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

2020 ◽  
Vol 70 (4) ◽  
pp. 290-299 ◽  
Author(s):  
H. Tahmasebi ◽  
S. Dehbashi ◽  
M.R. Arabestani
Keyword(s):  
Pseudomonas Aeruginosa ◽  
High Resolution ◽  
High Resolution Melting ◽  
Melting Curve ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
New Approach ◽  
High Resolution Melting Curve

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

2019 ◽  
Author(s):  
Sanaz Dehbashi ◽  
Hamed Tahmasebi ◽  
Mohammad Arabestani
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Melting Point ◽  
High Resolution ◽  
High Resolution Melting ◽  
Melting Curve ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
New Delhi ◽  
Analytical Sensitivity ◽  
High Resolution Melting Curve

Abstract 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.

Differentiation of five enterohepaticHelicobacterspecies by nested PCR with high-resolution melting curve analysis

Helicobacter ◽  
2016 ◽  
Vol 22 (2) ◽  
pp. e12362 ◽  
Author(s):  
Miaoli Wu ◽  
Dan Rao ◽  
Yujun Zhu ◽  
Jing Wang ◽  
Wen Yuan ◽  
...  
Keyword(s):  
High Resolution ◽  
Nested Pcr ◽  
High Resolution Melting ◽  
Melting Curve ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
High Resolution Melting Curve

scholarly journals Nested High-Resolution Melting Curve Analysis

2011 ◽  
Vol 13 (3) ◽  
pp. 263-270 ◽  
Author(s):  
Serge Carillo ◽  
Laurent Henry ◽  
Eric Lippert ◽  
François Girodon ◽  
Isabelle Guiraud ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Melting Curve ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
High Resolution Melting Curve
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