Design and Construction of a Single-Tube, LATE-PCR, Multiplex Endpoint Assay with Lights-On/Lights-Off Probes for the Detection of Pathogens Associated with Sepsis
Aims. The goal of this study was to construct a single tube molecular diagnostic multiplex assay for the detection of microbial pathogens commonly associated with septicemia, using LATE-PCR and Lights-On/Lights-Off probe technology. Methods and Results. The assay described here identified pathogens associated with sepsis by amplification and analysis of the 16S ribosomal DNA gene sequence for bacteria and specific gene sequences for fungi. A sequence from an unidentified gene in Lactococcus lactis subsp. cremoris served as a positive control for assay function. LATE-PCR was used to generate single-stranded amplicons that were then analyzed at endpoint over a wide temperature range in a specific fluorescent color. Each bacterial target was identified by its pattern of hybridization to Lights-On/Lights-Off probes derived from molecular beacons. Complex mixtures of targets were also detected. Conclusions. All microbial targets were identified in samples containing low starting copy numbers of pathogen genomic DNA, both as individual targets and in complex mixtures. Significance and Impact of the Study. This assay uses new technology to achieve an advance in the field of molecular diagnostics: a single-tube multiplex assay for identification of pathogens commonly associated with sepsis. 1. Introduction Globally, sepsis affects millions of people, killing at least 1 in 4 [1, 2]. Nucleic-acid-testing- (NAT-) based methods of pathogen identification have for some time been regarded as a desirable alternative to conventional culture-based diagnostic methods for analysis of septicemia specimens [3–23]. Early goal directed therapy within the first 3 hours of the clinical presentation of severe sepsis and septic shock has significantly improved outcomes [1, 24–31]. The use of molecular diagnostic methods is logical because they are rapid, sensitive, specific, and reproducible [32]. We have optimized linear-after-the-exponential PCR (LATE-PCR) to overcome the challenges inherent to construction of a highly informative single-tube multiplex assay for septicemia where an unlimited number of possible pathogens must be detected. LATE-PCR is a form of non-symmetric PCR that generates single-stranded DNA making it possible to probe and quantify these strands at endpoint [33–41]. Utilizing both the expanded temperature and fluorescent space now available for detection of targets at endpoint, LATE-PCR assays can readily be multiplexed [40]. Two conceptually distinct sepsis single tube multiplex assays have been designed and tested in our laboratory. One design used a
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