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Real-Time TaqMan PCR and Applications in Veterinary Medicine
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The intersection between the amplification plot and the threshold, where the threshold is defined as 10 times the standard deviation of the background fluorescence intensity and which is measured between cycle 3 and 15, is known as the cycle threshold, or CT, value (default settings of the SD software may be changed manually). The CT value is directly related to the amount of PCR product and therefore related to the initial amount of target DNA present in the PCR reaction. Figure 3 illustrates the single fluorescent components of the reaction.  Fig.3 The second of two ways in which a positive result from a TaqMan PCR analysis is visualised. The graph reflects the single fluorescent component of the reaction. The two fluorescent tags bound to the TaqMan probe are 6-carboxyfluorescein (FAM) and 6-carboxy-tetramethyl-rhodamine (TAMRA). A positive TaqMan result is reflected by an increase in the fluorescent intensity of FAM and a decrease in the fluorescent intensity of TAMRA. A positive TaqMan result is reflected by increasing the fluorescent intensity of the reporter dye, FAM, and by decreasing the fluorescent intensity of the second fluorescent tag, TAMRA. Other fluorescent components present in this procedure are ROX, which is mixed in the PCR buffer to a constant concentration and therefore may be used to normalise fluorescent signals when subtle differences in the volume of the PCR reaction mix occur. Background fluorescence is produced by the plastic of the 96-well plate as well as the optic devices of the detection unit.
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