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

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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.

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Abbreviations

MSP:

Methylation specific PCR

TaqMan real-time FQ-MSP:

TaqMan real-time fluorescence quantitative MSP

PER2:

Period 2

CNS:

Central nervous system

SCN:

Suprachiasmatic nucleus

AML:

Acute myeloid leukemia

CML:

Chronic myeloid leukemia

BSP:

Bisulfite sequencing

MS-HRM:

Methylation-sensitive high-resolution melting

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Funding

This work was supported by the Major scientific and technological innovation projects of Shandong Province of China (Grant No. 2019JZZY011018) and Yantai Science and Technology Plan Project of China (Grant No. 2019MSGY133) and Yantai Science and Technology Plan Project of China (Grant No. 2019YD004).

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Author notes

  1. Huihui Jiang and Xin Yang have equally contributed to this study.

Authors and Affiliations

  1. Qingdao University, Qingdao, 266000, Shandong, China

    Huihui Jiang, Hongyuan Wu & Liping Jiao

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

    Xin Yang & Chengming Sun

  3. Department of Laboratory Center, Qilu Hospital of Shandong University, Jinan, 250000, Shandong, China

    Miaomiao Mi

  4. Department of Laboratory Center, Qingdao Women and Children’s Hospital, Qingdao, 266000, Shandong, China

    Xiaonan Wei

  5. Binzhou Medical University, Yantai, 264000, Shandong, China

    Yu Xin

  6. Department of Laboratory Center, Yantaishan Hospital, Yantai, 264000, Shandong, China

    Shengjun Sun

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  1. Huihui Jiang
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Contributions

Material preparation, data collection and analysis were performed by HJ, XY, MM, XW, HW, YX, LJ and SS. The first draft of the manuscript was written by HJ and XY. CS critically revised the work. All authors have read and approved the manuscript, and all authors commented on previous versions of the manuscript.

Corresponding author

Correspondence to Chengming Sun.

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This study was approved by the Ethics Committee of Yantai Yuhuangding Hospital affiliated to Qingdao University with the protocol code of 2017210.

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Jiang, H., Yang, X., Mi, M. et al. Development and performance evaluation of TaqMan real-time fluorescence quantitative methylation specific PCR for detecting methylation level of PER2. Mol Biol Rep 49, 2097–2105 (2022). https://doi.org/10.1007/s11033-021-07027-z

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