■ Figure 12-2 Melt curve analysis of BK and JC viruses. BK and JC are differentiated from one another by differences in the Tm* of mthe probe specific for each viral sequence. Fluorescence from double-stranded DNA decreases with increasing temperature and DNA denaturation to single strands (top panel). Instrument software will present a derivative of the fluorescence (bottom panel) where the Tms (67o-68oC for BK and 73o-74oC for JC) are observed as peaks.
ment for probes in addition to primers increases the complexity of the design process. Instrument software and several Web sites offer computer programs that automatically design primers and probes on submitted sequences. Commercial primer and probe sets are also available for purchase in kit form. A variety of gene targets have been used for qPCR detection of a number of organisms. A list of examples of targets and probes is available in a comprehensive review by Espy et al.21
The genes that have been the targets for the design of primers include ribosomal RNA (rRNA), both 16S and 23S, and housekeeping genes such as groEL, rpoB, recA, and gyrB.21 16S rRNA is a component of the small subunit of the prokaryotic ribosome, and the 23S rRNA is a component of the large subunit of the prokaryotic ribo-some. Analysis of 16S rRNA is performed to determine the evolutionary and genetic relatedness of microorganisms and is driving changes in microorganism nomencla-ture.22 The rDNA that encodes the rRNA consists of alternating regions of conserved sequences and sequences that vary greatly from organism to organism. The conserved sequences encode the loops of the rRNA and can be used as a target to detect all or most bacteria. The se quences that have a great amount of heterogeneity encode the stems of the rRNA and can be used to detect a specific genus or species of bacteria.19 rRNA was the original target of many bacterial molecular-based assays, but because of the instability and difficulty in analyzing RNA, current assays amplify and detect rDNA sequences.
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