title: Colorimetric monitoring of rolling circle amplification for detection of H5N1 influenza virus using metal indicator.
abstract: A new colorimetric method for monitoring of rolling circle amplification was developed. At first H5N1 target hybrids with padlock probe (PLP) and then PLP is circularized upon the action of T4 ligase enzyme. Subsequently, the circular probe is served as a template for hyperbranched rolling circle amplification (HRCA) by utilizing Bst DNA polymerase enzyme. By improving the reaction, pyrophosphate is produced via DNA polymerization and chelates the Mg(2+) in the buffer solution. This causes change in solution color in the presence of hydroxy naphthol blue (HNB) as a metal indicator. By using pH shock instead of heat shock and isothermal RCA reaction not only the procedure becomes easier, but also application of HNB for colorimetric detection of RCA reaction further simplifies the assay. The responses of the biosensor toward H5N1 were linear in the concentration range from 0.16 to 1.20 pM with a detection limit of 28 fM.
authors.affiliation: Laboratory of Microanalysis, Institute of Bioc hemistry and Biophysics, University of Tehran, P.O. Box 131451384, Tehran, Iran; Nanobiomedicine Center of Excellence,g Nanoscience and Nanotechnology Research Center, University of Tehran, P.O. Box 131451384, Tehran, Iran.
authors.forename: Seyed Vahid
authors.initials: SV
authors.lastname: Hamidi
authors.affiliation: Laboratory of Microanalysis, Institute of Bioc hemistry and Biophysics, University of Tehran, P.O. Box 131451384, Tehran, Iran; Nanobiomedicine Center of Excellence,g Nanoscience and Nanotechnology Research Center, University of Tehran, P.O. Box 131451384, Tehran, Iran. Electronic address: ghourchian@ut.ac.ir.
authors.forename: Hedayatollah
authors.initials: H
authors.lastname: Ghourchian
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods; D001717:Birds; D003124:Colorimetry / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics; D011756:Diphosphates / Q000737:chemistry; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics / Q000302:isolation & purification*; D005585:Influenza in Birds / Q000175:diagnosis / Q000821:virology*; D007251:Influenza, Human / Q000175:diagnosis / Q000821:virology*; D057230:Limit of Detection; D008274:Magnesium / Q000737:chemistry; D009282:Naphthalenesulfonates / Q000737:chemistry; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification
chemical_list: D018076:DNA, Complementary; D011756:Diphosphates; D009282:Naphthalenesulfonates; D012367:RNA, Viral; C107241:diphosphoric acid; C540250:trisodium 3-hydroxy-4-((2Z)-2-(2-oxo-4-sulfonatonaphthalen-1-ylidene)hydrazinyl)naphthalene-2,7-disulfonate; D008274:Magnesium
keywords: Bst DNA polymerase; H(5)N(1) virus; Hydroxy naphthol blue; Rolling circle amplification; pH shock