Application of Rapid-Cycle Real-Time Polymerase Chain Reaction for the Detection of Microbial Pathogens: The Mayo-Roche Rapid Anthrax Test  James R. Uhl, MS, Constance A. Bell, PhD, Lynne M. Sloan, MT(ASCP), Mark J. Espy, MS, Thomas F. Smith, PhD, Jon E. Rosenblatt, MD, Franklin R. Cockerill, MD  Mayo Clinic Proceedings  Volume 77, Issue 7, Pages 673-680 (July 2002) DOI: 10.4065/77.7.673 Copyright © 2002 Mayo Foundation for Medical Education and Research Terms and Conditions

Figure 1 Conventional polymerase chain reaction (PCR) testing formats employ 3 and sometimes 4 steps. In this diagram, steps 1, 2, 3a, and 4 represent the traditional and most work-intensive approach, and steps 1, 2, and 3b represent newer testing formats that incorporate more user-friendly enzyme-linked hybridization capture assays to detect PCR product. cDNA = complementary DNA. Mayo Clinic Proceedings 2002 77, 673-680DOI: (10.4065/77.7.673) Copyright © 2002 Mayo Foundation for Medical Education and Research Terms and Conditions

Figure 2 A, Double-stranded “target” DNA is shown with red and green highlighted segments representing complementary sequences for polymerase chain reaction (PCR) primers. A-D, Strands labeled with a single asterisk indicate that the strand is the original target DNA. B, Double-stranded DNA is denatured by increasing the temperature in the reaction vessel. Polymerase chain reaction primers anneal to complementary target DNA (blue primer anneals to red complementary sequence, and yellow primer anneals to green complementary sequence). C, Double-stranded DNA is produced in a 5′ -3’ orientation from the 3’ end of the primers by using a thermostable DNA polymerase. “Shaggy-ended” double-stranded DNA results with new complementary strands containing primer-annealing sites for subsequent amplification reactions. Double asterisks represent new strands that are synthesized with this first amplification cycle. D, In this second PCR cycle, complementary DNA strands are synthesized for the 4 DNA strands that comprise the 2 double- stranded DNA structures formed in C. Of the 8 single-stranded DNA strands, 2 are of a length equal to and including the distance between the primer-annealing sites. These short DNA products accumulate exponentially with subsequent PCR cycles. Mayo Clinic Proceedings 2002 77, 673-680DOI: (10.4065/77.7.673) Copyright © 2002 Mayo Foundation for Medical Education and Research Terms and Conditions

Figure 3 Principles of TaqMan probes. The 5’ nuclease activity of Taq DNA polymerase is exploited to cleave a TaqMan probe during polymerase chain reaction (PCR). A, TaqMan probe is shown annealed to the target DNA. The probe contains a reporter dye at the 5’ end of the probe (green circle) and a quencher dye at the 3’ end of the probe (red circle). B, During PCR, a complementary strand of DNA is synthesized, and the 5’ exonuclease activity of Taq DNA polymerase excises the reporter dye. C, Fluorescence of the reporter dye (indicated by bright green starburst effect of reporter dye) occurs as a result of separation of the reporter dye from the quencher dye. Mayo Clinic Proceedings 2002 77, 673-680DOI: (10.4065/77.7.673) Copyright © 2002 Mayo Foundation for Medical Education and Research Terms and Conditions

Figure 4 Principles of FRET (fluorescence resonance energy transfer) probes using the LightCycler. A, Essential components using fluorescence-labeled oligonucleotides as FRET probes: 2 different oligonucleotides (labeled) and the amplification product. Probe 1 bears a fluorescein label at its 3’ end, whereas probe 2 is labeled with another label (LightCycler [LC] Red 640) at its 5’ end. The sequences of the 2 probes are selected so that they can hybridize to the amplified DNA fragment in a head-to-tail arrangement, thereby bringing the 2 fluorescent dyes into proximity (B). The first dye (fluorescein) is excited by the LightCycler's light source and emits green fluorescent light at a slightly longer wavelength. When the 2 dyes are in proximity, the energy thus emitted excites the LC Red 640 attached to the second probe that subsequently emits red fluorescent light at an even longer wavelength. This energy transfer, referred to as FRET, is highly dependent on the spacing between the 2 dye molecules. Only if the molecules are in proximity (between 1 and 5 nucleotides) is the energy transferred efficiently. The intensity of the light emitted by the LC Red 640 is measured in Channel 2 (640 nm) of the Light Cycler's optics. The increasing amount of measured fluorescence is proportional to the increasing amount of DNA generated during the ongoing PCR process. Since LC Red 640 emits a signal only when both oligonucleotides are hybridized, fluorescence is measured after the annealing step (B). Hybridization does not occur during the denaturation phase of the PCR (A), and fluorescence cannot be detected at 640 nm. After annealing, the temperature is raised, and the hybridization probe is displaced by the Taq DNA polymerase (C). At the end of the elongation step, the PCR product is double stranded, and the probes are too far apart to allow FRET (D). With permission from Roche Applied Science. Mayo Clinic Proceedings 2002 77, 673-680DOI: (10.4065/77.7.673) Copyright © 2002 Mayo Foundation for Medical Education and Research Terms and Conditions

Figure 5 Principle of molecular beacon probes. The molecular beacon probe in its hairpin form (top of the figure) is nonfluorescent because the stem hybrid keeps the fluorophore (reporter dye) close to the quencher dye. When the probe sequence in the loop hybridizes to its target (bottom of the figure), a rigid double helix is formed. This conformational reorganization separates the quencher from the reporter dye, restoring fluorescence (indicated by bright green starburst effect of reporter dye). Mayo Clinic Proceedings 2002 77, 673-680DOI: (10.4065/77.7.673) Copyright © 2002 Mayo Foundation for Medical Education and Research Terms and Conditions

Figure 6 Schematic of the LightCycler. Temperature cycling is achieved by alternating heated air provided by the heating coil and ambient air provided by the air inlet and fan. Composite plastic-glass capillary tubes provide a sealed vessel with a high surface- to-volume ratio, which, along with rapid air movement provided by the fan, allows rapid thermal exchange within the reaction fluid in the capillary tube. Fluorescence of polymerase chain reaction product is detected by photodiodes coupled with fluorimeters. Capillary tubes, positioned in a carousel, are periodically rotated over fluorimeters for fluorescent determinations. LED = light emitting diode. Mayo Clinic Proceedings 2002 77, 673-680DOI: (10.4065/77.7.673) Copyright © 2002 Mayo Foundation for Medical Education and Research Terms and Conditions

Figure 7 A, LightCycler Assay for detection of pagA gene with use of FRET (fluorescence resonance energy transfer) probes. As polymerase chain reaction (PCR) product accumulates, fluorescence of LightCycler (LC) Red 640 labeled probe increases because of the transfer of energy from the fluorescein-labeled probe at the temperature that both probes are hybridized to the amplified target DNA. The arrow indicates the crossing point (also referred to as crossover point or threshold cycle). Crossing points represent cycle numbers in which fluorescence levels of all samples are the same (eg, just above background). This specific cycle number at which a sample is positive (and thus the crossing point) depends on the initial amount of target in the sample—with increased amounts of initial material, fewer cycles will be needed to achieve the specific fluorescence value. Shown are the results of 3 samples. The orange line indicates a sample with a relatively high copy number of target DNA. The gold line is a control, and the green line is a sample with a relatively low copy number of target DNA. B, A confirmatory analysis (quality control step) is the evaluation of melting curves. In this case, the change in temperature is compared to the negative derivative of fluorescence designated as -d(F2/F1)/dT. F2 is the measurement of fluorescence emission for the LC Red 640 fluorophore, and F1 is the measurement of fluorescence emission for the fluorescein fluorophore. The same melting curve occurs for the 2 samples and the positive control shown in A, indicating that the DNA with identical sequences has been amplified for each of the 3 samples. Mayo Clinic Proceedings 2002 77, 673-680DOI: (10.4065/77.7.673) Copyright © 2002 Mayo Foundation for Medical Education and Research Terms and Conditions