Using Fluorescence-Based PCR
3.2. Fluorescence-Based PCR
1. Program the robot to set up PCR in a final volume of 25 µL consisting of 20 µL of ABsolute™ QPCR Mastermix, 1 µL of each primer (1 pmol final concentra- tion), 1 µL of probe (0.5 pM final concentration), and 2 µL of DNA.
2. PCR amplification is performed using the cycle, 95⬚C for 2 min, followed by 45 cycles of 95⬚C for 15 s, 53⬚C for 30 s, and 72⬚C for 2 min followed by a final extension at 72⬚C for 3 min.
3. To determine the assay cut-off value for negative and positive samples, five nega- tive controls and one of each positive control are used. Negative controls consist of PCR mix without target DNA, but include primers, and the final volume made to 25 µL with sterile distilled water. The positive control consists of PCR mix, 1 µL of each primer, and 2 µL of control DNA.
4. Wavelengths of 485/530 nm, 530/590 nm, and 590/645 nm are used to determine the fluorescence produced by the CtrA, PlyA, and BexA probes, respectively. A total of 100 endpoint readings are taken from each well. The average reading is determined by using the KC4 software and a cut-off value calculated using 1.4 standard deviations above the mean fluoresence for the five negative controls.
4. Notes
1. The method is relatively inexpensive compared with similar methods because the chemistry is widely available without the need for specialist equipment and, as such, can be performed manually using conventional set-up techniques and results read with a manually operated fluorescent plate reader possessing the appropriate filter set.
2. Liquid handling robots are available from various commercial manufacturers.
The actual system set-up often can be modified according to individual require- ments such that various assays can be performed on the same platform.
3. The methodology described assumes that the user has been fully trained on robot programming or the manufacturer has provided preprogrammed methods as requested by the user. The specific tasks performed by the robot can be modified as necessary.
4. PCR mastermix can be stored at 4°C for as long as 1 mo for ready access if reagents are used regularly. However, this storage is not recommended for occa- sional use and, instead reagents should preferably be stored at –20⬚C. They also should be protected from light. Repeated freeze–thawing should be avoided. All reagents should be maintained at 4⬚C while on the robotic platform. Failure to do so can result in reduced sensitivity and specificity of assays.
5. Genomic DNA extraction kits can be purchased commercially for manual or automated methods. A study compared a number of different methods to deter- mine the most suitable for automation by determining ease of use, sensitivity, and specificity among 96-well binding plate, 96-well filter plate, and metallic bead formats (19). Not all kits are specifically designed for all DNA extraction requirements, but most can be modified and validated accordingly.
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From: Methods in Molecular Biology, vol. 345: Diagnostic Bacteriology Protocols, Second Edition Edited by: L. O‘Connor © Humana Press Inc., Totowa, NJ