A new single-step quantitative pathogen detection system: template-tagging followed by multiplex asymmetric PCR using common primers and CE-SSCP.
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.
Shin
Gi Won
GW
School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Korea.
Cho
Yang Sook
YS
Hwang
Hee Sung
HS
Oh
Mi-Hwa
MH
Nam
Hong Gil
HG
Park
Jin Hyun
JH
Jung
Gyoo Yeol
GY
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
D017931:DNA Primers; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S