High Resolution Melting Analysis For Detection Research Articles

High-resolution melting analysis for detection of fusion allele of FUT2.

The secretor status of ABH antigens, determined by FUT2 polymorphisms, affects susceptibility to various infectious diseases. In addition to many SNPs responsible for the nonsecretor phenotype, five nonfunctional alleles (se) resulting from copy number variations have been reported. One of the five alleles generated by an unequal crossover between FUT2 and a pseudogene (SEC1), is sefus . This allele may be misidentified as a functional allele if only common inactivating SNPs are genotyped because it contains the 3' region of the functional FUT2. Therefore, accurate detection of sefus is desirable. For this purpose, a high-resolution melting (HRM) analysis is developed for detection of sefus in which a 284bp fragment of SEC1 and sefus but not FUT2, are amplified. This HRM analysis detected sefus reliably. Thus, an initial screening or prescreening for sefus using HRM analysis seems to be useful for association studies of FUT2.

The comparison of two whole genome amplification methods: A new method of pre-implantation genetic testing (PGT-M) for Southeast Asian deletion in alpha-thalassemia using high resolution melting analysis

Introduction In Southeast Asia, alpha-thalassemia with Southeast Asian (SEA) deletion type is the most common mutations of alpha-thalassemia-1 which can cause severe form known as Bart's hydrops fetalis. For couples which carrier of single gene defects, pre-implantation genetic testing for monogenic (PGT-M) plays a role in genetic testing for selecting of unaffected embryos prior implantation. Recently, high resolution melting (HRM) analysis is a simple, rapid and widely used for genotyping and gene scanning of genetic variation. In this study, we developed HRM analysis method for detection of SEA deletion in alpha-thalassemia and compared two WGA methods, SurePlex DNA Amplification System and REPLI-g Single Cell on the performance of HRM analysis and linkage analysis. Material and Methods Retrospective study was carried out in a single private fertility clinic from 2015 to 2017. The single cell embryos were biopsied on blastocyst stage from couples with disease- carrier of SEA deletion type. The trophectoderm biopsied cells were amplified by two whole genome amplification (WGA), SurePlex DNA Amplification System-PCR based method (Illumina, USA) and REPLI-g MDA Single Cell (QIAGEN, Germany). All WGA products were analyzed with two primer pairs of HRM analysis for detection of SEA deletion type in alpha-thalassemia disease and confirmed with the informative short tandem repeated (STRs) linkage analysis. Results A total of 53 single cell biopsied were amplified with SurePlex-WGA (n=33) and REPLI-g MDA (n=20) which used as template for HRM analysis. The HRM results revealed clearly distinguished between wild type and mutant type of SEA deletion mutation in alpha-thalassemia. Comparing between two WGA methods, the HRM amplification results efficiency and allele drop out (ADO) rate of REPLI- g MDA were 95% (19/20) and 31.57% (6/19), respectively. Meanwhile, for SurePlex-WGA showed 75.76% (25/33) and 28% (7/25), respectively. Furthermore, the HRM melting profile results of REPLI-g MDA showed higher concordance rate with the informative STRs linkage analysis results (63.16%, 12/18) than that of SurePlex-WGA (48%, 12/25). Conclusions HRM analysis is an alternative technique with high sensitivity and accuracy for detecting of alpha-thalassemia mutations. The REPLI-g MDA Single Cell method with HRM analysis is suitable for detection of alpha-thalassemia1 SEA type deletion in PGT-M and provided a good concordance with informative STRs linkage analysis. However, ADO from PCR amplification could be occurred and caused of misdiagnosis which might be affected to the interpretation results of the embryos disease- status. Thus, the mutation analysis method using HRM analysis combined with STRs linkage analysis are recommend in PGT-M to avoid the effected of ADO and increase the accuracy and reliable of the PGT-M results.

High-Resolution Melting and Real-Time Pcr for Quantification and Detection of Drug-Resistant HBV Mutants in a Single Amplicon

Antiviral therapy by nucleoside/nucleotide analogues (NAs) effectively reduces HBV replication in chronic hepatitis B (CHB) patients. Because long-term NA treatments will eventually select for drug-resistant mutants, early detection of mutants and frequent monitoring of viral loads is crucial for successful NA therapy. Because no efficient test for one-tube quantification and qualification of various HBV-resistant mutants exists, we propose to use high-resolution melting (HRM) analysis in combination with real-time PCR to achieve this unmet need. We developed a single amplicon for detecting HBV mutants resistant to lamivudine (LMV), adefovir (ADV) and entecavir (ETV), which are commonly used for CHB treatment. Our design consists of two steps: real-time PCR for viral quantification, and hybridization probe HRM analysis for detection of specific drug-resistant mutants. Assay quantification was accurate (R=0.98) for viral loads from 10(3) to 10(9) copies/ml. HRM analysis produced distinct melting temperatures that clearly distinguished the mutants, rtM204V/I (LMV), rtA181V and rtN236T (ADV), and rtT184G and rtM250V (ETV), from their respective wild types. The assay detected mutants at only 10-25% of the HBV population. The clinical applicability of this assay was tested in a pilot study with serial samples from patients receiving LMV treatment. Flexibility, speed and cost-efficiency are additional benefits unique to our assay. The clinical sample results further support the feasibility of applying our design to frequent and long-term monitoring of CHB patients receiving NA treatments in the clinical setting.

Multiplex real-time PCR and high-resolution melting analysis for detection of white spot syndrome virus, yellow-head virus, and Penaeus monodon densovirus in penaeid shrimp

A multiplex real-time PCR and high-resolution melting (HRM) analysis was developed to detect simultaneously three of the major viruses of penaeid shrimp including white spot syndrome virus (WSSV), yellow-head virus (YHV), and Penaeus monodon densovirus (PmDNV). Plasmids containing DNA/cDNA fragments of WSSV and YHV, and genomic DNAs of PmDNV and normal shrimp were used to test sensitivity of the procedure. Without the need of any probe, the products were identified by HRM analysis after real-time PCR amplification using three sets of viral specific primers. The results showed DNA melting curves that were specific for individual virus. No positive result was detected with nucleic acids from shrimp, Penaeus monodon nucleopolyhedrovirus (PemoNPV), Penaeus stylirostris densovirus (PstDNV), or Taura syndrome virus (TSV). The detection limit for PmDNV, YHV and WSSV DNAs were 40fg, 50fg, and 500fg, respectively, which was 10 times more sensitive than multiplex real-time PCR analyzed by agarose gel electrophoresis. In viral nucleic acid mixtures, HRM analysis clearly identified each virus in dual and triple infection. To test the capability to use this method in field, forty-one of field samples were examined by HRM analysis in comparison with agarose gel electrophoresis. For HRM analysis, 11 (26.83%), 9 (21.95%), and 4 (9.76%) were infected with WSSV, PmDNV, and YHV, respectively. Agarose gel electrophoresis detected lesser number of PmDNV infection which may due to the limit of sensitivity. No multiple infection was found in these samples. This method provides a rapid, sensitive, specific, and simultaneous detection of three major viruses making it as a useful tool for diagnosis and epidemiological studies of these viruses in shrimp and carriers.

High-resolution melting analysis for detection of familial ligand-defective apolipoprotein B-100 mutations

Familial ligand-defective apolipoprotein B-100 (FDB) is characterized by elevated plasma concentrations of LDL-cholesterol and apolipoprotein (apo) B, normal triglyceride and HDL-cholesterol levels, the presence of tendon xanthomas, and premature coronary artery disease. FDB cannot be clinically distinguished from heterozygous LDL-receptor-defective familial hypercholesterolaemia (FH) without genetic testing. Amplicons in exon 26 and exon 29 of the APOB gene were screened for established genetic variants including mutations and polymorphisms using high-resolution melting analysis. Six novel variants associated with FDB in hypercholesterolaemic Dutch patients (S3476L, S3488G, Y3533C, T3540M, I4350T, G4368D) were also studied. All positive controls, a total of 10 mutations in exon 26 and four mutations in exon 29, were readily detectable by melting curve analysis. In addition, a patient previously not known to be heterozygous for the H3543Y mutation was identified in a screen of hypercholesterolaemic subjects. The method was validated by comparison of high-resolution melting analysis with DNA sequence data in a 'blinded' manner in 35 consecutive patients attending a lipid disorders clinic. These patients were classified as 'definite FH' by the Dutch Lipid Clinic Network criteria. Five patients were found to be heterozygous for the R3500Q and one for H3543Y. We have established a novel, robust method of FDB mutation detection using high-resolution melting analysis in conjunction with DNA sequencing. Compared with existing methods it is not only more cost-effective, but is also capable of detecting new sequence changes and will have importance in cascade screening of affected subjects.

High-resolution melting analysis for detection of MYH9 mutations

May-Hegglin anomaly (MHA), Sebastian (SBS), Fechtner (FTNS) and Epstein (EPS) syndromes are rare autosomal dominant disorders with giant platelets and thrombocytopenia. Other manifestations of these disorders are combinations of the presence of granulocyte inclusions and deafness, cataracts and renal failure. Currently, MHA, SBS, FTNS and EPS are considered to be distinct clinical manifestation of a single illness caused by mutations of the MYH9 gene encoding the heavy chain of non-muscle myosin IIA (NMMHC-IIA). As the MYH9 gene has a high number of exons, it takes much time and material to use this method for the detection of MYH9 mutations. Recently, a new method has been introduced for scanning DNA mutations without the need for direct sequencing: high-resolution melting analysis (HRMA). Mutation detection with HRMA relies on the intercalation of the specific dye (LC Green plus) in double-strand DNA and fluorescence monitoring of PCR product melting profiles. In our study, we optimized the conditions and used HRMA for rapid screening of mutations in all MYH9 exons in seven affected individuals from four unrelated families with suspected MYH9 disorders. Samples identified by HRMA as positive for the mutation were analysed by direct sequencing. HRMA saved us over 85% of redundant sequencing.

High-Resolution Melting Analysis for Detection of Internal Tandem Duplications

High-resolution melting analysis (HRMA) is a recently introduced closed-tube fluorescence-based method for rapid mutation screening and detection. However, all of the targets by which this technique has been validated thus far have had single-base substitutions, deletions, or similarly small mutational deviations from the wild-type sequence. In the current study, we sought to determine the feasibility of utilization of HRMA for the detection of larger sequence aberrations, using internal tandem duplications (ITD) in the juxtamembrane domain of the FLT3 gene as a model system. This gene is important in the growth and differentiation of hematopoietic progenitors and ITDs in this gene have been identified in a subset of poor-prognosis acute myelogenous leukemias (AML). DNA extracted from 62 AML samples was analyzed on a prototype high-resolution melting instrument. The samples interrogated for the FLT3 ITDs were subjected to post-amplification denaturation with frequent and regular fluorescence acquisition. The fluorescence versus temperature melting graphs generated were analyzed for deviation from the profiles reproducibly obtained for the wild-type samples. Results by HRMA were compared to results obtained using capillary electrophoresis-based fragment analysis, temperature gradient capillary electrophoresis detection, and sequencing of ITDs. FLT3 ITDs were detected in 13 of 62 AML samples with 100% concordance between the detection methods. This study demonstrates the utility of HRMA to rapidly and accurately screen samples for the presence of large sequence aberrations including FLT3 ITDs.