Genetic analysis technology has been widely used, covering fields including medical testing, microbial qualitative and quantitative, and genetic modification testing. The comparability and reliability of gene quantification are related to people's health and even life safety, and are also the foundation of the development of life sciences. However, in the field of biometrics, the traceability of gene quantification has always been a technical challenge, and there is a lack of quantitative strategies with clear traceability. The lack of standard materials with accurate magnitudes affects the international and domestic mutual recognition of test results and hinders the expansion of application of gene analysis science and technology. Misjudgment of results will inevitably cause unnecessary economic costs. It even affects people's health. In short, the accuracy level of gene quantitative results does not match the vision of genomics application and precision medicine development. This project systematically studied new ways to trace DNA quantities, created sensitive, accurate and specific detection strategies, inspected uncertain sources, carried out the research and development of pioneering nucleic acid standard materials, realized the measurement calibration of multiple nucleic acid quantitative instruments, and promoted The capability verification of the testing laboratory has achieved significant benefits.
The project breaks out of the traditional model of using ultraviolet spectrophotometer to quantify nucleic acid molecules, innovatively carries out research on multiple nucleic acid quantification methods, and uses high-resolution inductively coupled plasma mass spectrometry to carry out quantitative research on phosphorus in nucleic acid molecules, thereby realizing The traceability of DNA quantities improves the reliability of results; The influence of primer design and target DNA configuration on the accuracy of dPCR quantitative results was discovered. Through the design of key primer sequences, a new PCR detection system was built to achieve high-sensitivity dPCR quantification; through DNA nanotechnology, a new interface reaction was built. The kinetic control mechanism has realized a high-sensitivity electrochemical nucleic acid biosensor, and the results have been applied to the detection of cancer markers. The key results of method research were published in the internationally renowned journals Methods (IF3.645), Biosens. Bioelectron. (IF6.409), published 2 international conference papers, 5 core Chinese journals, and authorized 1 invention patent. On the basis of systematic analysis of the uncertainty of quantitative methods, a multi-method joint determination mechanism was proposed, and a series of nucleic acid standard materials were developed: four Enterobacter sakazakii plasmid DNA standard materials (GBW(E)100305) were developed~8), 1 genetically modified soybean plasmid DNA standard material (GBW(E)100274), 12 pathogenic microorganism DNA standard material candidates. The concentration of this series of standard materials reaches 10E10 copies/μL, and 21 target genes are detected, of which the stability of certified standard materials can reach 24 months.
On the one hand, the DNA standard material developed has a high degree of accuracy and a relative expanded uncertainty of less than 5%(k=2), providing metrology support for the metrology calibration of a series of nucleic acid quantitative analysis instruments and breaking through the quantitative detection of DNA. The technical bottleneck of quantitative transmission is widely used in the metrology calibration of DNA quantitative analysis instruments such as real-time fluorescent quantitative PCR and micro-nucleic acid protein quantifiers. More than 600 metrology calibration services have been carried out and 200 users have been served. On the other hand, this set of standard materials provides reliable working standards and quality control basis for PCR-related testing and ensures the comparability and effectiveness of test results. Using the project's results in method research and standard materials, nucleic acid testing capabilities across Shanghai City have been carried out many times. 85 laboratories participated, achieving good results, ensuring the stability and reliability of quantitative gene testing results in the disease control system, and preventing large-scale public security incidents. Reflecting significant social benefits. The novelty search analysis proves that the project results have important practical application value, and the comprehensive technology has reached the international advanced level.