title: Solid-phase PCR for rapid multiplex detection of Salmonella spp. at the subspecies level, with amplification efficiency comparable to conventional PCR.
abstract: Solid-phase PCR (SP-PCR) has attracted considerable interest in different research fields since it allows parallel DNA amplification on the surface of a solid substrate. However, the applications of SP-PCR have been hampered by the low efficiency of the solid-phase amplification. In order to increase the yield of the solid-phase amplification, we studied various parameters including the length, the density, as well as the annealing position of the solid support primer. A dramatic increase in the signal-to-noise (S/N) ratio was observed when increasing the length of solid support primers from 45 to 80 bp. The density of the primer on the surface was found to be important for the S/N ratio of the SP-PCR, and the optimal S/N was obtained with a density of 1.49 × 1011molecules/mm2. In addition, the use of solid support primers with a short overhang at the 5' end would help improve the S/N ratio of the SP-PCR. With optimized conditions, SP-PCR can achieve amplification efficiency comparable to conventional PCR, with a limit of detection of 1.5 copies/μl (37.5 copies/reaction). These improvements will pave the way for wider applications of SP-PCR in various fields such as clinical diagnosis, high-throughput DNA sequencing, and single-nucleotide polymorphism analysis. Graphical abstract Schematic representation of solid-phase PCR.
authors.affiliation: National Food Institute, Technical University of Denmark, Mørkhøj Bygade 19, 2860, Søborg, Denmark.
authors.forename: Wai Hoe
authors.initials: WH
authors.lastname: Chin
authors.affiliation: Department of Micro- and Nanotechnology, Technical University of Denmark, 2800, Kongens Lyngby, Denmark. Sun.Yi@nanotech.dtu.dk.
authors.forename: Yi
authors.initials: Y
authors.lastname: Sun
authors.affiliation: National Food Institute, Technical University of Denmark, Mørkhøj Bygade 19, 2860, Søborg, Denmark.
authors.forename: Jonas
authors.initials: J
authors.lastname: Høgberg
authors.affiliation: National Food Institute, Technical University of Denmark, Mørkhøj Bygade 19, 2860, Søborg, Denmark.
authors.forename: Tran Quang
authors.initials: TQ
authors.lastname: Hung
authors.affiliation: Department of Micro- and Nanotechnology, Technical University of Denmark, 2800, Kongens Lyngby, Denmark.
authors.forename: Anders
authors.initials: A
authors.lastname: Wolff
authors.affiliation: National Food Institute, Technical University of Denmark, Mørkhøj Bygade 19, 2860, Søborg, Denmark.
authors.forename: Dang Duong
authors.initials: DD
authors.lastname: Bang
mesh_terms: D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000145:classification / Q000379:methods*; D012475:Salmonella / Q000235:genetics / Q000302:isolation & purification*; D012480:Salmonella Infections / Q000175:diagnosis* / Q000235:genetics / Q000382:microbiology
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial
keywords: High efficiency; Multiplex detection; Rapid diagnostics; Solid-phase PCR