Primer-DNA annealing temperature. This is generally dependent on the primer lenght and order and percentage of the nucleotides present composing the primers.
Number of cycles (the number of times that the denaturing, annealing and synthesis process occurs).
Primer-dimers: The formation of primer dimers can inhbit the amplificaiton process because few or no primers are available to start the process. Once can avoid primer dimer formation by designing primers that do not complement each other, meaning that they bind to the biological agent’s nucleic acid and not to each other.
Primer Design: Primers and probes are generally designed for the detection of a given genus (genus-specific primers) or for the detection of a single species (species-specific primers). The 18S or 16S ribosomal RNA genes can be used for the design of genus-specific primers and probes because they contain sequences that are highly conserved between members of the same genus, but the sequences are variable among different genera. Species-specific primers and probes can be designed to target the ineranl transcribed spacer regions and intergenic spacer of the nuclear rRNA gene, because these genes are highly variable areas within a genus or among populations.
The similarity (homology) in the DNA of clsoely related species makes the design of specis-specific primers and probes difficult. GenBank can be used to find known DNA sequences. Using a sequence homology program like Basic Local Alignment Search Tool (BLAST) algorithm unique DNA regions can be found on the target sequence. A primer and probe design software package such as Primer Express (Applied Biosystems, Foster City, CA) can then be used in which parameters such as melting temperature, amplicon size, base content and primer/probe lenght can be selected. Validation studies are needed to ensure that there is no cross-reactivity with other species and to determine the lower detection limit of the assay with the primer and probe set. These studies comprise testing n umerous strains of the target organisms, as well as related and non-related microorganisms. In addition to specificty testing, the PCR should be optimized for maxzimum amplificaiton of DNA which involves testing various concentrations of the primers and other PCR reagents with DNA from the target organism.
Qauntitation is achieved through the amplifcation of standards containing known concentrations of DNA with real-tiem PCR. A range of 10 to 10e5 templates per reaction rpvoides a standard curve over five orders of magnitude. Automated QPCR systems provide sofware that will construct a standard curve of CT (the PCR cycle at which fluorescence is first detected) versus concentration. This # is inversely proportional to the concentration of the initial DNA template. The concentration of the unkown samples can be extrapolated form the standard curve by the sofware and reproted as the mean of two replicates.
Reagent storage: Avoid repeatedly freezing and thawing the reagents. This degrades nucleotides and primers.