Wid

u ID

Cycle number

Figure 8-13 Quantitation using real-time PCR. A, In the example, four samples containing known amounts of target are amplified by real-time PCR. The inverse log of their fluorescence Is plotted against the cycle number and their respective Cj is determined; the fewer the number of targets, the greater the Cy value. B, Similarly, the clinical specimen is also run by real-time PCR and its Cj. value is determined. C, The log of the nucleic concentration and the respective Cr value for each specimen run containing a known amount of target or nucleic acid are plotted to generate a standard curve. By knowing the Cj- value of the clinical specimen, the concentration of target in the original sample can then be determined.

Log of the concentration the direct detection in clinical specimens of MRSA and beta-hemolytic group B streptococci, respectively, have recently received FDA clearance. The Gene Ohm kits are presently approved for real-time amplification using the SmartCycler (Cepheid).

AMPLIFICATION METHODS: NON-PCR-BASED

Although PCR was developed first and there are numerous PCR-based assays, rapid, sensitive, and specific detection of infectious agents by nucleic acid amplification can be achieved by a number of methods other than PCR These amplification formats can be divided into two broad categories: those that amplify the signal used to detect the target nucleic acid and those that directly amplify the target nucleic add but are not PCR-based. Examples of signal amplification methods used in infectious disease diagnostics are listed in Table 8-2. One such system is the ligase chain reaction (LCR). LCR uses two pairs of probes that span the target sequence of interest. Once annealed to the target sequence, a space remains between the probes that is enzymatically dosed using a ligase (i.e., a ligation reaction). On heating, the joined probes are rdeased as a single strand that is complementary to the target nuddc add; these newly synthesized strands are then used as the template for subsequent cydes of probe annealing and ligations.

Table 8-2 Examples of Commercially Available Signal Amplification Methods

Method

Manufacturer

Branched DNA(bDNA)

Bayer Diagnostics, Tarrytown, NY

Invader assays

Third Wave Molecular Diagnostics,

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Bacterial Vaginosis Facts

Bacterial Vaginosis Facts

This fact sheet is designed to provide you with information on Bacterial Vaginosis. Bacterial vaginosis is an abnormal vaginal condition that is characterized by vaginal discharge and results from an overgrowth of atypical bacteria in the vagina.

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