Single probe PCR melting curve analysis MTHFR C677T SNP sites

Kai-Yun Chen; Jiang-Xia Xu; Meng-Meng Wang; Die Hu; Fangfang Xie; Defa Huang; Jie Chen; Tong Yang; Jiajian Zhang; Fangli Song; Shao Huang; Tianyu Zhong

doi:10.1016/j.ab.2021.114102

Single probe PCR melting curve analysis MTHFR C677T SNP sites

Anal Biochem. 2021 Apr 15:619:114102. doi: 10.1016/j.ab.2021.114102. Epub 2021 Jan 13.

Authors

Kai-Yun Chen¹, Jiang-Xia Xu², Meng-Meng Wang³, Die Hu⁴, Fangfang Xie⁴, Defa Huang⁴, Jie Chen⁴, Tong Yang⁴, Jiajian Zhang⁵, Fangli Song⁵, Shao Huang⁵, Tianyu Zhong⁶

Affiliations

¹ Department of Drug Clinical Trial, The Fourth Affiliated Hospital of Nanchang University, Nanchang, Jiangxi Province, China.
² Department of Laboratory Medicine, The Fourth Affiliated Hospital of Nanchang University, Nanchang, Jiangxi Province, China.
³ Department of Pathology, The Fourth Affiliated Hospital of Nanchang University, Nanchang, Jiangxi Province, China.
⁴ The First School of Clinical Medicine, Gannan Medical University, Ganzhou, Jiangxi Province, China.
⁵ Jiangxi Shiningmed Com Ltd, China.
⁶ Department of Laboratory Medicine, First Affiliated Hospital of Gannan Medical University, Ganzhou, Jiangxi Province, China. Electronic address: zhongtianyu@126.com.

PMID: 33450284
DOI: 10.1016/j.ab.2021.114102

Abstract

Background: The detection and analysis of methylene tetrahydrofolate reductase (MTHFR) C677T single nucleotide polymorphism (SNP) from blood samples is time-consuming and costly. We aimed to establish a method to detect these SNPs by direct whole blood PCR and without DNA extraction.

Methods: Probes modified by different fluorescent groups on the same sequence were designed. Various MTHFR genotypes from direct blood PCR experiments were used to verify the similarity of the obtained and sequencing results. The SNP sites adjacent to the MTHFR C677T SNP were used to verify whether the method can accurately distinguish these sites.

Results: The ROX probe was found to be the most suitable for this study. We tested 291 samples with 1 μL whole blood as a template, and obtained 126, 43, and 122 cases of C677C, C677T, and C677 C/T genotypes, respectively. The melting curve was consistent with the sequencing results. The detection limit was approximately 1000 white blood cells/μL. Through PCR and the melting curve method, the adjacent sites were accurately distinguished.

Conclusion: We established a reliable, simple, rapid, and low-cost direct blood PCR method for the detection of MTHFR C677T SNPs. This could also be used as a potential diagnostic tool for a variety of diseases.

Keywords: Direct blood PCR; Folic acid; MTHFR C677T; Melting curve; TaqMan-probe.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Female
Humans
Male
Methylenetetrahydrofolate Reductase (NADPH2) / genetics*
Nucleic Acid Denaturation
Polymerase Chain Reaction*
Polymorphism, Single Nucleotide*

Substances

MTHFR protein, human
Methylenetetrahydrofolate Reductase (NADPH2)