Hi Elizabeth, I will suggest you go with Taqman qPCR but you need to be careful for DNA purification methods because you are comparing the pathogen concentration from different parts of the plant so DNA purification methods from different tissue can make a significant difference and error can drive you wrong way. I will also recommend you to use the multi-copy gene for primers and probe designing to reduce the chances of error. Let me know if you need any other clarification.

Yes you have to do a real-time PCR with SYBR green. If you want to do quantification you need a known amount of your targeted PCR product (either the purified PCR product or the PCR product cloned into a plasmid). You also need to have a 'plant' PCR to normalize. In all cases your DNAs have to be very clean.

go for real-time PCR to really measure the amount.

Thanx guys, I have settled for real time pcr, i wiil keep you posted incase am stuck!

I would also read "Improved Quantitative PCR using nested primers" by Haff. It is a fairly old paper (1994) but gives a good insight into how quantification of nested PCR is possible. Obviously it is also important to consider whether your nested PCR is a single or two-step procedure.

Dear Elizabeth Kusia,

As mentioned by M. Arif in the above suggestion the DNA extraction from different plant parts would definitely different which would give you a false pathogen concentration. I suggest you to do the real time PCR but compare your pathogen copy no. in reference with a plant house keeping gene to get a correct picture.

best of luck

Hi Elizabeth: Use real PCR, as suggested, but do include a house keeping gene as reference to normalize your QPCR results, as it can account for the variations in quantification due to enzymatic processes involved during RT and amplification step, as well as different amounts of DNA extracted from different parts of the plant. This approach is used as a standard in RTQ-PCR based quantification of oncogenes in various cancers for assessing the effect of different anti-cancer drugs, during different phases of the treatment, as well as in different tissues. Same methodology is used as FDA approved tests for various human viral pathogens like HCV etc. Be sure to select any valid housekeeping gene or other factor for normalizing your QPCR results. 

Hi Elizabeth: I do not know the type of plant you are dealing with but the quality and quantity of your DNA must not be compromised. I suggest you use CTAB method for DNA extraction and quantify qualitatively using  agarose electrophoresis and quantitatively  and qualitatively with nanodrop machine. Ensure you use samples with  values of 260:280nm greater than 1.8.  Taqman real time PCR is one of the best short for this experiment, but ensure to use plant "house keeping genes" to eliminate bias in the assay. Goodluck