High-Resolution Melting Curve Analysis to Predict Extended Blood Group Phenotypes among Thai Donors and Patients

Background: High-resolution melting (HRM) analysis is an alternative method for red cell genotyping. Differences in melting curves between homozygous and heterozygous genotypes can predict phenotypes in blood group systems based on single-nucleotide polymorphisms. This study aimed to implement HRM analysis to predict additional extended blood group phenotypes in Thai donor and patient populations. Methods: Blood samples obtained from 300 unrelated Thai blood donors and 23 patients with chronic transfusions were included. HRM analysis was developed and validated in genotyping of KEL*01 and KEL*02, JK*01 and JK*02, FY*01, FY*02, and FY*02 N.01, DI*01 and DI*02, GYPB*03 and GYPB*04, RHCE*E and RHCE*e, and DO*01 and DO*02. Then genotyping results from HRM and polymerase chain reaction with sequence-specific primer (PCR-SSP) and phenotyping results were compared. Results: The validated genotyping results in known DNA controls by HRM analysis agreed with DNA sequencing. The genotyping results among 300 donors in 15 alleles by HRM analysis were in complete concordance with those obtained by serological testing and PCR-SSP. The sensitivity and specificity of the HRM assay were both 100%. Among patients, 13 had alloantibodies that possessed predicted antigen-negative phenotypes corresponding to those antibody specificities, and the highest probability of genotyped-matched donors was given to the remaining patients. Conclusions: We developed and implemented the HRM analysis assay for red cell genotyping to predict extended blood group antigens in Thai donor and patient populations. The data from this study may help inform about and support transfusion care of Thai patients to reduce the risk of alloimmunisation.

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Identification of Four Mud Crab Species (Genus Scylla) based on High-resolution Melting (HRM) Analysis

10.21203/rs.3.rs-420600/v1 ◽

2021 ◽

Author(s):

Yongliang Li ◽

Ronghua Li ◽

Xiayue Chen ◽

Chunlin Wang ◽

Changkao Mu ◽

...

Keyword(s):

High Resolution ◽

High Resolution Melting ◽

Melting Curve ◽

Scylla Serrata ◽

Scylla Paramamosain ◽

Melting Curve Analysis ◽

Single Pair ◽

Crab Species ◽

Mud Crab ◽

Hrm Analysis

Abstract In this study, we described the successful identification of four mud crab species (genus Scylla) by high-resolution melting curve analysis (HRM) of single nucleotide polymorphisms (SNPs). The complete mitochondrion genome sequences of Scylla serrata, Scylla paramamosain, Scylla olivacea and Scylla tranquebarica were aligned and screened for distinct fragments. A single pair of primers were designed to amplify the 203-bp consensus amplicon. As a result, a total of 96 samples from the four mud crab species were clearly separated. The melting profiles from HRM analysis were found to be distinct across the species tested, and a number of 28 SNP sites were confirmed by sequencing. This new identification method will be a useful tool for discrimination of the four Scylla species and contribute to the effective breeding management of these species.

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Rapid screening for targeted genetic variants via high-resolution melting curve analysis

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2016-0603 ◽

2017 ◽

Vol 55 (4) ◽

Cited By ~ 2

Author(s):

Allison B. Chambliss ◽

Molly Resnick ◽

Athena K. Petrides ◽

William A. Clarke ◽

Mark A. Marzinke

Keyword(s):

Cytochrome P450 ◽

High Resolution ◽

Genetic Variants ◽

High Resolution Melting ◽

Melting Curve ◽

Rapid Screening ◽

Melting Curve Analysis ◽

Nucleotide Polymorphisms ◽

Cytochrome P450 Enzymes ◽

Confirmatory Testing

Abstract Background: Current methods for the detection of single nucleotide polymorphisms (SNPs) associated with aberrant drug-metabolizing enzyme function are hindered by long turnaround times and specialized techniques and instrumentation. In this study, we describe the development and validation of a high-resolution melting (HRM) curve assay for the rapid screening of variant genotypes for targeted genetic polymorphisms in the cytochrome P450 enzymes CYP2C9, CYP2C19, and CYP3A5. Methods: Sequence-specific primers were custom-designed to flank nine SNPs within the genetic regions of aforementioned drug metabolizing enzymes. PCR amplification was performed followed by amplicon denaturation by precise temperature ramping in order to distinguish genotypes by melting temperature (T Results: Intra-assay (n=5) precision of T Conclusions: The presented assay provides a rapid method for the screening for genetic variants in targeted CYP450 regions with a result of ‘reference’ or ‘variant’ available within 2 h from receipt of extracted DNA. The method can serve as a screening approach to rapidly identify individuals with variant sequences who should be further investigated by reflexed confirmatory testing for aberrant cytochrome P450 enzymatic activity. Rapid knowledge of variant status may aid in the avoidance of adverse clinical events by allowing for dosing of normal metabolizer patients immediately while identifying the need to wait for confirmatory testing in those patients who are likely to possess pharmacogenetically-relevant variants.

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High-Resolution Melting Curve Analysis of Genomic and Whole-Genome Amplified DNA

Clinical Chemistry ◽

10.1373/clinchem.2008.109744 ◽

2008 ◽

Vol 54 (12) ◽

pp. 2055-2058 ◽

Cited By ~ 27

Author(s):

Michael H Cho ◽

Dawn Ciulla ◽

Barbara J Klanderman ◽

Benjamin A Raby ◽

Edwin K Silverman

Keyword(s):

High Resolution ◽

High Resolution Melting ◽

Melting Curve ◽

Curve Analysis ◽

Melting Curve Analysis ◽

Dna Melting ◽

Whole Genome ◽

Nucleotide Polymorphisms ◽

Wild Type ◽

High Resolution Melting Curve

Abstract Background: High-resolution melting curve analysis is an accurate method for mutation detection in genomic DNA. Few studies have compared the performance of high-resolution DNA melting curve analysis (HRM) in genomic and whole-genome amplified (WGA) DNA. Methods: In 39 paired genomic and WGA samples, 23 amplicons from 9 genes were PCR amplified and analyzed by high-resolution melting curve analysis using the 96-well LightScanner (Idaho Technology). We used genotyping and bidirectional resequencing to verify melting curve results. Results: Melting patterns were concordant between the genomic and WGA samples in 823 of 863 (95%) analyzed sample pairs. Of the discordant patterns, there was an overrepresentation of alternate melting curve patterns in the WGA samples, suggesting the presence of a mutation (false positives). Targeted resequencing in 135 genomic and 136 WGA samples revealed 43 single nucleotide polymorphisms (SNPs). All SNPs detected in genomic samples were also detected in WGA. Additional genotyping and sequencing allowed the classification of 628 genomic and 614 WGA amplicon samples. Heterozygous variants were identified by non–wild-type melting pattern in 98% of genomic and 97% of WGA samples (P = 0.11). Wild types were correctly classified in 99% of genomic and 91% of WGA samples (P < 0.001). Conclusions: In WGA DNA, high-resolution DNA melting curve analysis is a sensitive tool for SNP discovery through detection of heterozygote variants; however, it may misclassify a greater number of wild-type samples.

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

Journal of the Hellenic Veterinary Medical Society ◽

10.12681/jhvms.16425 ◽

2018 ◽

Vol 69 (1) ◽

pp. 783 ◽

Cited By ~ 1

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.

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One-Step Triplex High-Resolution Melting Analysis for Rapid Identification and Simultaneous Subtyping of Frequently Isolated Salmonella Serovars

Applied and Environmental Microbiology ◽

10.1128/aem.07668-11 ◽

2012 ◽

Vol 78 (9) ◽

pp. 3352-3360 ◽

Cited By ~ 15

Author(s):

Josef Zeinzinger ◽

Ariane T. Pietzka ◽

Anna Stöger ◽

Christian Kornschober ◽

Renate Kunert ◽

...

Keyword(s):

High Resolution ◽

Salmonella Enterica ◽

High Resolution Melting ◽

Melting Curve ◽

Cost Effective ◽

Melting Curve Analysis ◽

Nucleotide Polymorphisms ◽

Content Type ◽

High Resolution Melting Curve ◽

Gene Scanning

ABSTRACTSalmonellosis is one of the most important food-borne diseases worldwide. For outbreak investigation and infection control, accurate and fast subtyping methods are essential. A triplex gene-scanning assay was developed and evaluated for serotype-specific subtyping ofSalmonella entericaisolates based on specific single-nucleotide polymorphisms in fragments offljB,gyrB, andycfQ. Simultaneous gene scanning offljB,gyrB, andycfQby high-resolution melting-curve analysis of 417Salmonellaisolates comprising 46 different serotypes allowed the unequivocal, simple, and fast identification of 37 serotypes. Identical melting-curve profiles were obtained in some cases fromSalmonella entericaserotype Enteritidis andSalmonella entericaserotype Dublin, in all cases fromSalmonella entericaserotype Ohio andSalmonella entericaserotype Rissen, fromSalmonella entericaserotype Mbandaka andSalmonella entericaserotype Kentucky, and fromSalmonella entericaserotype Bredeney,Salmonella entericaserotype Give, andSalmonella entericaserotype Schwarzengrund. To differentiate the most frequentSalmonellaserotype, Enteritidis, from someS. Dublin isolates, an additional single PCR assay was developed for specific identification ofS. Enteritidis. The closed-tube triplex high-resolution melting-curve assay developed, in combination with anS. Enteritidis-specific PCR, represents an improved protocol for accurate, cost-effective, simple, and fast subtyping of 39Salmonellaserotypes. These 39 serotypes represent more than 94% of all human and more than 85% of all nonhumanSalmonellaisolates (including isolates from veterinary, food, and environmental samples) obtained in the years 2008 and 2009 in Austria.

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Identification and Differentiation of Monilinia Species Causing Brown Rot of Pome and Stone Fruit using High-Resolution Melting (HRM) Analysis

Phytopathology ◽

10.1094/phyto-01-16-0016-r ◽

2016 ◽

Vol 106 (9) ◽

pp. 1055-1064 ◽

Cited By ~ 12

Author(s):

Antonios Papavasileiou ◽

Panagiotis B. Madesis ◽

George S. Karaoglanidis

Keyword(s):

High Resolution ◽

High Resolution Melting ◽

Melting Curve ◽

Brown Rot ◽

Rapid Identification ◽

Stone Fruit ◽

Universal Primers ◽

Melting Curve Analysis ◽

Fungal Isolation ◽

Hrm Analysis

Brown rot is a devastating disease of stone fruit caused by Monilinia spp. Among these species, Monilinia fructicola is a quarantine pathogen in Europe but has recently been detected in several European countries. Identification of brown rot agents relies on morphological differences or use of molecular methods requiring fungal isolation. The current study was initiated to develop and validate a high-resolution melting (HRM) method for the identification of the Monilinia spp. and for the detection of M. fructicola among other brown rot pathogens. Based on the sequence of the cytb intron from M. laxa, M. fructicola, M. fructigena, M. mumecola, M. linhartiana, and M. yunnanensis isolates originating from several countries, a pair of universal primers for species identification and a pair of primers specific to M. fructicola were designed. The specificity of the primers was verified to ensure against cross-reaction with other fungal species. The melting curve analysis using the universal primers generated six different HRM curve profiles, each one specific for each species. Τhe HRM analysis primers specific to M. fructicola amplified a 120-bp region with a distinct melt profile corresponding to the presence of M. fructicola, regardless of the presence of other species. HRM analysis can be a useful tool for rapid identification and differentiation of the six Monilinia spp. using a single primer pair. This novel assay has the potential for simultaneous identification and differentiation of the closely related Monilinia spp. as well as for the differentiation of M. fructicola from other common pathogens or saprophytes that may occur on the diseased fruit.

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