Validating the qPCR

1. Validation and optimization of qPCR

Aim of this step: Find the best qPCR reaction conditions for your samples with specific PCR-mix.

As for any qPCR assay, measuring the PCR efficiency is a crucial part of testing and suitable efficiency value is proof of successful validation. For this assay test the qPCR efficiency of parasite 7-1 primers (see table below) with your samples.

For efficiency tsting, make a dilution series eg. 3-fold and run each dilution as a sample in qPCR in triplicate and with 8 or more points. It is likely that parasite DNA concentrations are too low to a make dilution series for efficiency determination. To overcome this problem, you can make a PCR with primers from Hellgren et al. (2004) or parasite 7-1 primers to generate enough DNA to make a dilution series, which can be used to determine efficiency. qPCR efficiency should ideally be 85-105 %. If you get higher or lower values, try to optimize reaction conditions eg. MgCl₂ concentration and/or annealing temperature to reach recommend efficiency (see picture below). To optimize annealing temperature we recommend to use a qPCR cycler with a gradient feature.

The melting curve is an efficient way to analyze PCR-products after qPCR. In this assay it is used to seperate unspecific product from specific. The melting peak should be sharp and smooth (see picture below). Melting peak should also have a consistent melting temperature; any deviations can be caused by unspecific product. We also recommend analysing the PCR product in an agarose gel to verify the correct (~250 bp) fragment length. Once efficiency, melting peaks and fragment size are within satisfactory limits your can continue to identify the parasites.