Detection of IDH1 and IDH2 Mutation in Formalin-fixed Paraffin-embedded Gliomas Using Allele-specific COLD-PCR and Probe Melting Curve Analysis
| Autor: | Haitao Lan, Tianhai Ji, Dan Liu, Wensheng Yang, Qing Zhang, Kun Liu, Wei Shao |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
Male
Histology IDH1 Biopsy DNA Mutational Analysis Biology medicine.disease_cause Polymerase Chain Reaction IDH2 Melting curve analysis Pathology and Forensic Medicine law.invention 03 medical and health sciences symbols.namesake 0302 clinical medicine law medicine Humans Polymerase chain reaction Sanger sequencing COLD-PCR Mutation Paraffin Embedding Brain Neoplasms Glioma Molecular biology Isocitrate Dehydrogenase Neoplasm Proteins Medical Laboratory Technology Isocitrate dehydrogenase 030220 oncology & carcinogenesis symbols Female 030217 neurology & neurosurgery |
| Zdroj: | Applied Immunohistochemistry & Molecular Morphology. 26:e93-e100 |
| ISSN: | 1541-2016 |
| Popis: | Many tumors, especially gliomas, contain an isocitrate dehydrogenase (IDH) mutation that can be used for clinical diagnosis and prognosis. Our study aimed to develop a new reliable detection assay for IDH1 and IDH2 mutations for clinical diagnosis based on the allele-specific (AS) coamplification with lower denaturing-polymerase chain reaction (COLD-PCR) and probe melting curve analysis (PMCA). The method includes 3 elements allowing for the sensitive detection of low-abundance mutations: (1) PCR amplification of the target fragments with AS primers; (2) COLD-PCR; and (3) PMCA for differentiating the different mutations after amplification. We conducted a blinded study with 45 paraffin-embedded gliomas specimens and 13 fresh specimens screened for IDH mutations using Sanger sequencing. Concordance between the results of our AS-COLD-PCR/PMCA assay and Sanger sequencing was 100%. Our assay appeared to be superior to direct sequencing with a much higher sensitivity of 0.4% mutations. In summary, our assay is a cost-effective, convenient, and sensitive method for detecting IDH mutations and could be applied in the clinical setting to assess small brain biopsies. |
| Databáze: | OpenAIRE |
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