Development of a new High Resolution Melting (HRM) assay for identification and differentiation of Mycobacterium tuberculosis complex samples

Landolt, Patricia; Stephan, Roger; Scherrer, Simone

doi:10.1038/s41598-018-38243-6

Cited by 25 publications

(26 citation statements)

References 32 publications

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“…Some recent microbiological applications that used this technology have been reported. Various studies in Switzerland (Landolt et al ), USA (Esteves et al ), Italy (Pugliese et al ), and China (Lu et al ) demonstrated the potential of HRM to differentiate between bacterial species rapidly.…”

Section: Resultsmentioning

confidence: 99%

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New approach to identify colistin‐resistant Pseudomonas aeruginosa by high‐resolution melting curve analysis assay

Tahmasebi

Dehbashi

Arabestani

2020

Lett Appl Microbiol

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Colistin‐resistant Pseudomonas aeruginosa (CRPA), as a health care system threat, is increasing globally. This study aimed was to determine CRPA by high‐resolution melting curve (HRM) analysis method. The HRM method was done on standard strains of P. aeruginosa and CRPA strains. Ninefold serial dilutions of known genomic DNA (gDNA) and plasmid DNA (pDNA) concentrations, extracted from P. aeruginosa NCTC13715 and P. aeruginosa NCTC10728 were prepared and tested by melting curve analysis and HRM assay. Data analysis was performed using the Step‐One Plus Software v2.3 and hrm Software v3.0.1. The results of the melt curve analysis and HRM showed a very similar melt peak for all positive controls with a melt temperature that ranged from 88·1°C to 88·6°C for the 16srRNA, 90·0°C to 90·05°C for the mcr‐1 and 91·2°C to 91·7°C for the pmrA gene. Furthermore, the data indicated that the HRM approach has the sensitivity to detect 100 CFU per ml for the 16srRNA gene, 101 CFU per ml for the pmrA gene, and 103 CFU per ml for the mcr‐1 gene. According to our findings, it was concluded that the HRM method could detect 100 to 103 CFU per ml of P. aeruginosa gDNA and pDNA. Therefore, CRPA strains can be identified with high sensitivity and specificity by HRM assay. Significance and Impact of the Study The highlight of our research is about the detection of bacteria resistance genes to antibiotics by advanced molecular methods, which means high‐resolution melting curve (HRM) analysis. We determined that the HRM method in identifying colistin‐resistant P. aeruginosa has high accuracy and speed, and with high sensitivity and specificity.

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Section: Resultsmentioning

confidence: 99%

“…Human sources of errors cannot be excluded (Tong and Giffard 2012). In some studies, it has been emphasized how primer and magnesium concentration, as well as annealing temperature, affects the Tm specificity and overall efficiency of the Real-time HRM assay (Esteves et al 2018;Landolt et al 2019).…”

Section: Analytical Sensitivity and Specificity Of Primersmentioning

confidence: 99%

New approach to identify colistin‐resistant Pseudomonas aeruginosa by high‐resolution melting curve analysis assay

Tahmasebi

Dehbashi

Arabestani

2020

Lett Appl Microbiol

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“…4 School of Paramedical, Hamadan University of Medical Sciences, Hamadan, Iran. 5 Nutritious Research Center, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran.…”

Section: Acknowledgmentsmentioning

confidence: 99%

“…The accuracy and efficiency are the advantages of this method. The major challenge to develop this method is applying the most specific melt temperature for each target [5].…”

Section: Introductionmentioning

confidence: 99%

Development of high-resolution melting curve analysis in rapid detection of vanA gene, Enterococcus faecalis, and Enterococcus faecium from clinical isolates

et al. 2020

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Background: High-resolution melting analysis (HRMA) is a novel molecular technique based on the real-time PCR that can be used to detect vancomycin resistance Enterococcus (VRE). The purpose of this study was to identify VRE species with HRMA in clinical isolates.Results: Out of 49 Enterococcus isolates, 11 (22.44%) E. faecium isolates and 19 (38.77%) E. faecalis isolates were detected. Average melting temperatures for divIVA in E.faecalis, alanine racemase in E.faecium, and vanA in VRE strains were obtained as 79.9 ± 0.5°C, 85.4 ± 0.5°C, and 82.99 ± 0.5°C, respectively. Furthermore, the data showed that the HRMA method was sensitive to detect 10 0 CFU/ml for the divIVA, alanine racemase, and vanA genes. Also, out of 49 Enterococcus spp., which were isolated by HRMA assay, 8 isolates (16.32%) of E. faecium and 18 isolates (36.73%) of E. faecalis were detected. The vanA gene was reported in 2 isolates (25%) of E. faecium and 9 isolates (50%) of E. faecalis. Conclusions: This study demonstrated that using the HRMA method, we can detect E. faecium, E. faecalis, and the vanA gene with high sensitivity and specificity.

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“…HRM is used to characterize samples according to their dissociation behavior as they transition from dsDNA to ssDNA with increasing temperature and uorescence detection. HRM melt curves is a function of the amplicon DNA sequence, which allows discrimination of amplicons with different nucleotide sequence based on melt curve shape, regardless of the amplicon Tm [8,9]. The melt curve analysis is generally used in conjunction with HRM.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of High-Resolution Melting Curve Analysis (HRM) assay for Detection of Pseudomonas aeruginosa PASGNDM699: A dangerous New Delhi metallo-β-lactamase (NDM) strain

Dehbashi¹,

Tahmasebi²,

Arabestani³

2019

Preprint

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Background: New Delhi metallo-β-lactamase (NDM-1) is a broad spectrum β-lactamase that is able to inactivate all β-lactams except aztreonam, as is typical of metallo-β-lactamases. NDM-1 producers in Pseudomonas aeruginosa, especially PASGNDM699 strain, cause a range of infections such as urinary tract, diarrhoea and soft tissue infections. The aim of this study was to Standardization of High-Resolution Melting Curve Analysis (HRM) assay for detection of P. aeruginosa, especially PASGNDM699 strain. Methods: The HRM method was done on standard strains of P. aeruginosa strains. 9-fold Serial dilutions of known DNA concentrations, extracted from standard isolates were prepared and tested by Real Time Melting curve and HRM assay. Data analysis was performed using the StepOne Software v2.3 and HRM Software v3.0.1 (Applied Biosystems, Ltd). Results: Based on the results of the Real Time PCR assay and melt curve analysis, melting point temperatures of the N-1, N-2 and N-3 amplicon for isolates identified as NDM strains were 87.57°C, 76.92°C and 82.97°C, respectively. Furthermore, melting point temperatures of the blaVIM, blaSPM and blaSIM amplicon for isolates identified as MBL strains were 84.56°C, 85.35°C and 86.62°C, respectively. Due to the analytical specificity of the primers, all dilutions with a similar Tm and melt peaks were obtained in the melting curves. Moreover, the analytical sensitivity of NDM primer were able to detected 100CFU/mL, 103CFU/mL and 104CFU/mL of standard DAN by N-1, N-2 and N-3 primers, respectively. Also, according to analytical sensitivity of MBL primers, blaVIM was able detected of 100CFU/mL, blaSPM primer 105CFU/mL and blaSIM primer 102CFU/mL of PASGNDM699 strain. HRM results showed that N-1 primers with 55 bp and blaVIM primers with 124 bp had the highest sensitivity and specificity for P. aeruginosa PASGNDM699 strain identification. Conclusion: The data from our study indicated that the sensitivity and specificity of the HRM method linked to the primer length and the fluorescent dye. Further, we can identify antibiotic resistance in substrates such as P. aeruginosa PASGNDM699 by software analysis and melting curve analysis.

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Development of a new High Resolution Melting (HRM) assay for identification and differentiation of Mycobacterium tuberculosis complex samples

Cited by 25 publications

References 32 publications

New approach to identify colistin‐resistant Pseudomonas aeruginosa by high‐resolution melting curve analysis assay

New approach to identify colistin‐resistant Pseudomonas aeruginosa by high‐resolution melting curve analysis assay

Development of high-resolution melting curve analysis in rapid detection of vanA gene, Enterococcus faecalis, and Enterococcus faecium from clinical isolates

Evaluation of High-Resolution Melting Curve Analysis (HRM) assay for Detection of Pseudomonas aeruginosa PASGNDM699: A dangerous New Delhi metallo-β-lactamase (NDM) strain

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