4.6. Qualitative RT-PCR for honey bee and pathogen targets
Detection by PCR can be “qualitative”, i.e. recording only the presence or absence of the target cDNA, by analysing the accumulated “end-point” PCR products after the PCR is completed. The sensitivity of the assay can be raised or lowered as desired by, respectively, increasing or decreasing the number of amplification cycles. Usually PCR does not exceed 40 cycles, which is theoretically sufficient to detect a single molecule of the target DNA in the original template, when analysing the end products by agarose gel electrophoresis. Consider the following rough calculation:
- Assuming perfect doubling with each amplification cycle.
- 20 molecules (i.e. 1 molecule) prior to PCR = 240 molecules after 40 cycles of PCR.
- 240 molecules of a 100 bp DNA fragment (mw ~ 61,700 g/mol)
= 1.1 x 1012 molecules x 1 mol/6.0221415 x 1023 molecules
= 1.8 x 10-12 mol x 61,700 g/mol
= 1.1 x 10-7 g = 110 ng DNA
Normally, 20 ng DNA is easily visible as a single band on an ethidium bromide-stained agarose gel. Even when allowing for imperfections in the amplification, 40 cycles are therefore theoretically more than sufficient to detect a single molecule in a reaction.
However, such extreme sensitivity is rarely required in practical or even most experimental settings. Furthermore, by aiming for absolute detection at the level of a single molecule of target DNA, the detection system becomes axiomatically susceptible to high rates of detection error: both false positives (accidental amplification of contaminating molecules) and false negatives (non-detection of a single molecule due to amplification insufficiencies).
By raising the detection threshold a few orders of magnitude, to around 1,000 molecules per reaction (~210 molecules prior to PCR) it is possible to produce detectable amounts of target DNA (~240 molecules) with 30 cycles of amplification (210+30 molecules), again assuming perfect doubling each cycle. This avoids most of the risk of both types of detection errors, since chance contamination events of singular molecules (false positive results) are now below the detection threshold and there is sufficient initial target DNA in the reaction to avoid accidental non-detection (false negative results). A few more cycles beyond 30 can be added to compensate for the imperfections in the PCR efficiency. This means that 35 amplification cycles should be the upper limit for most practical applications. Beyond 35 cycles, the rapidly increasing risk of detection errors outweighs the marginal gains in sensitivity.