Im an MRes student new genetics and qPCR, and am developing a species-specific primer/probe set to detect Najas flexilis, a rare aquatic macrophyte in Scotland. The aim is to detect the plant by eDNA filtered from freshwater. However, I am running into efficiency problems with qPCR. Can you help? If so, firstly, allow me to explain the development steps.
Using Primer3 software, I developed a pair of primers with Tm ~ 53/55 C. The forward primer showed high hairpin stability in silico. After running a gradient PCR, it was discovered that 59 C gave the greatest concentration of amplicon, perhaps because the melt temperature was too high for hairpin formation (?).
The probe that was designed with the primer pair had Tm ~62 C. A qPCR, using Environmental Master Mix (different to the master mix used in the aforementioned gradient standard PCR), was undertaken. The thermal protocol used was based on work for Great Crested Newts, rather than that recommended in the master mix manual, as recommended by my supervisor:
50 C for 5 minutes
95 C for 10 Minutes
Then 55 Cycles of:
95 C for 30 seconds
59 C for 1 Minute.
The qPCR resulted in amplification of eDNA extracted from filtered water, and of a dilution series extracted from a live tissue sample. The efficiency was only 44%. I'm keen to increase the efficiency of the reaction, perhaps by reducing inhibition, or modifying the thermal profile based on the primer/probe set designed. This in turn will improve the potential to detect Najas flexilis using eDNA extracted from freshwater this summer.
Qiagen Soil Kits were used in the extraction of live plant tissue used in the qPCR dilution series, which, like Environmental Master Mix (EMM), helps reduce inhibition. I will soon test for inhibition using an IPC (waiting for it to arrive), but suspect the probe design is the problem, or the thermal cycling condition.
The amplicon is 193 bp, higher than the maximum recommended 150 bp. The high hairpin stability of the forward primer is may still be affecting the amplification.
From what I have detailed above, can anyone recommend next steps to increase efficiency, before I begin analysing samples collected in the field?
I was thinking of running a gradient PCR using Environmental Master Mix and see if the greater concentration occurs at a Tm that is different to 59 C.
I also thought of qPCR dilution series in triplicate, at different temperatures. My instincts suggest that 57 C might be a better Tm, as it is 5 C lower than the Probe Tm.
Also, I thought that perhaps the the thermal cycling could have three steps, as in standard pcr, with a 72 C elongation step added to aid in elongation 193 bp.
In truth, I am unsure, but before delving into the unknown, thought I would ask those who might know.
Any thoughts would be greatly appreciated.