Considerations when developing PCR diagnostic assays
- Conventional vs real-time PCR
- While conventional PCR may be less expensive to run, most diagnostic and regulatory labs are shifting to real time PCR due to increased sensitivity and the less time that is involved to run a sample
In general, the target amplicons for real time PCR should be 200 bp or less, although larger sizes (ca 400-500 bp) have been used with SYBR green
- There are several different chemistries for real-time PCR
SYBR green is a dye that intercalates into double stranded DNA and fluoresces when exposed to a specific wavelength of light. This method relies on the specificity of the primers only
Other methods utilize an additional probe that hybridizes to the target sequence to enhance specificity of the marker system
TaqMan appears to be the most commonly used
Molecular Beacons and Scorpion are other options
- Real-time PCR guidelines
- Additional information on the use of real-time PCR, the different chemistries that can be used, as well as considerations and recommendations to help in the design of probes and specific primers can be found on the M. Tevfik Dorak website.
- The same author also wrote a book on real-time PCR (Dorak, M. T., 2006. Real-Time PCR (BIOS Advanced Methods Series). Oxford: Taylor & Francis) that contains background information that would be helpful when using of Real-Time PCR.
Assays should include an internal control amplification
This is to ensure the DNA quality and prevent false negatives (if the control does not amplify then there are problems with the DNA quality)
- Other plant genes have been used as internal controls
May need to reduce the concentration of primers and probe otherwise it may out compete with the pathogen amplification due to having a much higher concentrations of plant DNA present in the sample
It is important to check to make sure multiplexing with the internal control does not influence the amplification efficiency of the species-specific target
- Genus specific marker to diagnose if a Phytophthora spp. is present
- Rather then rely strictly on a species specific marker it would be helpful to have a genus specific marker as well that sequence analysis would identify the species that was present.
- This would help identify if there is another Phytophthora spp. present in the sample. While this may not be of primary importance if the objective is to identify a species that is under regulatory control, it would provide additional information about other species present in the ecosystem being sampled
- When evaluating a technique developed by another lab, follow the procedure exactly before modification
- In our lab we have found that using a different Taq polymerase or amplification buffer can cause nonspecific background amplification of other species not previously reported
- This was particularly true when using different master mixes for TaqMan real time PCR (ABI vs BioRad)
- Different thermal cyclers can have a different block calibration, so an annealing temperature reported for a marker system with one thermal cycler may not be the same temperature needed to get the reported specificity on another thermal cycler. This needs to be experimentally verified.
- The biggest limitation to the sensitivity of a molecular diagnostic assay is the quality of template DNA
- Depending on the plant species, addition of plant DNA to purified pathogen DNA prior to amplification can prevent PCR amplification
- Many plants have PCR inhibitors that can reduce the amplification efficiency or prevent amplification unless the DNA extraction procedure used removes them from the sample.
- Some procedures reduce the impact of this by diluting the DNA samples prior to running the assay, but this reduces the sensitivity of the assay by reducing the target DNA concentration.
- The DNA extraction procedures that will be used on the host tissue under study should be experimentally tested to ensure the resulting DNA quality is high enough to use in subsequent diagnostic assays.