Figure 1 Effect of increasing concentration differences between targets
in multiplex qPCR reactions. Dilution series of multicopy targets (A-C) or internal control target cry1 (D-F) were made in the presence of the other HM781-36B mouse targets detected in each qPCR at a Selleckchem AICAR constant concentration near the detection limit. Triplicate multiplex qPCR measurements were performed and mean Cq values with 95% confidence limits are shown for each target. Significant concentration differences are possible between the pathogen specific targets and the internal control target, as these organisms could be mixed in very different quantities. Inhibition of the internal control (IC) by excess pathogen DNA is not a problem as the function of the IC is to exclude false negative results (a positive pathogen signal makes an additional IC signal irrelevant). In contrast, it is essential that inhibition of pathogen targets by the internal control is prevented. To determine the boundaries within which IC B. thuringiensis DNA could be added to pathogen DNA without interfering with the detection of low pathogen concentrations, a dilution series of the IC target amplicon (cry1 gene) was made in the presence of a constant and low concentration of pathogen targets and
measured by the multiplex qPCRs. As shown in Figure 1D-F, the amplification of 20 copies of pathogen targets was inhibited (increasing Cq) if more than 200 copies of the internal control target were present for B. anthracis or more than 2000 copies for Y. pestis and F. tularensis. BAY 80-6946 Moreover, rare targets were still detectable at much higher excess ratios of internal
control, even though at higher Cq values. Discussion Multiplexing and the reduction of false negative and false positive results In this report, we describe the development of multiplex qPCRs for the rapid and reliable detection of B. anthracis, F. tularensis and Y. pestis. The assays include a signature sequence from B. thuringiensis which allows the use of its spores as combined internal control for both DNA extraction and subsequent DNA amplification. As Bacillus spores are among the Megestrol Acetate most resistant of microbial structures, DNA extraction from such spores can be considered to be a reliable indicator for successful DNA extraction from other microbes. Application of internal controls is especially important when measuring environmental samples because these tend to contain various sorts of PCR inhibitors. The internal control helps preventing false negative results, which are further reduced by the sensitivity of the methods and by the recognition of multiple signatures per organism. Multiplexing reduces the chance that the pathogen escapes detection due to modification or loss of plasmids or genes (natural or by manipulation).