Design of Melting Curve Analysis (MCA) by Real-Time Polymerase Chain Reaction Assay for Rapid Distinction of Staphylococci and Antibiotic Resistance

Heydari, Narges; Alikhani, Mohammad Yousef; Tahmasebi, Hamed; Asghari, Babak; Arabestani, Mohammad Reza

doi:10.5812/archcid.81604

Cited by 6 publications

(5 citation statements)

References 24 publications

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“…Interestingly, the Ct vs. DNA relationship's slope varied little across the nine-fold dilutions tested, ranging from −3.589 to −3.955. According to previous studies, this can be justified because short fragments bind less fluorescent and are compensated by a higher primer concentration [29] , [30] . However, sometimes the peak height of the short amplicon increases in a different replicate.…”

Section: Discussionmentioning

confidence: 99%

Optimization and development of high-resolution melting curve analysis (HRMA) assay for detection of New Delhi metallo-β-lactamase (NDM) producing Pseudomonas aeruginosa

et al. 2022

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<abstract> New Delhi metallo-β-lactamase-1 (NDM-1) producing <italic>Pseudomonas aeruginosa</italic> strain detection plays a vital role in confirming bacterial disease diagnosis and following the source of an outbreak for public health. However, the standard method for NDM-1 determination, which relies on the features of the colony of the bacteria cultured from the patient's specimen, is time-consuming and lacks accuracy and sensitivity. This study aimed to standardize a high-resolution melting curve analysis (HRMA) assay to detect NDM producing <italic>P. aeruginosa</italic>. For optimization and development of the HRMA method, a reference strain of <italic>P. aeruginosa</italic> was used. For evaluating the broad range PCR data, ABI Step One-Plus Manager Software version 3.2 and Precision Melt Analysis Software 3.02 (Applied Biosystems) were used. Based on the results, expected results were obtained for all tested strains, with high analytical sensitivity and specificity. Temperature melting analyses of the HRMA time PCR assays showed the Tm at 89.57 °C, 76.92 °C and 82.97 °C for N-1, N-2 and N-3 genes, respectively. Also, melting point temperatures of the <italic>bla</italic>VIM, <italic>bla</italic>SPM and <italic>bla</italic>SIM amplicons for isolates identified as MBL strains were 84.56 °C, 85.35 °C and 86.62 °C, respectively. The amplification results using negative control genomes as templates were negative, showing the specificity of the designed assays. Our study's data indicated that the sensitivity and specificity of the HRMA method are linked to the primer length and the fluorescent dye. We can further identify antibiotic resistance in NDMproducing <italic>P. aeruginosa</italic> by software analysis and melting curve analysis. </abstract>

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Section: Discussionmentioning

confidence: 99%

Optimization and development of high-resolution melting curve analysis (HRMA) assay for detection of New Delhi metallo-β-lactamase (NDM) producing Pseudomonas aeruginosa

et al. 2022

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“…In addition to this, the presence of another pathogen CoNS possessing mecA gene and its influence on NAAT methods based detection cannot be overlooked (31). The methicillin resistance target gene mecA is often found in two organisms namely, CoNS and S. aureus ,(28) and hence merely detecting mecA is not sufficient to distinguish between MRSA and methicillin resistant CoNS.…”

Section: Discussionmentioning

confidence: 99%

“…Primer sequences (Table 2) specific to the conservative domains of Staphylococcus genus ( 16S ), S. aureus ( ITS ), and mecA gene were used for the assay. (31) For MCA assay, qPCR reactions were setup to check amplification of 16S, ITS , and mecA genes in the samples with discordant results between the two NAAT diagnostic methods. The reactions were carried out in real-time q PCR machine CFX96® (BioRad Technologies, USA).…”

Section: Methodsmentioning

confidence: 99%

Probing the dissonance among the diagnostic outputs of multiple approaches used for detection of Methicillin-resistant Staphylococcus aureus (MRSA)

Dahiya

Sikidar

Sharma

et al. 2020

Preprint

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Methicillin-resistant staphylococcus aureus (MRSA) is an extremely infectious hospital acquired bacterial pathogen often found in post-surgical patients globally. Early detection of such pathogens is a critical requirement to eliminate or reduce the incidence of antimicrobial resistance as well as for effective management of the disease. Despite the development of multiple biochemical, microbiological and nucleic acid amplification techniques (NAATs), conventional culture methods are widely used clinically owing to high variability between the methods, technical skills, and infrastructural needs. Further, multiple reports suggest a significant variation among diagnostic output for MRSA detection. This work attempts to probe the discordance among the diagnostic output of three commonly used methods while trying to understand the underlying cause of variability. MRSA detection on 217 clinical pus isolates was carried out using three different methods namely, conventional culture method, qPCR-based amplification, and a modern LAMP-based detection approach. Also, to confirm the presence of MRSA and distinguish from coagulase-negative staphylococci (CoNS), as well as to investigate the observed differences between qPCR and LAMP outputs, melt curve analysis was performed on discordant samples. LAMP-based MRSA detection was found to be the optimum method. In summary, this study evaluates the diagnostic efficiency of the different detection methods, while probing for possible explanations for the observed differences.

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“…CT > 40, was obtained for P. aeruginosa PASGNDM699 strain. According to Lalonde et al [21] and Heydari el al [22] studies, this can be justi ed because short fragment binds less uorescent and compensated by its higher primer concentration. However, sometimes the peak height of short amplicon increases in different replicate.…”

Section: Discussionmentioning

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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Design of Melting Curve Analysis (MCA) by Real-Time Polymerase Chain Reaction Assay for Rapid Distinction of Staphylococci and Antibiotic Resistance

Cited by 6 publications

References 24 publications

Optimization and development of high-resolution melting curve analysis (HRMA) assay for detection of New Delhi metallo-β-lactamase (NDM) producing <i>Pseudomonas aeruginosa</i>

Optimization and development of high-resolution melting curve analysis (HRMA) assay for detection of New Delhi metallo-β-lactamase (NDM) producing <i>Pseudomonas aeruginosa</i>

Probing the dissonance among the diagnostic outputs of multiple approaches used for detection of Methicillin-resistant Staphylococcus aureus (MRSA)

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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