scholarly journals Cost-effective and fast KIR gene-content genotyping by multiplex melting curve analysis

HLA ◽  
2018 ◽  
Vol 92 (6) ◽  
pp. 384-391
Author(s):  
Leonardo M. Amorim ◽  
Tiago H. S. Santos ◽  
Jill A. Hollenbach ◽  
Paul J. Norman ◽  
Wesley M. Marin ◽  
...  
Keyword(s):  
Melting Curve ◽  
Cost Effective ◽  
Curve Analysis ◽  
Gene Content ◽  
Melting Curve Analysis ◽  
Kir Gene

scholarly journals Detection of Eight Respiratory Bacterial Pathogens Based on Multiplex Real-Time PCR with Fluorescence Melting Curve Analysis

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Liuyang Hu ◽  
Bing Han ◽  
Qin Tong ◽  
Hui xiao ◽  
Donglin Cao
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Bacterial Pathogens ◽  
Melting Curve ◽  
Cost Effective ◽  
Culture Method ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Fluorescence Melting Curve Analysis

Background and Objective. Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Pseudomonas aeruginosa, and Mycobacterium tuberculosis are primary respiratory bacterial pathogens contributing to morbidity and mortality in developing countries. This study evaluated the diagnostic performance of multiplex real-time PCR with fluorescence melting curve analysis (MCA) assay, which was used to detect eight respiratory bacterial pathogens simultaneously. Methods. A total of 157 sputum specimens were examined by multiplex real-time with fluorescence MCA, and the results were compared with the conventional culture method. Results. Multiplex real-time PCR with fluorescence MCA specifically detected and differentiated eight respiratory bacterial pathogens by different melting curve peaks for each amplification product within 2 hours and exhibited high repeatability. The limit of detection ranged from 64 to 102 CFU/mL in the multiplex PCR system. Multiplex real-time PCR with fluorescence MCA showed a sensitivity greater than 80% and a 100% specificity for each pathogen. The kappa correlation of eight bacteria ranged from 0.89 to 1.00, and the coefficient of variation ranged from 0.05% to 0.80%. Conclusions. Multiplex real-time PCR with fluorescence MCA assay is a sensitive, specific, high-throughput, and cost-effective method to detect multiple bacterial pathogens simultaneously.

Rapid detection of non-deletional mutations causing α-thalassemia by multicolor melting curve analysis

2016 ◽  
Vol 54 (3) ◽  
Author(s):  
Qiuying Huang ◽  
Xudong Wang ◽  
Ning Tang ◽  
Chunjiang Zhu ◽  
Tizhen Yan ◽  
...  
Keyword(s):  
Rapid Detection ◽  
Sanger Sequencing ◽  
Melting Curve ◽  
High Sensitivity ◽  
Cost Effective ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Sensitivity Specificity

AbstractThe assay consisted of one pair of primers specific for theThe assay had a reproducibility of 100%, could detect gDNA of different genotype as low as 1 ng per reaction, and had an overall accuracy of 100% when compared with RDB analysis and Sanger sequencing.The developed assay is rapid, robust, and cost-effective while maintaining high sensitivity, specificity, and throughput.

Rapid and cost effective detection of small mutations in the DMD gene by high resolution melting curve analysis

2009 ◽  
Vol 19 (6) ◽  
pp. 383-390 ◽  
Author(s):  
Rowida Almomani ◽  
Nienke van der Stoep ◽  
Egbert Bakker ◽  
Johan T. den Dunnen ◽  
Martijn H. Breuning ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Melting Curve ◽  
Cost Effective ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
High Resolution Melting Curve ◽  
Dmd Gene

scholarly journals Barcoding Melting Curve Analysis for Rapid, Sensitive, and Discriminating Authentication of Saffron (Crocus sativusL.) from Its Adulterants

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Chao Jiang ◽  
Liang Cao ◽  
Yuan Yuan ◽  
Min Chen ◽  
Yan Jin ◽  
...  
Keyword(s):  
Curcuma Longa ◽  
Melting Curve ◽  
Cost Effective ◽  
Carthamus Tinctorius ◽  
Curve Analysis ◽  
Crocus Sativus ◽  
Melting Curve Analysis ◽  
Cost Effective Method ◽  
Premium Price ◽  
Zea May

Saffron (Crocus sativusL.) is one of the most important and expensive medicinal spice products in the world. Because of its high market value and premium price, saffron is often adulterated through the incorporation of other materials, such asCarthamus tinctoriusL. andCalendula officinalisL. flowers,HemerocallisL. petals,Daucus carotaL. fleshy root,Curcuma longaL. rhizomes,Zea mayL., andNelumbo nuciferaGaertn. stigmas. To develop a straightforward, nonsequencing method for rapid, sensitive, and discriminating detection of these adulterants in traded saffron, we report here the application of a barcoding melting curve analysis method (Bar-MCA) that uses the universal chloroplast plant DNA barcoding regiontrnH-psbAto identify adulterants. When amplified at DNA concentrations and annealing temperatures optimized for the curve analysis, peaks were formed at specific locations for saffron (81.92°C) and the adulterants:D. carota(81.60°C),C. tinctorius(80.10°C),C. officinalis(79.92°C),Dendranthema morifolium(Ramat.) Tzvel. (79.62°C),N. nucifera(80.58°C),Hemerocallis fulva(L.) L. (84.78°C), andZ. mays(84.33°C). The constructed melting curves for saffron and its adulterants have significantly different peak locations or shapes. In conclusion, Bar-MCA could be a faster and more cost-effective method to authenticate saffron and detect its adulterants.

scholarly journals Development and validation of cost-effective one-step multiplex RT-PCR assay for detecting the SARS-CoV-2 infection using SYBR Green melting curve analysis

2021 ◽  
Author(s):  
Shovon Lal Sarkar ◽  
A. S. M. Rubayet Ul Alam ◽  
Prosanto Kumar Das ◽  
Md. Hasan Ali Pramanik ◽  
Hassan M. Al-Emran ◽  
...  
Keyword(s):  
Internal Control ◽  
Melting Curve ◽  
Cost Effective ◽  
Curve Analysis ◽  
Sybr Green ◽  
Taqman Probe ◽  
Melting Curve Analysis ◽  
Rt Pcr ◽  
One Step ◽  
Commercial Kit

TaqMan probe-based expensive commercial real-time (RT) PCR kits are being used in COVID-19 diagnosis. The unprecedented scale of SARS-CoV-2 infections has urgently needed to meet the challenge of testing more persons at a reasonable cost. This study developed a rapid, simple, and cost-effective alternative diagnostic method based on melting curve analysis of SYBR green multiplex assay with a host-specific internal control. A total of 90 randomly selected samples were used for comparing the assay with an available commercial kit to analyse the variation and validity of this in-house developed method. Our customized designed primers specifically detected the virus as similar to commercial kit manufactured by Sansure Biotech Inc. We optimized separately the N, E, S, and RdRp genes by SYBR Green RT-PCR method based on melting curve analysis. Afterwards, a multiplex COVID-19 diagnosis method targeting N and E genes of the virus along with the β-actin gene of the host as an internal control has been established. The total run-time of our proposed method was less than 90 minutes. The cost of each sample processing was less than $2. Overall, this one-step and one-tube method can revolutionize the COVID-19 diagnosis in developing countries.

scholarly journals Rapid genotypic detection ofBacillus anthracisand theBacillus cereusgroup by multiplex real-time PCR melting curve analysis

2005 ◽  
Vol 43 (2) ◽  
pp. 301-310 ◽  
Author(s):  
Kijeong Kim ◽  
Juwon Seo ◽  
Katherine Wheeler ◽  
Chulmin Park ◽  
Daewhan Kim ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Melting Curve ◽  
Curve Analysis ◽  
Melting Curve Analysis

scholarly journals Diagnosis of α-1-Antitrypsin Deficiency: An Algorithm of Quantification, Genotyping, and Phenotyping

2006 ◽  
Vol 52 (12) ◽  
pp. 2236-2242 ◽  
Author(s):  
Melissa R Snyder ◽  
Jerry A Katzmann ◽  
Malinda L Butz ◽  
Ping Yang ◽  
D Brian Dawson ◽  
...  
Keyword(s):  
Laboratory Testing ◽  
Null Allele ◽  
Melting Curve ◽  
Curve Analysis ◽  
Laboratory Evaluation ◽  
Melting Curve Analysis ◽  
Genotype Assay ◽  
Antitrypsin Deficiency ◽  
Phenotype Analysis ◽  
A1at Deficiency

Abstract Background: Laboratory testing in suspected α-1-antitrypsin (A1AT) deficiency involves analysis of A1AT concentrations and identification of specific alleles by genotyping or phenotyping. The purpose of this study was to define and evaluate a strategy that provides reliable laboratory evaluation of A1AT deficiency. Methods: Samples from 512 individuals referred for A1AT phenotype analysis were analyzed by quantification, phenotype, and genotype. A1AT concentrations were measured by nephelometry. Phenotype analysis was performed by isoelectric focusing electrophoresis. The genotype assay detected the S and Z deficiency alleles by a melting curve analysis. Results: Of the 512 samples analyzed, 2% of the phenotype and genotype results were discordant. Among these 10 discordant results, 7 were attributed to phenotyping errors. On the basis of these data we formulated an algorithm, according to which we analyzed samples by genotyping and quantification assays, with a reflex to phenotyping when the genotype and quantification results were not concordant. Retrospective analyses demonstrated that 4% of samples submitted for genotype and quantitative analysis were reflexed to phenotyping. Of the reflexed samples, phenotyping confirmed the genotype result in 85% of cases. In the remaining 15%, phenotyping provided further information, including identifying rare deficiency alleles and suggesting the presence of a null allele, and allowed for a more definitive interpretation of the genotype result. Conclusions: The combination of genotyping and quantification, with a reflex to phenotyping, is the optimal strategy for the laboratory evaluation of A1AT deficiency.

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