高分辨率熔解曲线分析（HRMA）是一种简单而敏感的基于粪便的 DNA 检测方法，可用于检测结直肠肿瘤中的突变。

High-resolution melting assay (HRMA) is a simple and sensitive stool-based DNA Test for the detection of mutations in colorectal neoplasms.

机构信息

Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, China.

出版信息

Clin Colorectal Cancer. 2012 Dec;11(4):280-90. doi: 10.1016/j.clcc.2012.04.001. Epub 2012 May 18.

DOI:10.1016/j.clcc.2012.04.001

PMID:22609129

Abstract

BACKGROUND

Stool-based DNA testing for colorectal cancer is becoming a favored alternative to existing DNA screening tests. However, current methods of analysis often become more complicated and costly with increased sensitivity. The high-resolution melting assay (HRMA) is a simple and rapid mutation scanning method with low cost and superb accuracy. In this study, we verified the accuracy of HRMA for screening KRAS/TP53 mutations in stool-isolated DNA from patients with colorectal cancer.

MATERIALS AND METHODS

Comparing to direct DNA sequencing, the accuracy of HRMA was verified by detecting KRAS/TP53 mutations in 2 independent stages. In study stage I, both tissue and stool samples from colorectal neoplasm patients were analyzed. In study stage II, stool samples from patients with colorectal neoplasms, and normal controls in clinical screening settings were examined.

RESULTS

In study stage I, the HRMA identified 14 of 17 target mutations (82.4%) in stools from cancer patients, and 4 of 5 (80.0%) target mutations in stools from advanced adenoma patients. The mutation detection rate in fecal samples (45.0%; 18/40) and referred tissue samples (55.0%; 22/40) was highly consistent (κ = 0.79). The HRMA detected 1% mutant DNA in a background of wild type DNA. In study stage II, the HRMA assay detected 58.8% (20/34) mutations in tumor samples, 41.5% (17/41) in advanced adenomas samples, and 3.33% (2/60) in age-matched normal control samples. The results from HRMA and DNA sequencing revealed 100% sensitivity and specificity in both tissue and stool samples.

CONCLUSION

HRMA is a simple, reliable, and sensitive method for detecting DNA mutations in the stool samples from patients with colorectal neoplasms.

摘要

背景

基于粪便的结直肠癌 DNA 检测正成为现有 DNA 筛查检测的一种首选替代方法。然而，随着灵敏度的提高，目前的分析方法往往变得更加复杂和昂贵。高分辨率熔解曲线分析（HRMA）是一种简单、快速的突变扫描方法，具有低成本和超高准确性。在本研究中，我们验证了 HRMA 检测结直肠癌患者粪便分离 DNA 中 KRAS/TP53 突变的准确性。

材料与方法

通过在 2 个独立阶段检测 KRAS/TP53 突变，来验证 HRMA 的准确性。在研究阶段 I 中，对结直肠肿瘤患者的组织和粪便样本进行了分析。在研究阶段 II 中，对结直肠肿瘤患者和临床筛查人群中的正常对照者的粪便样本进行了检测。

结果

在研究阶段 I 中，HRMA 在癌症患者的粪便中鉴定出 17 个目标突变中的 14 个（82.4%），在高级腺瘤患者的粪便中鉴定出 5 个目标突变中的 4 个（80.0%）。粪便样本（45.0%，18/40）和送检组织样本（55.0%，22/40）的突变检测率高度一致（κ=0.79）。HRMA 在野生型 DNA 背景下检测到 1%的突变 DNA。在研究阶段 II 中，HRMA 检测到肿瘤样本中的 58.8%（20/34）突变、高级腺瘤样本中的 41.5%（17/41）突变、年龄匹配的正常对照样本中的 3.33%（2/60）突变。HRMA 和 DNA 测序的结果在组织和粪便样本中均显示出 100%的灵敏度和特异性。