Figure 4
Molecular beacons can detect DNA between 1 and 106A. phagocytophilum in a duplex assay when the human DNA is also present. Amplification plots of APH1387 and ACTA1 genes in PCR assays using the human DNA representing 105 ACTA1 copies spiked with ten-fold dilutions from 1 to 106 plasmid copies containing APH1387 were used to estimate quantities of A. phagocytophilum (A) and human (C) DNA by employing both Aph1387 and ACTA1 molecular beacons. The assay quantified amplicons from both the APH1387 and the ACTA1 genes in the same PCR assay tubes. A high coefficient of correlation (r2 = 0.985) between the Ct values and the bacterial numbers obtained from the standard curve (B) indicates that the molecular beacons can quantify burden of this intracellular pathogen in the infected human cells using multiplex assay system under the standardized conditions in a sensitive and specific manner even though sensitivity of detection is slightly higher than one.