title: The isothermal amplification detection of double-stranded DNA based on a double-stranded fluorescence probe.
abstract: Here we have developed a novel method of isothermal amplification detection of double-stranded DNA (dsDNA) based on double-stranded fluorescence probe (ds-probe). Target dsDNA repeatedly generated single-stranded DNA (ssDNA) with polymerase and nicking enzyme. The ds-probe as a primer hybridized with ssDNA and extended to its 5'-end. The displaced ssDNA served as a new detection target to initiate above-described reaction. Meanwhile, the extended ds-probe could dynamically dissociate from ssDNA and self-hybridize, converting into a turn-back structure to initiate another amplification reaction. In particular, the ds-probe played a key role in the entire experimental process, which not only was as a primer but also produced the fluorescent signal by an extension and displacement reaction. Our method could detect the pBluescript II KS(+) plasmid with a detection limit of 2.3 amol, and it was also verified to exhibit a high specificity, even one-base mismatch. Overall, it was a true isothermal dsDNA detection strategy with a strongly anti-jamming capacity and one-pot, only requiring one ds-probe, which greatly reduced the cost and the probability of contamination. With its advantages, the approach of dsDNA detection will offer a promising tool in the field of point-of-care testing (POCT).
authors.affiliation: Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China.
authors.forename: Chao
authors.initials: C
authors.lastname: Shi
authors.affiliation: Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China.
authors.forename: Fanjin
authors.initials: F
authors.lastname: Shang
authors.affiliation: Shandong Provincial Key Laboratory of Biochemical Analysis, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China.
authors.forename: Mei
authors.initials: M
authors.lastname: Pan
authors.affiliation: Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China.
authors.forename: Sen
authors.initials: S
authors.lastname: Liu
authors.affiliation: Shandong Provincial Key Laboratory of Biochemical Analysis, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China. Electronic address: mcp169@163.com.
authors.forename: Cuiping
authors.initials: C
authors.lastname: Ma
mesh_terms: D015374:Biosensing Techniques; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D004277:DNA, Single-Stranded / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D057230:Limit of Detection; D009693:Nucleic Acid Hybridization
chemical_list: D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D004247:DNA
keywords: Double-stranded fluorescence probe; Isothermal amplification; POCT; Turn-back structure; dsDNA detection