用于检测PER2甲基化水平的TaqMan实时荧光定量甲基化特异性PCR的开发与性能评估

Development and performance evaluation of TaqMan real-time fluorescence quantitative methylation specific PCR for detecting methylation level of PER2.

作者信息

Jiang Huihui, Yang Xin, Mi Miaomiao, Wei Xiaonan, Wu Hongyuan, Xin Yu, Jiao Liping, Sun Shengjun, Sun Chengming

机构信息

Qingdao University, Qingdao, 266000, Shandong, China.

Department of Laboratory Center, Yantai Yuhuangding Hospital Affiliated to Qingdao University, 20 Yuhuangding East Road, Yantai, 264000, Shandong, China.

出版信息

Mol Biol Rep. 2022 Mar;49(3):2097-2105. doi: 10.1007/s11033-021-07027-z. Epub 2021 Dec 1.

DOI:10.1007/s11033-021-07027-z

PMID:34854010

Abstract

BACKGROUND

PER2 gene methylation is closely related to the occurrence and progress of some cancers, but there is no method to quantitatively detect PER2 methylation in conventional laboratories. So, we established a TaqMan real-time fluorescence quantitative methylation specific PCR (TaqMan real-time FQ-MSP) assay and use it for quantitative detection of PER2 methylation in leukemia patients.

METHODS

According to the PER2 sequence searched by GenBank, a CpG sequence enrichment region of the PER2 gene promoter was selected, and the methylated and unmethylated target sequences were designed according to the law of bisulfite conversion of DNA to construct PER2 methylation positive and negative reference materials. Specific primers and probe were designed. The reference materials were continuously diluted into gradient samples by tenfold ratio to evaluate the analytical sensitivity, specificity, accuracy and reproducibility of the method, and the analytical sensitivity of TaqMan real-time FQ-MSP assay was compared with that of the conventional MSP assay. At the same time, the new-established TaqMan real-time FQ-MSP assay and the conventional MSP assay were used to detect the PER2 methylation level of 81 patients with leukemia, and the samples with inconsistent detection results of the two assays were sent to pyromethylation sequencing to evaluate the clinical detection performance.

RESULTS

The minimum detection limit of TaqMan real-time FQ-MSP assay for detecting PER2 methylation level established in this study was 6 copies/uL, and the coefficient of variation(CV) of intra-assay and inter-assay was less than 3%. Compared with the conventional MSP assay, it has higher analytical sensitivity. For the samples with inconsistent detection results, the results of pyrosequencing and TaqMan real-time FQ-MSP assay are consistent.

CONCLUSION

TaqMan real-time FQ-MSP assay of PER2 methylation established in this study has high detection performance and can be used for the detection of clinical samples.

摘要

背景

PER2基因甲基化与某些癌症的发生和进展密切相关，但传统实验室尚无定量检测PER2甲基化的方法。因此，我们建立了一种TaqMan实时荧光定量甲基化特异性PCR（TaqMan实时荧光定量甲基化特异性PCR）检测方法，并将其用于白血病患者PER2甲基化的定量检测。

方法

根据GenBank搜索到的PER2序列，选择PER2基因启动子的一个CpG序列富集区，根据DNA亚硫酸氢盐转化规律设计甲基化和未甲基化靶序列，构建PER2甲基化阳性和阴性参考物质。设计特异性引物和探针。将参考物质按10倍比例连续稀释成梯度样本，以评估该方法的分析灵敏度、特异性、准确性和重复性，并将TaqMan实时荧光定量甲基化特异性PCR检测方法的分析灵敏度与传统甲基化特异性PCR检测方法进行比较。同时，采用新建立的TaqMan实时荧光定量甲基化特异性PCR检测方法和传统甲基化特异性PCR检测方法检测81例白血病患者的PER2甲基化水平，将两种检测方法检测结果不一致的样本送去焦磷酸测序以评估临床检测性能。

结果

本研究建立的TaqMan实时荧光定量甲基化特异性PCR检测方法检测PER2甲基化水平的最低检测限为6拷贝/μL，批内和批间变异系数（CV）均小于3%。与传统甲基化特异性PCR检测方法相比，其分析灵敏度更高。对于检测结果不一致的样本，焦磷酸测序结果与TaqMan实时荧光定量甲基化特异性PCR检测方法结果一致。