Standard curves for molecular beacon-based real-time PCR detection of targets invA, fliC and prot6E. The plots illustrate the relationship of known number of target DNA copies per reaction to the threshold cycle of detection (CT) for each this website molecular beacon reaction. The CT is directly proportional to the log of the input copy equivalents, as
demonstrated by the standard curves generated. Detection of S. enterica alleles in bacterial samples by molecular beacon-based uniplex real-time PCR The molecular beacon-based real-time PCR assay designed in this study was tested on environmental and food samples of S. Enteritidis
click here and S. Typhimurium (Table 1), as well as several commercially available bacterial strains (Table 2) and various Salmonella serovars obtained from a reference laboratory for Salmonella (Table 3). All samples were investigated first by uniplex assays to detect invA, prot6E and fliC (Table 4). In the reaction for detection of invA, all 44 Salmonella samples were positive and all 18 non-Salmonella samples were undetectable. Positive results (≤ 10 copies of DNA per reaction) had CT values ranging from 15 to 25. In the prot6E reaction, all 21 S. Enteritidis samples gave positive PCR results and all 41 non-Enteritidis samples were negative. Positive samples for the prot6E gene had CT values ranging between 15 and 18 with one exception, the commercially available specimen of S. Enteritidis (Table 3) for which fluorescence
detection significantly increased around cycle 30. Mannose-binding protein-associated serine protease Finally, in the fliC reaction, all 17 S. Typhimurium samples gave positive PCR results and all 45 non-Typhimurium samples were negative. Positive results had CT values ranging from 15 to 18 cycles. These results showed that the primers and beacons for each reaction work well individually and that they amplify and detect their target sequence with very high specifiCity and sensitivity. The CT values exhibited by the samples in these experiments, compared to the plot of the standards of known concentration, indicated that the extracted DNA from the bacterial samples was higher than the range of concentrations tested by the standards (>107 copies per reaction). Therefore 100-fold dilutions of all extracted DNA samples were prepared for use in the two-step duplex assay, so that the resulting CT values would fall within the range seen on the standard curves.