• Asian Pacific Journal of Cancer Prevention
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• 제14권11호
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• pp.6619-6623
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• 2013

Background:The aim of the research was to explore a cost effective, fast, easy to perform, and sensitive method for epidermal growth factor receptor (EGFR) mutation testing. Methods: High resolution melting analysis (HRM) was introduced to evaluate the efficacy of the analysis for dectecting EGFR mutations in exons 18 to 21 using formalin-fixed paraffin-embedded (FFPE) tissues and plasma free DNA from 120 patients. Results: The total EGFR mutation rate was 37.5% (45/120) detected by direct sequencing. There were 48 mutations in 120 FFPE tissues assessed by HRM. For plasma free DNA, the EGFR mutation rate was 25.8% (31/120). The sensitivity of HRM assays in FFPE samples was 100% by HRM. There was a low false-positive mutation rate but a high false-negative rate in plasma free DNA detected by HRM. Conclusions: Our results show that HRM analysis has the advantage of small tumor sample need. HRM applied with plasma free DNA showed a high false-negative rate but a low false-positive rate. Further research into appropriate methods and analysis needs to be performed before HRM for plasma free DNA could be accepted as an option in diagnostic or screening settings.

• 한국수산과학회지
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• 제47권3호
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• pp.241-246
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• 2014

Viruses in the genus Megalocytivirus have been subdivided into four subgroups. Among these subgroups 2 and 4, represented by the red sea bream iridovirus (RBIV) and the olive flounder iridovirus (FLIV), respectively, are non-exotic. subgroups 1 and 3, represented by the red sea bream iridovirus (RSIV) and the infectious spleen and kidney necrosis virus (ISKNV), respectively, have not been detected in Korea and are known as exotic. Shellfish are filter-feeders, and can thus filter and accumulate Megalocytivirus in their digestive glands, allowing us to track viral contamination in surrounding aquatic environment. In this study, we developed a high-resolution melting (HRM) analysis to differentiate among subgroups of Megalocytivirus accumulated in shellfish, and confirmed the convenience and efficiency of this method. More than two subgroups of Megalocytivirus were found in the digestive gland of a single shellfish. We classified all Megalocytivirus viruses from shellfish in Korea into subgroups 2 and 4, although proportions of subgroups were different among regions. Compared to nucleotide sequencing analysis, HRM analysis is a simple and rapid method for differentiating of Megalocytivirus subgroups.

• BMB Reports
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• 제55권11호
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• pp.553-558
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• 2022

Hepatocellular carcinoma (HCC) is dangerous cancer that often evades early detection because it is asymptomatic and an effective detection method is lacking. For people with chronic liver inflammation who are at high risk of developing HCC, a sensitive detection method for HCC is needed. In a meta-analysis of The Cancer Genome Atlas pan-cancer methylation database, we identified a CpG island in the USP44 promoter that is methylated specifically in HCC. We developed methylation-sensitive high-resolution melting (MS-HRM) analysis to measure the methylation levels of the USP promoter in cell-free DNA isolated from patients. Our MS-HRM assay correctly identified 40% of patients with early-stage HCC, whereas the α-fetoprotein test, which is currently used to detect HCC, correctly identified only 25% of early-stage HCC patients. These results demonstrate that USP44 MS-HRM analysis is suitable for HCC surveillance.

• 한국약용작물학회지
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• 제23권6호
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• pp.439-445
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• 2015

Background : Correct identification of Panax species is important to ensure food quality, safety, authenticity and health for consumers. This paper describes a high resolution melting (HRM) analysis based method using internal transcribed spacer (ITS) and 5.8S ribosomal DNA barcoding regions as target (Bar-HRM) to obtain barcoding information for the major Panax species and to identify the origin of ginseng plant. Methods and Results : A PCR-based approach, Bar-HRM was developed to discriminate among Panax species. In this study, the ITS1, ITS2, and 5.8S rDNA genes were targeted for testing, since these have been identified as suitable genes for use in the identification of Panax species. The HRM analysis generated cluster patterns that were specific and sensitive enough to detect small sequence differences among the tested Panax species. Conclusion : The results of this study show that the HRM curve analysis of the ITS regions and 5.8S rDNA sequences is a simple, quick, and reproducible method. It can simultaneously identify three Panax species and screen for variants. Thus, ITS1HRM and 5.8SHRM primer sets can be used to distinguish among Panax species.

• 환경생물
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• 제40권2호
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• pp.199-205
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• 2022

Two tree frogs, Dryophytes suweonensis and Dryophytes japonicus, inhabiting Korea, are morphologically similar and share the same habitats. Therefore, they are identified mainly through their calls, especially for males. Dryophytes suweonensis is registered as an endangered (IUCN: EN grade) and protected species in South Korea. Thus, it is necessary to develop a method to rapidly identify and discriminate the two species and establish efficient protection and restoration plans. We identified significant genetic variation between them by sequencing a maternally-inherited mitochondrial 12S ribosomal DNA region. Based on the sequence data, we designed a pair of primers containing 7bp differences for high resolution melting(HRM) analysis to rapidly and accurately characterize their genotypes. The HRM analysis using genomic DNA showed that the melting peak for D. suweonensis was 76.4±0.06℃, whereas that of D. japonicus was 75.0±0.05℃. The differential melt curve plot further showed a distinct difference between them. We also carried out a pilot test for the application of HRM analysis based on immersing D. suweonensis in distilled water for 30 min to generate artificial environmental DNA(eDNA). The results showed 1.10-1.31℃ differences in the melting peaks between the two tree frog samples. Therefore, this HRM analysis is rapid and accurate in identifying two tree frogs not only using their genomic DNA but also using highly non-invasive eDNA.

• Asian Pacific Journal of Cancer Prevention
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• 제17권12호
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• pp.5147-5152
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• 2016

Background: Investigations of methods for detection of mutations have uncovered major weaknesses of direct sequencing and pyrosequencing, with their high costs and low sensitivity in screening for both known and unknown mutations. High resolution melting (HRM) analysis is an alternative tool for the rapid detection of mutations. Here we describe the accuracy of HRM in screening for KRAS and BRAF mutations in metastatic colorectal cancer (mCRCs) samples. Materials and Methods: A total of 1000 mCRC patients in Mehr Hospital, Tehran, Iran, from Feb 2008 to May 2012 were examined for KRAS mutations and 242 of them were selected for further assessment of BRAF mutations by HRM analysis. In order to calculate the sensitivity and specificity, HRM results were checked by pyrosequencing as the golden standard and Dxs Therascreen as a further method. Results: In the total of 1,000 participants, there were 664 (66.4%) with wild type and 336 (33.6%) with mutant codons 12 and/or 13 of the KRAS gene. Among 242 samples randomly checked for the BRAF gene, all were wild type by HRM. Pyrosequencing and Dxs Therascreen results were in line with those of the HRM. In this regard, the sensitivity and specificity of HRM were evaluated as 100%. Conclusion: The findings suggest that the HRM, in comparison with DNA sequencing, is a more appropriate method for precise scanning of KRAS and BRAF mutations. It is also possible to state that HRM may be an attractive technique for the detection of known or unknown somatic mutations in other genes.

• 한국균학회지
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• 제50권3호
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• pp.225-233
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• 2022

Pyogo (Shiitake, Lentinula edodes) is one of the most important edible mushrooms because of its outstanding nutritive and medicinal value. In the registration and protection procedure for newly developed mushroom cultivars, the application of molecular markers that can supplement the morphological characteristic-based distinction has been strongly requested. Sanjo 701 and Chamaram, newly developed at the Federation Forest Mushroom Research Center of Korea, have been characterized as innovative cultivars suitable for customer demands because of their high yields and cultivation rates. However, no technical tools can protect the rights to these important cultivars. In this study, using comparative genomic information from 23 commercially available pyogo cultivars, we identified single nucleotide polymorphisms (SNPs) that accurately differentiated Sanjo701 and Chamaram from the other cultivars. We also developed high-resolution melting analysis (HRM)-based SNP markers that discriminate among the tested 23 pyogo cultivars. The developed SNP markers can be utilized for rapid, accurate identification of pyogo cultivars with low genetic diversity and to prevent cultivar contamination caused by illegally distributed inocula. In addition, these markers can serve as a crucial scientific basis for securing the right to conserve new cultivars in international markets.

• 농업과학연구
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• 제46권1호
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• pp.27-31
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• 2019

It is well documented that intensive selection in dairy cattle for economic value such as increased milk yield led to a decline in reproductive performance. Recent studies using genome-wide association studies (GWASs) discovered candidate genes involved in the lower fertility including embryo development and conception rates. However, the information, which showed a lower reproductive performance, is limited to dairy cattle, especially Holstein, and the candidate genes were not examined in the Korean native cattle Hanwoo which has been intensively selected and bred for meat in the last few decades. We selected the candidate genes WBP1 and PARM1 reported to be associated with cow and/or heifer conception in dairy cattle and analyzed the genotype because those genes have non-synonymous single nucleotide polymorphisms (SNPs). To determine the single base change, we used the high resolution melting (HRM) assay which is rapid and cost-effective for a small number of genes. We found that most heifers with higher conception (1: service per conception) have the AA genotype coding Threonine rather than Proline in the WBP1 gene. We did not detect an association for a SNP in PARM1 in our analysis. In conclusion, the genetic variation of WBP1 can be used as a selective marker gene to improve reproductive performance, and HRM assay can be used to identify common SNP genotypes rapidly and cost effectively.

• Asian Pacific Journal of Cancer Prevention
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• 제15권2호
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• pp.891-894
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• 2014

This study is aimed to investigate the role of paired boxed gene 1 (PAX1) methylation analysis by methylation-sensitive high-resolution melting (MS-HRM) in the detection of high grade lesions in atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion (ASC-H) and compared its performance with the Hybrid Capture 2 (HC2) human papillomavirus (HPV) test. In our study, 130 cases with a diagnosis of ASC-H from the cervical cytological screening by Thinprep cytologic test (TCT) technique were selected for triage. Their cervical scrapings were collected and evaluated by using PAX1 methylation analysis (MS-HRM) and high-risk HPV DNA test (HC2), followed by colposcopy and cervical biopsy. Chi-square test were used to test the differences of PAX1 methylation or HPV infection between groups. In the detection of CIN2+, the sensitivity, specificity, the PPV, NPV and the accuracy of PAX1 MS-HRM assay and high-risk HPV (HR-HPV) tests were respectively 80.6% vs 67.7%, 94.9% vs 54.5%, 83.3%, vs 31.8%, 94.0% vs 84.4%, and 91.5% vs 57.7%. The PAX1 MS-HRM assay proved superior to HR-HPV testing in the detection of high grade lesions (CIN2+) in ASC-H. This approach could screen out the majority of high grade lesion cases of ASC-H, and thus could reduce the referral rate to colposcopy.

• Asian-Australasian Journal of Animal Sciences
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• 제26권1호
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• pp.30-35
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• 2013

The myostatin (MSTN) gene, as a negative regulator of skeletal muscle growth, has been proposed to be associated with production traits in farm animals. In the present study, a T/C variant at -125 bp (relative to ATG start codon) of 5'regulatory region of rabbit MSTN was identified by direct sequencing. Two hundred and twenty two rabbits, which were randomly sampled from 3 breeds (Ira rabbits, Champagne rabbits and Tianfu black rabbits), were genotyped by high-resolution melting (HRM). Comparing the genotyping results of 47 samples with direct sequencing, the HRM showed high sensitivity (0.96) and high specificity (0.98). In the three rabbit breeds, the allele C was the predominant allele. The polymorphic site showed high heterozygosity (He = 0.48) and high effective number of alleles (Ne = 1.91). The genetic diversity was reasonably informative (0.25