title: Suspension bead array of the single-stranded multiplex polymerase chain reaction amplicons for enhanced identification and quantification of multiple pathogens.
abstract: Rapid identification of single and multiple infectious agents is vital in clinical settings and during biothreat attack. This study assesses the assay of single-stranded multiplex polymerase chain reaction (PCR) amplicons by suspension bead array (SSMP-SBA) for multiple pathogens identification in a single-tube reaction. A 15-plex assay for identification of 11 highly infectious pathogens was developed to evaluate the performance of SSMP-SBA. Pathogen-specific amplicons were obtained by sequential amplification of genomic DNAs using gene-specific primers tagged with artificial unique sequences and unique primers of which the reverse primer was modified by biotin and phosphorothioate. The SSMP products generated by T7 exonuclease-mediated DNA hydrolysis were hybridized to 15 sets of beads coupled with gene-specific and control oligonucleotide probes for pathogen identification and quantification by flow cytometry. This method was validated via assessment of 57 reference strains and one clinical bacterial isolate. All 11 pathogens can be detected by the 15-plex SSMP-SBA assay, and this design significantly enhanced the signal-to-noise ratio and improved the assay performance. This assay achieves similar sensitivity to our in-house real-time PCR system with the limit of detection equivalent to 5-100 genome copies and a linear dynamic range crossing three to five logs. In the validation assay, a 100% accuracy rate was achieved when the pathogens were among the target species. Notably, the species of pathogens were accurately identified from the samples with multiple infections. SSMP-SBA presents superior performance with multiplexing capability in a single-tube reaction and provides a new approach for detection and species identification of multiple pathogen infections.
authors.affiliation: Department of Biotechnology and Laboratory Science in Medicine, Infection and Immunity Center, National Yang-Ming University, Taipei, Taiwan, Republic of China.
authors.forename: Hui-Ling
authors.initials: HL
authors.lastname: Hsu
authors.forename: Hsin-Hsien
authors.initials: HH
authors.lastname: Huang
authors.forename: Chung-Chih
authors.initials: CC
authors.lastname: Liang
authors.forename: Hung-Chi
authors.initials: HC
authors.lastname: Lin
authors.forename: Wen-Tssann
authors.initials: WT
authors.lastname: Liu
authors.forename: Feng-Ping
authors.initials: FP
authors.lastname: Lin
authors.forename: Jyh-Hwa
authors.initials: JH
authors.lastname: Kau
authors.forename: Kuang-Hui
authors.initials: KH
authors.lastname: Sun
mesh_terms: D017848:Blood-Borne Pathogens / Q000145:classification / Q000302:isolation & purification*; D002002:Brucella / Q000235:genetics* / Q000302:isolation & purification; D002006:Brucellosis / Q000175:diagnosis / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D005260:Female; D006801:Humans; D008875:Middle Aged; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction; D059629:Signal-To-Noise Ratio
chemical_list: D004269:DNA, Bacterial