A DNA molecular diagnostic technology with LAMP-like sensitivity based on one pair of hairpin primers-mediated isothermal polymerization amplification

• Published in: Analytica chimica acta Vol. 1134; pp. 144 - 149
• Main Authors: Yin, Yao, Wu, Zuoci, Li, Guangping, Huang, Jin, Guo, Qiuping, Meng, Xiangxian
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 16.10.2020
• Subjects: DNA - genetics; Hairpin primers; Humans; Isothermal amplification; Molecular diagnostic; Nucleic Acid Amplification Techniques; Pathology, Molecular; Polymerization; Schistosoma japonicum; Sensitivity and Specificity; Technology; Hairpin primers; Molecular diagnostic; Schistosoma japonicum; Isothermal amplification
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/j.aca.2020.05.062

With the rapid development of isothermal amplification technology, DNA molecular diagnosis has become an important reference for clinical treatment. In this work, we have designed a DNA molecular diagnostic technology with LAMP-like sensitivity for nucleic acid analysis and detection based on only one pair of hairpin primers. This DNA molecular diagnostic technology consists of Bst DNA polymerase and one pair of hairpin primers, which are designed easily by adding a stem-loop structure to a target binding domain. When the target is present, the polymerization reaction between the hairpin primers and the target generates a specific dumbbell DNA similar to LAMP, which triggers cyclic amplification reactions to extend a series of long dsDNA products with repeated sequences by inserting fluorescent dye Eva Green observed the increase in fluorescence signal. In our method, using the hairpin primers-mediated isothermal polymerization amplification, we can specifically monitor 3–5 copies of the target nucleic acid in the system without labeling and temperature cycling in the reaction. In addition, serum samples from 13 patients with suspected schistosomiasis were targeted; we further demonstrated the ability of the technology to detect complex clinic samples, and its potentially inestimable applicability in clinic early molecular diagnostic research. In this work, we designed a simple DNA molecular diagnostic technology with LAMP-like sensitivity based on one pair of hairpin primers-mediated isothermal polymerization amplification. The hairpin primers are designed easily by adding a stem-loop structure to a target binding domain. By quickly forming a dumbbell DNA structure, which not only trigger product extension and polymerization, but also can achieve 3–5 copies of sensitivity, without the need to label and temperature cycling in the reaction. By inserting Eva Green to emit fluorescence, it can be used as a DNA molecular diagnostic sensor. [Display omitted] •The method can more easily form a dumbbell DNA structure.•Developed method can achieve a 3-5 copies sensitivity.•This design can form a DNA sensor by EVG to emit fluorescence.•The method can detect serum samples.