title: Polymerase Spiral Reaction (PSR): A novel isothermal nucleic acid amplification method.
abstract: In this study, we report a novel isothermal nucleic acid amplification method only requires one pair of primers and one enzyme, termed Polymerase Spiral Reaction (PSR) with high specificity, efficiency, and rapidity under isothermal condition. The recombinant plasmid of blaNDM-1 was imported to Escherichia coli BL21, and selected as the microbial target. PSR method employs a Bst DNA polymerase and a pair of primers designed targeting the blaNDM-1 gene sequence. The forward and reverse Tab primer sequences are reverse to each other at their 5' end (Nr and N), whereas their 3' end sequences are complementary to their respective target nucleic acid sequences. The PSR method was performed at a constant temperature 61 °C-65 °C, yielding a complicated spiral structure. PSR assay was monitored continuously in a real-time turbidimeter instrument or visually detected with the aid of a fluorescent dye (SYBR Greenı), and could be finished within 1 h with a high accumulation of 10(9) copies of the target and a fine sensitivity of 6 CFU per reaction. Clinical evaluation was also conducted using PSR, showing high specificity of this method. The PSR technique provides a convenient and cost-effective alternative for clinical screening, on-site diagnosis and primary quarantine purposes.
authors.affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, 100071, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Liu
authors.affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, 100071, China.
authors.forename: Derong
authors.initials: D
authors.lastname: Dong
authors.affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, 100071, China.
authors.forename: Zhan
authors.initials: Z
authors.lastname: Yang
authors.affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, 100071, China.
authors.forename: Dayang
authors.initials: D
authors.lastname: Zou
authors.affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, 100071, China.
authors.forename: Zeliang
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, 100071, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Yuan
authors.affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, 100071, China.
authors.forename: Liuyu
authors.initials: L
authors.lastname: Huang
mesh_terms: D001419:Bacteria / Q000145:classification / Q000201:enzymology / Q000235:genetics; D001424:Bacterial Infections / Q000382:microbiology; D001483:Base Sequence; D052160:Benzothiazoles; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004256:DNA Polymerase I / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D003959:Diamines; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009930:Organic Chemicals / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011804:Quinolines; D015203:Reproducibility of Results; D013045:Species Specificity; D013696:Temperature; D001618:beta-Lactamases / Q000235:genetics
chemical_list: D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D004247:DNA; D004256:DNA Polymerase I; D001618:beta-Lactamases; C552864:beta-lactamase NDM-1