo nucleotide calibrators while performing HRM. DNA polymorphisms were previously identified using capillary electrophoresis analysis (CE). The result showed that CE detected more genotypes than HRM in the same loci regardless of the level of polymorphism at the SSR loci. We demonstrate current limitations of the HRM method for the analysis of SSR loci.

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“…Genotyping of the PCR products was performed on the Roche LightCycler 480 Fluorescence Real-Time PCR System (Roche, Rotkreuz, Switzerland) using melting temperature shift (Tm-shift) according to the manufacturer's instructions [12, 13]. Tm-shift method uses two allele-specific primers and one reverse primer to amplify the polymorphic region encoding the targeted variant, and genotypes can be determined by inspection of a melting curve [14, 15] (Figure 1). To verify the repeatability and stability of experiment, 5% of random samples and 18 control samples (including 9 negative and 9 positive controls) were used for quality control.…”

Section: Methodsmentioning

confidence: 99%

An Association Study between Genetic Polymorphism in the Interleukin-6 Receptor Gene and Coronary Heart Disease

Zhou

Chen

et al. 2014

BioMed Research International

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The goal of our study is to test the association of IL6R rs7529229 polymorphism with CHD through a case-control study in Han Chinese population and a meta-analysis. Our result showed there is a lack of association between IL6R rs7529229 polymorphism and CHD on both genotype and allele levels in Han Chinese (P > 0.05). However, a meta-analysis among 11678 cases and 12861 controls showed that rs7529229-C allele was significantly associated with a decreased risk of CHD, especially in Europeans (P < 0.0001, odds ratio = 0.93, 95% confidential interval = 0.89–0.96). Since there is significant difference among different populations, further studies are warranted to test the contribution of rs7529229 to CHD in other ethnic populations.

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Use of High-Resolution Melting and Melting Temperature-shift assays for specific detection and identification of Bacillus anthracis based on single nucleotide discrimination

Cited by 15 publications

References 33 publications

Application of Different Molecular Techniques for Characterization of Catalase‐Positive Cocci Isolated from Sucuk

Application of Different Molecular Techniques for Characterization of Catalase‐Positive Cocci Isolated from Sucuk

Limitation of high-resolution melting curve analysis for genotyping simple sequence repeats in sheep

An Association Study between Genetic Polymorphism in the Interleukin-6 Receptor Gene and Coronary Heart Disease

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