Therefore, to maintain sensitivity of the assay and accurate

quantification of B. burgdorferi in the infected tissues using molecular beacons, the samples should be diluted to obtain 200 ng or less total DNA per reaction. Figure 4 A serial dilution of mouse joint DNA is detectable by Nirogacestat Nidogen molecular beacons. Amplification plots of five-fold dilution of mouse DNA used STAT inhibitor in PCR assays with Nidogen molecular beacon for detection of nidogen amplification products are shown (A). A standard curve (B) and high coefficient of correlation (r2 = 0.998) indicates that the Nidogen molecular beacon is effective in detecting 200 ng to the low level (1 ng) of mouse DNA. Sensitivity and specificity of detection of qPCR amplicons is not affected by multiplex analysis Quantity of B. burgdorferi in the infected tissues has been determined using conventional monoplex assays in which spirochete-specific primers and detection reagent (SYBR Green Vactosertib datasheet dye or TaqMan probe) are incorporated in the qPCR assay. This quantification involves simultaneous isolation of host and pathogen

DNA. Therefore, the sensitivity of the detection of the spirochetes could be affected in multiplex analyses. Molecular beacons can simultaneously detect more than one amplicon, i.e., both the pathogen and the host, in the same reaction tube. To examine if sensitivity of detection by molecular beacons diminishes in multiplex analyses, a comparative analysis of the serially diluted B. burgdorferi in the mouse tissues was conducted in monoplex and multiplex assay systems. Uninfected C3H mouse tissue DNA (105 nidogen copies) was spiked with DNA from 106 B. burgdorferi followed by ten-fold dilution in same concentration of mouse

DNA. Both set of primers, for recA and nidogen amplification, were added in each reaction. Only one molecular beacon was used at a time for monoplex assays while both RecA3 and Nidogen molecular beacons were included in multiplex assays. Sensitivity of detection of B. Y-27632 burgdorferi was high both in monoplex (Figure 5A) and multiplex assays (Figure 5B). Although a slight delay in Ct values was observed in multiplex relative to monoplex system (Figure 5), both monoplex and multiplex analyses show good correlation and are able to detect as little as one copy number of B. burgdorferi. Hence, the presence of primers and a molecular beacon for nidogen amplicon does not affect sensitivity of detection of B. burgdorferi. Thus, a multiplex assay system can be employed to accurately quantify Lyme spirochetes in infected mammalian tissues. Figure 5 Multiplex analysis does not affect sensitivity of detection of B. burgdorferi by molecular beacons. A comparison of monoplex (A) and multiplex (B) assay systems of different dilutions of B. burgdorferi spiked in the mouse DNA containing 105 nidogen copies indicates that multiplex analysis does not affect the sensitivity of spirochete detection.