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. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA.