title: Isothermal and rapid detection of pathogenic microorganisms using a nano-rolling circle amplification-surface plasmon resonance biosensor.
abstract: Rolling circle amplification (RCA) of DNA is a sensitive and cost effective method for the rapid identification of pathogens without the need for sequencing. In this study, a surface plasmon resonance DNA biosensor based on RCA with a gold (Au) nanoparticle surface was established for isothermal identification of DNA. The probes included a specific padlock probe, a capture probe (CP), which is bound to biotin, and an Au nanoparticle-modified probe, which hybridizes with the RCA products. The CP was assembled on gold nanoparticles to increase its ability to bind and hybridize. The linear padlock probe, which was designed to circularize by ligation upon recognition of the bacterial pathogen-specific sequence in 16S rDNA, hybridizes to fully complementary sequences within the CP. Upon recognition, each target gene DNA is distinguished by localization onto the corresponding channel on the chip surface. Then, the immobilized CPs act as primers to begin the in situ solid-phase RCA reaction, which produces long single-stranded DNA. The RCA products fixed on the chip surface cause significant surface plasmon resonance angle changes. We demonstrated that six different bacterial pathogens can be identified simultaneously and that 0.5 pM of synthetic oligonucleotides and 0.5 pg μl(-1) of genomic DNA from clinical samples can be detected by this method with low background signals. Therefore, the multiplex diagnostic method provides a highly sensitive and specific approach for the rapid identification of positive samples.
authors.affiliation: Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing 400038, PR China.
authors.forename: Dachuan
authors.initials: D
authors.lastname: Shi
authors.affiliation: Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing 400038, PR China.
authors.forename: Junfu
authors.initials: J
authors.lastname: Huang
authors.affiliation: Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing 400038, PR China.
authors.forename: Zhengran
authors.initials: Z
authors.lastname: Chuai
authors.affiliation: Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing 400038, PR China.
authors.forename: Dong
authors.initials: D
authors.lastname: Chen
authors.affiliation: Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing 400038, PR China.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Zhu
authors.affiliation: Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing 400038, PR China.
authors.forename: Huan
authors.initials: H
authors.lastname: Wang
authors.affiliation: Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing 400038, PR China.
authors.forename: Jia
authors.initials: J
authors.lastname: Peng
authors.affiliation: Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing 400038, PR China.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Wu
authors.affiliation: Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing 400038, PR China. Electronic address: Dr.Q.Huang@gmail.com.
authors.forename: Qing
authors.initials: Q
authors.lastname: Huang
authors.affiliation: Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing 400038, PR China. Electronic address: fu_weiling@sina.com.
authors.forename: Weiling
authors.initials: W
authors.lastname: Fu
mesh_terms: D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification / Q000472:pathogenicity; D001483:Base Sequence; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D006046:Gold; D006801:Humans; D053768:Metal Nanoparticles; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000235:genetics; D020349:Surface Plasmon Resonance / Q000379:methods*
chemical_list: D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D006046:Gold
keywords: Au nanoparticles; Multiplex detection; Padlock probe; Pathogen; Rolling circle amplification; Surface plasmon resonance