A High Resolution Melting Point Analysis Based Simplex PCR Assay Enables Reliable Identification of Clinically Relevant Candida Species

Abstract Background: Single-nucleotide polymorphisms and single-base deletions within the cytochrome P450 2D6 (CYP2D6) gene have been associated with a poor metabolizer (PM) phenotype and display a frequency of 7–10% in the Caucasian population. Methods: We developed a reliable and rapid procedure to identify five major PM-associated mutations (CYP2D6*4, *7, and *8) and deletions (CYP2D6*3 and *6) by real-time PCR with subsequent fluorometric melting point analysis of the PCR product. These polymorphisms within the CYP2D6 gene were detected by use of two primer pairs and five different pairs of hybridization probes. DNA extracted from whole blood of 323 individuals was analyzed, and results were compared with genotypes obtained by allele-specific multiplex PCR. In case of uncertain results, additional sequence analysis was performed. Results: Genotyping results by real-time PCR were 100% reliable, whereas conventional allele-specific multiplex PCR produced uncertain results for 12.1% of samples, as confirmed by sequence analysis. Costs for reagents and consumables were considerably higher for the real-time PCR technology, but labor time was reduced by 2 h compared with allele-specific PCR. The allele frequencies in the population investigated were 0.186 for allele *4, 0.026 for allele *5, 0.009 for allele *3, 0.031 for allele *6, and 0.002 for allele *8. The defective CYP2D6*7 allele was not found. In addition, three additional mutations were detected, one of them displaying a PM genotype. Conclusion: Genotyping of CYP2D6 by real-time PCR with fluorometric melting point analysis is a rapid and reliable method.

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Molecular identification of Candida isolates by Real‐time PCR‐high‐resolution melting analysis and investigation of the genetic diversity of Candida species

Journal of Clinical Laboratory Analysis ◽

10.1002/jcla.23444 ◽

2020 ◽

Vol 34 (10) ◽

Author(s):

Esmaeel Eghtedar Nejad ◽

Pooya Ghasemi Nejad Almani ◽

Mohammad Ali Mohammadi ◽

Samira Salari

Keyword(s):

Genetic Diversity ◽

High Resolution ◽

Real Time ◽

Molecular Identification ◽

Real Time Pcr ◽

Candida Species ◽

High Resolution Melting ◽

High Resolution Melting Analysis ◽

Melting Analysis

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Simple Technique for Internal Control of Real-Time Amplification Assays

Clinical Chemistry ◽

10.1373/clinchem.2003.027961 ◽

2004 ◽

Vol 50 (5) ◽

pp. 819-825 ◽

Cited By ~ 35

Author(s):

Siegfried Burggraf ◽

Bernhard Olgemöller

Keyword(s):

Melting Point ◽

Real Time ◽

Internal Control ◽

Real Time Pcr ◽

False Negative ◽

Hybridization Probe ◽

Melting Point Analysis ◽

Point Analysis ◽

Pcr Assays ◽

Time Amplification

Abstract Background: In real-time PCR assays, the most accurate way to identify false-negative results, e.g., those caused by PCR inhibitors, is to add to samples an internal control that will be coamplified with the target (e.g., pathogen) DNA. Current internal control procedures, however, which usually involve the introduction of a DNA fragment, are complex, time-consuming, and expensive. Methods: Single-stranded oligonucleotides, which contain little more than primer and probe binding sites, were used as internal controls in real-time PCR assays. Mismatches were included in the probe-binding region of the internal control oligonucleotide (ICO) to prevent probe–control hybridization during the fluorescence acquisition step of the PCR. Amplified ICOs were detected by melting point analysis. ICOs could be added directly to the sample material before DNA extraction. Results: To demonstrate the feasibility of the new approach, we designed ICOs for the LightCycler hybridization probe assays for Mycobacterium tuberculosis complex, hepatitis B virus, herpes simplex virus, and varicella zoster virus. In each case, the controls did not interfere with detection of the pathogen, but were clearly detectable during a subsequent melting point analysis. Conclusions: A single-stranded oligonucleotide that mimics the target region of the pathogen but is clearly distinguishable from the target during melting point analysis can serve as a simple, cost-effective internal control for real-time amplification assays. Such control oligonucleotides are easy to design and inexpensive. A costly second probe system is not necessary. Moreover, the internally controlled assay uses only one fluorescence detection channel of the instrument, leaving the second channel free for multiplex applications.

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Reliable Identification of the Type III Collagen Gene Polymorphism rs1800255 with the Use of High Resolution Melting Analysis

Laboratory Medicine ◽

10.1309/lmua5cs39fulvwem ◽

2009 ◽

Vol 40 (10) ◽

pp. 604-606 ◽

Cited By ~ 1

Author(s):

Sabrina L. Lince ◽

Kirsten B. Kluivers ◽

Jeroen R. Dijkstra ◽

Marjolein J.W. Janssen ◽

Mark E. Vierhout ◽

...

Keyword(s):

High Resolution ◽

Gene Polymorphism ◽

High Resolution Melting ◽

High Resolution Melting Analysis ◽

Type Iii Collagen ◽

Collagen Gene ◽

Reliable Identification ◽

Type Iii ◽

Melting Analysis

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High-resolution melting PCR assay, applicable for diagnostics and screening studies, allowing detection and differentiation of several Babesia spp. infecting humans and animals

Parasitology Research ◽

10.1007/s00436-017-5576-x ◽

2017 ◽

Vol 116 (10) ◽

pp. 2671-2681 ◽

Cited By ~ 6

Author(s):

Wioletta Rozej-Bielicka ◽

Aleksander Masny ◽

Elzbieta Golab

Keyword(s):

High Resolution ◽

High Resolution Melting ◽

Pcr Assay ◽

Babesia Spp

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A REPORT ABOUT BENZOXAZINONE MATERIAL

Chemistry & Material Sciences Research Journal ◽

10.51594/cmsrj.v2i1.91 ◽

2020 ◽

Vol 2 (1) ◽

pp. 30-41

Author(s):

Abdul Ahad

Keyword(s):

Melting Point ◽

Acetyl Chloride ◽

Melting Points ◽

Capillary Method ◽

Analytical Studies ◽

Melting Point Analysis ◽

Point Analysis ◽

Amino Phenol

In this study, the objective was to investigate the Benzoxazinone material using the uncorrected and open capillary method for conducting and reporting the melting points. Laboratory grade and analytical grade reagents were used for conducting the synthesis and analytical studies based on with or without modification appropriately as and were required. Results showed that First of all the Synthesis of 2H-1, 4- benzoxazin-3(4H)-one was carried out by reacting 2- amino phenol with chloro acetyl chloride in dichloromethane in presence of triethylamine and then the bromo substitution was done by reacting with dibromoethane. Piperazine substituents were prepared in laboratory and then the title compounds were synthesized. One additional benzoyl substitution was also done. The entire synthesized compounds were primarily characterized by running T.L.C. and melting point analysis.

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

10.21203/rs.2.16996/v1 ◽

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.

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