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A melting curve analysis--based PCR assay for one-step genotyping of ?-thalassemia mutations a multicenter validation.

ABSTRACT: The increasing number of disease-causing mutations demands a simple, direct, and cost-effective diagnostic genotyping technique capable of detecting multiple mutations. This study validated the efficacy of a novel melting curve analysis-based genotyping assay (MeltPro HBB assay) for 24 ?-thalassemia mutations in the Chinese population. The diagnostic potential of this assay was evaluated in 1022 pretyped genomic DNA samples, including 909 clinical cases of ?-thalassemia minor or major, using a double-blind analysis in a multicenter validation study. Reproducibility of the assay was 100%, and the limit of detection was 10 pg per reaction. All 24 ?-thalassemia mutations were accurately genotyped, and ?-thalassemia genotypes were correctly determined in all 1022 samples, yielding overall sensitivity and specificity of 100%. The concordance rate was 99.4% between this assay and the reference method. It was concluded that the MeltPro HBB assay is useful for reliable genotyping of multiple ?-thalassemia mutations in clinical settings and may have potential as a versatile method for rapid genotyping of known mutations because of its high throughput, accuracy, ease of use, and low cost.

SUBMITTER: Xiong F

PROVIDER: S-EPMC3123802 | biostudies-other | 2011 Jul

REPOSITORIES: biostudies-other

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A melting curve analysis--based PCR assay for one-step genotyping of β-thalassemia mutations a multicenter validation.

Xiong Fu F Huang Qiuying Q Chen Xiaoyun X Zhou Yuqiu Y Zhang Xinhua X Cai Ren R Chen Yajun Y Xie Jiansheng J Feng Shanwei S Wei Xiaofeng X Xiao Qizhi Q Zhang Tianlang T Luo Shiqiang S Yang Xuehuang X Hao Ying Y Qu Yanxia Y Li Qingge Q Xu Xiangmin X

The Journal of molecular diagnostics : JMD 20110506 4

The increasing number of disease-causing mutations demands a simple, direct, and cost-effective diagnostic genotyping technique capable of detecting multiple mutations. This study validated the efficacy of a novel melting curve analysis-based genotyping assay (MeltPro HBB assay) for 24 β-thalassemia mutations in the Chinese population. The diagnostic potential of this assay was evaluated in 1022 pretyped genomic DNA samples, including 909 clinical cases of β-thalassemia minor or major, using a d ...[more]

PMID: 21704277

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Project description:BackgroundKRAS mutational analysis is the standard of care prior to initiation of treatments targeting the epidermal growth factor receptor (EGFR) in patients with metastatic colorectal cancer. Sensitive methods are required to reliably detect KRAS mutations in tumor samples due to admixture with non-mutated cells. Many laboratories have implemented sensitive tests for KRAS mutations, but the methods often require expensive instrumentation and reagents, parallel reactions, multiple steps, or opening PCR tubes.MethodsWe developed a highly sensitive, single-reaction, closed-tube strategy to detect all clinically significant mutations in KRAS codons 12 and 13 using the Roche LightCycler® instrument. The assay detects mutations via PCR-melting curve analysis with a Cy5.5-labeled sensor probe that straddles codons 12 and 13. Incorporating a fast COLD-PCR cycling program with a critical denaturation temperature (Tc) of 81°C increased the sensitivity of the assay >10-fold for the majority of KRAS mutations.ResultsWe compared the COLD-PCR enhanced melting curve method to melting curve analysis without COLD-PCR and to traditional Sanger sequencing. In a cohort of 61 formalin-fixed paraffin-embedded colorectal cancer specimens, 29/61 were classified as mutant and 28/61 as wild type across all methods. Importantly, 4/61 (6%) were re-classified from wild type to mutant by the more sensitive COLD-PCR melting curve method. These 4 samples were confirmed to harbor clinically-significant KRAS mutations by COLD-PCR DNA sequencing. Five independent mixing studies using mutation-discordant pairs of cell lines and patient specimens demonstrated that the COLD-PCR enhanced melting curve assay could consistently detect down to 1% mutant DNA in a wild type background.ConclusionsWe have developed and validated an inexpensive, rapid, and highly sensitive clinical assay for KRAS mutations that is the first report of COLD-PCR combined with probe-based melting curve analysis. This assay significantly improved diagnostic accuracy compared to traditional PCR and direct sequencing.

Dataset Information

A melting curve analysis--based PCR assay for one-step genotyping of ?-thalassemia mutations a multicenter validation.

Publications

A melting curve analysis--based PCR assay for one-step genotyping of β-thalassemia mutations a multicenter validation.

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