Peng Qi, Liao Shengbin, He Yuejing, Liang Liyun, Su Xiaohua, Rao Chunbao, Li Wenrui, Li Siping, Lu Xiaomei
Department of Medical and Molecular Genetics, Dongguan Institute of Pediatrics, Dongguan, China.
Medical Laboratory, Dongguan Eighth People's Hospital, Dongguan, China.
J Clin Lab Anal. 2020 Aug;34(8):e23337. doi: 10.1002/jcla.23337. Epub 2020 May 15.
Polymorphisms (rs1801133 or C677T; rs1801131 or A1298C) of the MTHFR gene and rs1801394 (A66G) of the MTRR gene are important genetic determinants of folate metabolism. A convenient, sensitive, and reliable method is required to detect polymorphisms for the precise supplementation of folate.
A rapid detection method based on molecular beacon probes that can detect rs1801133, rs1801131, and rs1801394 simultaneously was developed in this study. Specific primers and probes were designed, and the amplification system and conditions were optimized. We applied our method to a group of 500 unrelated women of gestational age in the Dongguan region of Guangdong Province in China. The clinical performance of this assay was evaluated by testing 94 samples in comparison with Sanger sequencing.
The molecular-beacon-based PCR assay we established is extremely sensitive, with a detection limit of 2 ng/μL of genomic DNA, and validated by direct sequencing in a blind study with 100% concordance.
The results demonstrate that our molecular-beacon-based asymmetric PCR assay is an easy, reliable, high-yield, and cost-effective method for the simultaneous detection of three polymorphisms related to folate metabolism. It could help evaluate the risk of perinatal-neonatal neural tube malformation, pregnancy hypertension, and other diseases and guide the individualized supplementation of folic acid. Data on the spectrum of mutations in the Dongguan District in this study are beneficial for guiding the supplementation of folic acid.
亚甲基四氢叶酸还原酶（MTHFR）基因多态性（rs1801133或C677T；rs1801131或A1298C）和甲硫氨酸合成酶还原酶（MTRR）基因的rs1801394（A66G）是叶酸代谢的重要遗传决定因素。需要一种方便、灵敏且可靠的方法来检测多态性，以便精确补充叶酸。
本研究开发了一种基于分子信标探针的快速检测方法，可同时检测rs1801133、rs1801131和rs1801394。设计了特异性引物和探针，并优化了扩增体系和条件。我们将该方法应用于中国广东省东莞市地区的一组500名无亲缘关系的育龄妇女。通过与桑格测序法比较检测94个样本，评估了该检测方法的临床性能。
我们建立的基于分子信标的聚合酶链反应（PCR）检测方法极其灵敏，基因组DNA的检测限为2 ng/μL，并在一项盲法研究中通过直接测序进行了验证，一致性为100%。
结果表明，我们基于分子信标的不对称PCR检测方法是一种简便、可靠、高产且经济高效的方法，可同时检测与叶酸代谢相关的三种多态性。它有助于评估围产期-新生儿神经管畸形、妊娠高血压和其他疾病的风险，并指导叶酸的个体化补充。本研究中东莞市地区的突变谱数据有利于指导叶酸补充。
