A Method for Detection of Nicotinamide Adenine Dinucleotide Based on Enzymatic Isothermal Amplification Strategy

Nicotinamide adenine dinucleotide (NAD+) is a key indicator reflecting the body′s physiological state, and its accurate detection is of great significance for scientific research and clinical diagnosis. However, traditional detection methods have limitations of long detection cycles and high operation cost, which make it difficult to meet the needs of wide application. Based on the cofactor dependence of ligase and DNA isothermal amplification technology, a novel NAD+ detection system was developed in this study. The detection process of the system included three steps: Taq DNA ligase catalyzed the ligation of oligonucleotides in the presence of NAD+; Nt.BstNBI endonuclease and Bst polymerase worked synergistically to achieve isothermal amplification, generating a large number of single-stranded DNA; the fluorescent signal was triggered by molecular beacons to complete the qualitative and quantitative analysis of NAD+. Experimental results showed that this detection method could complete the detection within 1‒2 h, with a detection limit of 3.16 pmol/L, significantly improving the detection speed and sensitivity. This method effectively broke through the limitations of traditional technologies in many aspects, provided efficient and economical technical support for medical diagnosis, environmental monitoring, food safety and other fields, and had broad industrialization prospects.