scholarly journals Three-gene PCR and high-resolution melting analysis for differentiating vertebrate species mitochondrial DNA for forensic and biodiversity research pipelines

2019 ◽  
Author(s):  
Daniel O. Ouso ◽  
Moses Y. Otiende ◽  
Maamun Jeneby ◽  
Joseph W. Oundo ◽  
Joel L. Bargul ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Low Cost ◽  
Forensic Identification ◽  
Vertebrate Species ◽  
East African ◽  
Biodiversity Research ◽  
Large Numbers ◽  
Routine Surveillance ◽  
Pcr Products

AbstractReliable molecular identification of vertebrate species from morphologically unidentifiable tissue is critical for the prosecution of illegally-traded wildlife products, conservation-based biodiversity research, and identification of blood-meal hosts of hematophagous invertebrates. However, forensic identification of vertebrate tissue relies on the sequencing of mitochondrial cytochrome oxidase I (COI) ‘barcode’ genes, which remains costly for purposes of screening large numbers of unknown samples during routine surveillance. Here, we adopted a rapid, low-cost approach to differentiate 10 domestic and 24 wildlife species that are common in the East African illegal wildlife products trade based on their unique high-resolution melting profiles from COI, cytochrome b, and 16S ribosomal RNA gene PCR products. Using the approach, we identified (i) giraffe among covertly sampled meat from Kenyan butcheries, and (ii) forest elephant mitochondrial sequences among savannah elephant reference samples. This approach is being adopted for high-throughput pre-screening of potential bushmeat samples in East African forensic science pipelines.

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.

scholarly journals A High Resolution Melting Analysis-Based Genotyping Toolkit for the Peach (Prunus persica) Chilling Requirement

2020 ◽  
Vol 21 (4) ◽  
pp. 1543
Author(s):  
Lin Chou ◽  
Shih-Jie Huang ◽  
Chen Hsieh ◽  
Ming-Te Lu ◽  
Chia-Wei Song ◽  
...  
Keyword(s):  
High Resolution ◽  
Prunus Persica ◽  
High Resolution Melting ◽  
Physical Map ◽  
Low Cost ◽  
Principal Component ◽  
Chilling Requirement ◽  
Nucleotide Polymorphisms ◽  
Genotyping Platform ◽  
Peach Breeding

The chilling requirement (CR) is the main factor controlling the peach floral bud break and subsequent reproductive growth. To date, several peach CR quantitative trait loci (QTLs) have been identified. To improve the accessibility and convenience of this genetic information for peach breeders, the aim of this study was to establish an easy-to-use genotype screening system using peach CR molecular markers as a toolkit for marker-assisted selection. Here, we integrated 22 CR-associated markers from three published QTLs and positioned them on the Prunus persica physical map. Then, we built a PCR-based genotyping platform by using high-resolution melting (HRM) analysis with specific primers and trained this platform with 27 peach cultivars. Due to ambiguous variant calls from a commercial HRM software, we developed an R-based pipeline using principal component analysis (PCA) to accurately differentiate genotypes. Based on the PCA results, this toolkit was able to determine the genotypes at the CR-related single nucleotide polymorphisms (SNPs) in all tested peach cultivars. In this study, we showed that this HRM-PCA pipeline served as a low-cost, high-throughput, and non-gel genotyping solution. This system has great potential to accelerate CR-focused peach breeding.

scholarly journals Traceability of PDO Olive Oil “Terra di Bari” Using High Resolution Melting

2015 ◽  
Vol 2015 ◽  
pp. 1-7 ◽  
Author(s):  
Cinzia Montemurro ◽  
Monica Marilena Miazzi ◽  
Antonella Pasqualone ◽  
Valentina Fanelli ◽  
Wilma Sabetta ◽  
...  
Keyword(s):  
High Resolution ◽  
Olive Oil ◽  
High Resolution Melting ◽  
Virgin Olive Oil ◽  
Cost Effective ◽  
Extra Virgin Olive Oil ◽  
Hrm Analysis ◽  
Olive Cultivars ◽  
Pcr Products ◽  
Set Up

The aim of the research was to verify the applicability of microsatellite (SSR) markers in High Resolution Melting (HRM) analysis for the identification of the olive cultivars used in the “Terra di Bari” PDO extra virgin olive oil. A panel of nine cultivars, widespread in Apulia region, was tested with seventeen SSR primer pairs and the PCR products were at first analysed with a Genetic Analyzer automatic sequencer. An identification key was obtained for the nine cultivars, which showed an unambiguous discrimination among the varieties constituting the “Terra di Bari” PDO extra virgin olive oil: Cima di Bitonto, Coratina, and Ogliarola. Subsequently, an SSR based method was set up with the DCA18 marker, coupled with HRM analysis for the distinction of the Terra di Bari olive oil from non-Terra di Bari olive oil using different mixtures. Thus, this analysis enabled the distinction and identification of the PDO mixtures. Hence, this assay provided a flexible, cost-effective, and closed-tube microsatellite genotyping method, well suited to varietal identification and authentication analysis in olive oil.

scholarly journals Sensitivity and Specificity of Single-Nucleotide Polymorphism Scanning by High-Resolution Melting Analysis

2004 ◽  
Vol 50 (10) ◽  
pp. 1748-1754 ◽  
Author(s):  
Gudrun H Reed ◽  
Carl T Wittwer
Keyword(s):  
Single Nucleotide Polymorphism ◽  
High Resolution ◽  
Sensitivity And Specificity ◽  
High Resolution Melting ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Product Size ◽  
Pcr Products ◽  
Melting Analysis

Abstract Background: Screening for heterozygous sequence changes in PCR products, also known as “mutation scanning”, is an important tool for genetic research and clinical applications. Conventional methods require a separation step. Methods: We evaluated the sensitivity and specificity of homogeneous scanning, using a saturating DNA dye and high-resolution melting. Heterozygous single-nucleotide polymorphism (SNP) detection was studied in three different sequence backgrounds of 40%, 50%, and 60% GC content. PCR products of 50–1000 bp were generated in the presence of LCGreen™ I. After fluorescence normalization and temperature overlay, melting curve shape was used to judge the presence or absence of heterozygotes among 1632 cases. Results: For PCR products of 300 bp or less, all 280 heterozygous and 296 wild-type cases were correctly called without error. In 672 cases between 400 and 1000 bp with the mutation centered, the sensitivity and specificity were 96.1% and 99.4%, respectively. When the sequence background and product size with the greatest error rate were used, the sensitivity of off-center SNPs (384 cases) was 95.6% with a specificity of 99.4%. Most false negatives occurred with SNPs that were compared with an A or T wild type sequence. Conclusions: High-resolution melting analysis with the dye LCGreen I identifies heterozygous single-base changes in PCR products with a sensitivity and specificity comparable or superior to nonhomogeneous techniques. The error rate of scanning depends on the PCR product size and the type of base change, but not on the position of the SNP. The technique requires only PCR reagents, the dye LCGreen I, and 1–2 min of closed-tube, post-PCR analysis.

Abstract 2115: Increased sensitivity ofKRASmutation detection by High-Resolution Melting analysis of COLD-PCR products

2010 ◽  
Author(s):  
Iben L. Daugaard ◽  
Lasse S. Kristensen ◽  
Tina Kjeldsen ◽  
Stephen Hamilton Dutoit ◽  
Henrik Hager ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
High Resolution Melting Analysis ◽  
Pcr Products ◽  
Melting Analysis ◽  
Increased Sensitivity

scholarly journals Identification of new avian Infectious Bronchitis virus variants in Iranian poultry flocks by High Resolution Melting curve analysis

2018 ◽  
Vol 69 (1) ◽  
pp. 783 ◽  
Author(s):  
A. MASOUDIAN ◽  
N. SHEIKHI ◽  
M. H. BOZORGMEHRI-FARD
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Melting Curve ◽  
Melting Curve Analysis ◽  
Nucleotide Sequencing ◽  
Infectious Bronchitis ◽  
Hrm Analysis ◽  
High Resolution Melting Curve ◽  
Performance Reduction ◽  
Pcr Products

Avian Infectious bronchitis (IB) is a common coronavirus infection of chickens and responsible for performance reduction and increasing mortality due to subsequent respiratory, renal and/or reproductive disorders. Classification of causative agent is necessary to plan successful vaccination strategies to prevent the infection due to poor inter-strains cross-reaction. To identify dominant circulating strains in Iran, a Real-time PCR combined with 3’ Un-Translated Region (3’ UTR) High Resolution Melting (HRM) analysis designed as a rapid and reliable method for IB Virus (IBV) detection and differentiation. Samples collected from 20-suspected flocks and after PCR products, HRM curves of samples as well as 6 commercial IB live vaccines with 2 standard strains, were analyzed as references. IBV genomes detected in 11 samples while according to HRM analysis and calculating Genotype Confidence Percentage (GCP), 6 positive specimens identified as 793/B field strains and the left 5 found as new IBV variant strains. Then obtained PCR products sent for nucleotide sequencing to determine genotype relativity. All five infectious agents, related to QX-like type and indicating circulation of new variants in Iran as a probable cause of vaccination failures and consequent economical losses.

scholarly journals Validation of a Novel Molecular Assay to the Diagnostic of COVID-19 Based on Real Time PCR with High Resolution Melting

2021 ◽  
Author(s):  
Beatriz Iandra da Silva Ferreira ◽  
Natalia Lins da Silva Gomes ◽  
Wagner Luis da Costa Nunes Pimentel Coelho ◽  
Vanessa Duarte Costa ◽  
Vanessa Cristine de Souza Carneiro ◽  
...  
Keyword(s):  
High Resolution ◽  
Internal Control ◽  
High Resolution Melting ◽  
Low Cost ◽  
Cost Effective ◽  
Alternative Methods ◽  
Molecular Diagnostic ◽  
Molecular Biology Technique ◽  
Clinical Specimens ◽  
P Gene

With the emergence of the Covid-19 pandemic, the world faced an unprecedented need for RT-qPCR-based molecular diagnostic tests, leading to a lack of kits and inputs, especially in developing countries. Hence, the costs for commercial kits and inputs were overrated, stimulating the development of alternative methods to detect SARS-CoV-2 in clinical specimens. The availability of the complete SARS-CoV-2 genome at the beginning of the pandemic facilitated the development of specific primers and standardized laboratory protocols for Covid-19 molecular diagnostic. High-sensitive and cost-effective molecular biology technique based on the Melting Temperature differences between purine and pyrimidine bases can be used to the detection and genotyping of pathogens in clinical specimens. Here, a RT-qPCR assays with High Resolution Melting (HRM-RTqPCR) was developed for different regions of the SARS-CoV-2 genome (RdRp, E and N) and an internal control (human RNAse P gene). The assays were validated using synthetic sequences from the viral genome and clinical specimens (nasopharyngeal swabs, serum and saliva) of sixty-five patients with severe or moderate COVID-19 from different states in Brazil, in comparison to a commercial TaqMan RT-qPCR assay, as gold standard. The sensitivity of the HRM-RTqPCR assays targeting N, RdRp and E were 94.12, 98.04 and 92.16%, with 100% specificity to the 3 targets, and diagnostic accuracy of 95.38, 98.46 and 93.85%, respectively. Thus, the HRM-RTqPCR emerges as an alternative and low-cost methodology to increase the molecular diagnostic of patients suspicious for Covid-19, especially in restricted-budget laboratories.

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