linear amplification portion of the reaction where conditions are optimal and fluorescence accumulates in proportion to the amplicon.
With most instrument analyses, the value used for quantitative measurement is the PCR cycle number in which the fluorescence reaches a threshold value of 10 times the standard deviation of baseline fluorescence emission; this cycle number is referred to as the threshold cycle (CT) or crossing point and is inversely proportional to the starting amount of target present in the clinical sample (see Mackay in Additional Reading). In other words, the CT is the cycle number in which the fluorescent signal rises above background (the threshold value previously defined) and is dependent on the amount of target at the beginning of the reaction. Thus, to quantitate the target in a clinical specimen, a standard curve is generated in which known amounts of target are prepared and then subjected to real-time PCR along with the clinical sample containing an unknown amount of target. A standard curve is generated using the Cj-values for each of the known amounts of target amplified. By taking the CT value of the clinical specimen and using the standard curve, the amount of target in the original sample can then be determined (Figure 8-13). Quantitative nudeic add methods are used to monitor response to therapy, detect the development of drug resistance, and predict disease progression.
The introduction of commerdally available analyte-spedfic reagents (ASRs) followed soon after the
Figure 8-12 Melting curve analyses performed using the LighfCycler HSV1/2 netection Kit. DNA was extracted and subjected to real-time PCR using the LightCycler to detect the presence of HSV DNA. Following amplification, melting curve analysis was performed in which amplified product was cooled to below 55° C and then the temperature slowly raised. The Tm is the temperature at which half of the DNA is single-stranded and is specific for the sequence of the particular DNA product. The specific melting temperature is determined at 640 nm (channel F2 on the cycler) for the clinical samples and the positive and negative controls. For illustration purposes, melting curve analyses are "overlaid" relative to one another in this figure for three clinical samples and the HSV-1 and HSV-2 positive or-"template" control. The clinical specimens containing HSV-1 DNA (red line) or HSV-2 (green line) result in a melting peak at 54° C (the Tm) or 67' C (the Tm), respectively. The LightCycler positive or template control containing HSV-1 and HSV-2 DNA, displayed as a purple line, shows two peaks at 54° C and 67 C, respectively. The clinical sample that is negative (brown line) for both HSV-1 and HSV-2 shows no peaks.
introduction of real-time PCR. ASRs represent a new regulatory approach by the FDA in which reagents (e.g., antibodies, specific receptor proteins, ligands, oligo-nudeotides such as DNA or RNA probes or primers, and many reagents used in in-house PCR assays) in this broad category can be used in multiple diagnostic applications. ASR-labeled reagents carry the "For Research Use Only" .labd, and the manufacturer is prohibited from promoting any applications for these reagents or providing redpes for using the reagents. On a cautionary note, because they are not FDA-deared, ASR assays cannot be reimbursed by Medicare carriers. Because rulings vary on a state-by-state basis, one is advised to check into Medicare reimbursement before development and introduction of an ASR assay. The high-complexity CIIA laboratory must take full responsibility for developing, validating, and offering the diagnostic assay that employs these reagents. This new regulation essentially allows for new diagnostic methods to become available more quickly, particularly those methods targeted toward smaller patient populations. Importandy, because good manufacturing practices are mandated, ASRs provide more standardized products for the performance of amplification assays. ASRs are available for a number of organisms such as beta-hemo-lytic group A and B streptococd, methicillin-resistant 5. aureus (MRSA), Bordeiella pertussis, vancomycin-resistant enterococa, hepatitis A virus, and Epstein-Barr virus. Finally, molecular kits such as the ID1-MRSA and IDI-StrepB (GeneOhm Saences, San Diego, Calif) for
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