title: A new single-step quantitative pathogen detection system: template-tagging followed by multiplex asymmetric PCR using common primers and CE-SSCP.
abstract: Rapid diagnosis of bacterial infection is important for patient management and appropriate therapy during the early phase of bacteria-induced disease. Among the existing techniques for identifying microbial, CE-SSCP combined with 16S ribosomal RNA gene-specific PCR has the benefits of excellent sensitivity, resolution, and reproducibility. However, even though CE-SSCP can separate PCR products with high-resolution, multiplex detection and quantification are complicated by primer-dimer formation and non-specific amplification. Here, we describe a novel technique for multiplex detection and quantification of pathogens by template-tagging followed by multiplex asymmetric PCR and subsequent CE-SSCP. More specifically, we reverse transcribed 16S ribosomal RNAs from seven septicemia-inducing pathogens, tagged the templates with common end sequences, and amplified them using common primers. The resulting amplicons could be successfully separated by CE-SSCP and quantified by comparison to an internal standard. This method yielded results that illustrate the potential of this system for diagnosing infectious disease.
authors.affiliation: School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Korea.
authors.forename: Gi Won
authors.initials: GW
authors.lastname: Shin
authors.forename: Yang Sook
authors.initials: YS
authors.lastname: Cho
authors.forename: Hee Sung
authors.initials: HS
authors.lastname: Hwang
authors.forename: Mi-Hwa
authors.initials: MH
authors.lastname: Oh
authors.forename: Hong Gil
authors.initials: HG
authors.lastname: Nam
authors.forename: Jin Hyun
authors.initials: JH
authors.lastname: Park
authors.forename: Gyoo Yeol
authors.initials: GY
authors.lastname: Jung
mesh_terms: D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification*; D001424:Bacterial Infections / Q000382:microbiology*; D017931:DNA Primers / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods*; D016014:Linear Models; D016133:Polymerase Chain Reaction / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S