scholarly journals Modified low cost SNP genotyping technique using cycle threshold (Ct) & melting temperature (Tm) values in allele specific real-time PCR

2015 ◽  
Vol 142 (5) ◽  
pp. 555 ◽  
Author(s):  
BVishnu Bhat ◽  
DBenet Bosco Dhas ◽  
AHiasindh Ashmi ◽  
SubashChandra Parija ◽  
N Banupriya
Keyword(s):  
Real Time ◽  
Melting Temperature ◽  
Real Time Pcr ◽  
Low Cost ◽  
Snp Genotyping ◽  
Cycle Threshold ◽  
Allele Specific

scholarly journals Single-copy detection of somatic variants from solid and liquid biopsy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ana-Luisa Silva ◽  
Paulina Klaudyna Powalowska ◽  
Magdalena Stolarek ◽  
Eleanor Ruth Gray ◽  
Rebecca Natalie Palmer ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Real Time Pcr ◽  
Clinical Decision Making ◽  
High Sensitivity ◽  
Clinical Decision ◽  
Single Copy ◽  
Turnaround Time ◽  
Copy Detection ◽  
Allele Specific

AbstractAccurate detection of somatic variants, against a background of wild-type molecules, is essential for clinical decision making in oncology. Existing approaches, such as allele-specific real-time PCR, are typically limited to a single target gene and lack sensitivity. Alternatively, next-generation sequencing methods suffer from slow turnaround time, high costs, and are complex to implement, typically limiting them to single-site use. Here, we report a method, which we term Allele-Specific PYrophosphorolysis Reaction (ASPYRE), for high sensitivity detection of panels of somatic variants. ASPYRE has a simple workflow and is compatible with standard molecular biology reagents and real-time PCR instruments. We show that ASPYRE has single molecule sensitivity and is tolerant of DNA extracted from plasma and formalin fixed paraffin embedded (FFPE) samples. We also demonstrate two multiplex panels, including one for detection of 47 EGFR variants. ASPYRE presents an effective and accessible method that simplifies highly sensitive and multiplexed detection of somatic variants.

Complement C3 Genotyping of Slow and Fast Variants by Real Time PCR-High Resolution Melting

2012 ◽  
Vol 10 (3) ◽  
pp. 329-334 ◽  
Author(s):  
D.M. Valero-Hervás ◽  
P. Morales ◽  
M.J. Castro ◽  
P. Varela ◽  
M. Castillo-Rama ◽  
...  
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Real Time Pcr ◽  
High Resolution Melting ◽  
Low Cost ◽  
Complement C3 ◽  
Amplification Refractory Mutation System ◽  
Rt Pcr ◽  
Dna Amount ◽  
Mutation System

“Slow” and “Fast” C3 complement variants (C3S and C3F) result from a g.304C>G polymorphism that changes arginine to glycine at position 102. C3 variants are associated with complement-mediated diseases and outcome in transplantation. In this work C3 genotyping is achieved by a Real Time PCR - High Resolution Melting (RT-PCR-HRM) optimized method. In an analysis of 49 subjects, 10.2% were C3FF, 36.7% were C3SF and 53.1% were C3SS. Allelic frequencies (70% for C3S and 30% for C3F) were in Hardy-Weinberg equilibrium and similar to those published previously. When comparing RT-PCR-HRM with the currently used Tetraprimer-Amplification Refractory Mutation System PCR (T-ARMS-PCR), coincidence was 93.8%. The procedure shown here includes a single primer pair and low DNA amount per reaction. Detection of C3 variants by RT-PCR-HRM is accurate, easy, fast and low cost, and it may be the method of choice for C3 genotyping.

Ultrafast real-time PCR with integrated melting curve analysis and duplex capacities using a low-cost polymer lab-on-a-chip system

2015 ◽  
Author(s):  
Rainer Gransee ◽  
Tristan Schneider ◽  
Deniz Elyorgun ◽  
Xenia Strobach ◽  
Tobias Schunck ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Low Cost ◽  
Melting Curve ◽  
Lab On A Chip ◽  
Curve Analysis ◽  
Melting Curve Analysis

scholarly journals Prediction of sofosbuvir response using interleukin-6 serum level and single nucleotide polymorphism of interferon lambda- 4

2020 ◽  
Vol 14 (01) ◽  
pp. 80-88
Author(s):  
Amal E Saafan ◽  
Ashraf Abobaker ◽  
Mohamed S Abbas ◽  
Ahmed El-Gendy
Keyword(s):  
Serum Level ◽  
Real Time ◽  
Real Time Pcr ◽  
Dual Therapy ◽  
Snp Genotyping ◽  
Nucleotide Polymorphism ◽  
Time Intervals ◽  
Genotype 4 ◽  
Single Nucleotide ◽  
Taqman Probes

Introduction: In Egypt, 15% of the populations are suffering from chronic hepatitis C especially genotype 4. Sofosbuvir was approved by FDA in December 2013 for treatment of HCV genotypes 2 and 3 in combination with Ribavirin, and for genotypes 1 and 4 in combination with Peg-IFN. Recently, polymorphism of different genes and plasma levels of IL-6 were utilized for better prediction of HCV clearance. This study aimed at early prediction of the efficacy of HCV treatment with Sofosbuvir (Sovaldi) and comparing the antiviral efficacy of dual and triple Sovaldi combination therapy. Methodology: Blood samples were collected from 100 HCV positive patients and detected by real time PCR at three time intervals. SNP genotyping of INFL-4 gene was estimated by using real-time PCR with predesigned primers and Taqman probes. IL-6 serum level was estimated before, during and after the end of the treatment using ELISA assay based on human IL-6 KIT. Results: SNP genotyping of INFL-4 gene showed that 13.1% of patients carried ∆G/∆G, 30.4% patients had TT/TT and 56.5% patients possessed heterozygote allele ∆G/TT. Clinical data displayed that 13 patients were got relapsed at SVR 12. Serum level of IL-6 was noticed higher in HCV patients than healthy ones. Noteworthy, it was increased during treatment then decreased to a minimal level than begining of treatment. Conclusion: SNP in INFL-4 gene has displayed no effect in response to Sofosbuvir. Dual therapy had the same effect like triple therapy, so interferon could be withdrawn from the treatment regimen.

scholarly journals Allele-Specific Real-Time PCR System for Detection of Subpopulations of Genotype 1a and 1b Hepatitis C NS5B Y448H Mutant Viruses in Clinical Samples

2011 ◽  
Vol 49 (9) ◽  
pp. 3168-3174 ◽  
Author(s):  
Andrew S. Bae ◽  
Karin S. Ku ◽  
Michael D. Miller ◽  
Hongmei Mo ◽  
Evguenia S. Svarovskaia
Keyword(s):  
Hepatitis C ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Samples ◽  
Genotype 1A ◽  
Allele Specific

scholarly journals A Novel Broad Allele-Specific TaqMan Real-Time PCR Method To Detect Triazole-Resistant Strains of Aspergillus fumigatus, Even with a Very Low Percentage of Triazole-Resistant Cells Mixed with Triazole-Susceptible Cells

2019 ◽  
Vol 57 (9) ◽  
Author(s):  
Qian Wang ◽  
Dimitrios P. Kontoyiannis ◽  
Ruoyu Li ◽  
Wei Chen ◽  
Dingfang Bu ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Real Time ◽  
Real Time Pcr ◽  
High Efficiency ◽  
Gene Mutations ◽  
Health Concern ◽  
Content Type ◽  
Clinical Strains ◽  
Allele Specific ◽  
Resistant Strains

ABSTRACT Invasive aspergillosis caused by triazole-resistant strains of Aspergillus fumigatus is a growing public health concern, as is the occurrence of mixed infections with triazole-resistant and -susceptible A. fumigatus strains. Therefore, it is crucial to develop robust methods to identify triazole-resistant strains of A. fumigatus, even in mixtures of triazole-resistant and -susceptible strains of A. fumigatus. In this work, we developed a robust, highly selective, and broad-range allele-specific TaqMan real-time PCR platform consisting of 7 simultaneous assays that detect TR34 (a 34-bp tandem repeat in the promoter region), TR46, G54W (a change of G to W at position 54), G54R, L98H, Y121F, and M220I mutations in the cyp51A gene of A. fumigatus. The method is based on the widely used TaqMan real-time PCR technology and combines allele-specific PCR with a blocking reagent (minor groove binder [MGB] oligonucleotide blocker) to suppress amplification of the wild-type cyp51A alleles. We used this method to detect triazole-resistant clinical strains of A. fumigatus with a variety of cyp51A gene mutations, as well as the triazole-resistant strains in mixtures of triazole-resistant and -susceptible strains of A. fumigatus. The method had high efficiency and sensitivity (300 fg/well, corresponding to about 100 CFU per reaction mixture volume). It could promptly detect triazole resistance in a panel of 30 clinical strains of A. fumigatus within about 6 h. It could also detect cyp51A-associated resistance alleles, even in mixtures containing only 1% triazole-resistant A. fumigatus strains. These results suggest that this method is robustly able to detect cyp51A-associated resistance alleles even in mixtures of triazole-resistant and -susceptible strains of A. fumigatus and that it should have important clinical applications.

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