Birth defects represent a significant challenge in global public health, with folic acid deficiency being a primary contributing factor. If pregnant women do not absorb enough folic acid, it can lead to serious problems such as delayed development and neural tube defects of embryonic, and megaloblastic anemia of pregnant women. 5,10-Methylentetrahydrofolate reductase (MTHFR) enzyme plays a central role in folate metabolism by catalyzing the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, which is the main circulating form of folate conversion into absorbable active folate in the human body. The common C677T polymorphism in the human MTHFR gene causes a reduction in MTHFR enzyme activity in individuals who are homozygous for the mutation. The enzyme structural changes caused by MTHFR 677C→T include nonsynonymous substitutions on the catalytic domain, where alanine is converted to valine. In heterozygous subjects, this substitution reduces enzyme activity by 35%, while in homozygous subjects, it reduces it by 70%.

Our project aims to develop a rapid, sensitive, and straightforward detection method for identifying the single nucleotide variant C677T in the MTHFR gene. This method will enable the determination of CC, CT, or TT genotypes through a one-tube reaction, facilitating early detection of folate metabolism disorder risks and contributing to the health of pregnant women and their fetuses.

In addition, we aim to popularize knowledge of folate metabolism among the public through communication, collaboration, and science education, thereby enhancing understanding and contributing to the health of women and newborns.

Our project can determine the specific genotypes (CC, CT, and TT) of the MTHFR 677 single nucleotide polymorphism through a single-tube reaction, which is easy to perform, time-efficient, and cost-effective. This method has been successfully applied to the detection of blood samples, with genetic typing results confirmed through comparison with sequencing data. The project demonstrated an accuracy of 100%, along with excellent precision.

Compared to existing detection techniques, our technology significantly simplifies experiments, including both experimental design and operational procedures. The TaqMan approach heavily relies on probe length and Tm values, which are determined by the target sequence. However, target gene sequences vary widely, with bases distributed randomly. Certain genes consistently fail to achieve suitable Tm values (e.g., fragments with high poly T or poly A content), making it difficult to design TaqMan-compatible probes. Our method completely eliminates this limitation. By replacing conventional temperature-controlled recognition with the specific recognition and cleavage of RNase H II, it enables highly flexible probe design. In addition, we only need to perform a simple blood pre-treatment, then place the sample into a qPCR machine for reaction and result output. According to the protocol, the experiment can be fully replicated without requiring extensive professional training.

By developing algorithmic modules that process qPCR fluorescence data, our system analyzes and statistically processes the information. Using predefined genotype models, it accurately identifies genetic profiles and generates standardized reports. This automated solution dramatically lowers operational barriers with a user-friendly interface. Operators need only follow step-by-step instructions to obtain precise results without requiring specialized training. More importantly, it eliminates time-consuming manual identification processes and reduces training costs for professionals. The system also prevents diagnostic errors caused by human error, significantly lowering overall expenses.

Our experimental methodology can be applied to other genes, such as BRAF V600E. Our project provides inspiring ideas for future clinical detection of SNP genes and has broad prospects.

Communication
In the professional field, we interviewed two doctors from the Obstetrics Department of Jiangsu Maternity and three professors from different regions of the world.
In the communication between universities, our team engaged in several online exchanges with different universities and attended the CCIC. These exchanges helped iGEM teams learn more about the synthetic biology competition they were participating in, and also promote our folate metabolism gene detection technology.
Through media platforms such as Bilibili, WeChat Official Accounts, RedNote and TikTok, our team conducted exchanges on competition experience and project progress with multiple universities including Nanjing Agricultural University, Hubei University, Beijing Institute of Technology, and Nanjing University, and published a number of theme articles.
To extend the benefits of scientific research to more people, we have collaborated with Southern Medical University and Huazhong Agricultural University to transform cutting-edge knowledge related to folic acid diagnosis into warm-hearted picture books, which accompany expectant parents.
In addition, our team collaborated with other teams to explore the role of functional nucleic acids, and together written the White Paper on Innovative Applications of Functional Nucleic Acids in iGEM.

Practical Exploration
This visit to Amp-Future (Changzhou) Biotech Co., Ltd. enabled team members to gain an in-depth understanding of genetic testing technologies and products, resolve experimental challenges, and clarify the connections between scientific research projects, enterprise applications, and market demands. This experience provided valuable insights for synthetic biology practice.

Community Education
Our team carried out biological science popularization activities for children and adolescents in Laifeng Street Community and Cuilin Villa Community. We combined the basic knowledge of synthetic biology with interesting practices. These activities not only broadened their scientific horizons but also sowed the seeds of exploring the mysteries of life, realizing the effective extension of science education at the community level.