Test the whole assay

8. Test whole assay with all steps

Aim of this step: combine all the previous steps to make the assay ready to analyse your samples.

(1) We recommend running all the steps in duplicates or triplicates starting from here. Make a PCR with parasite 7-1 primers, using a small number of cycles (10-15 is usually enough). Too high number of cycles in the first PCR causes the qPCR to plateau and makes quantification impossible.

(2) Use restrictions enzymes to digest PCR product with three restriction mixes to differentiate parasites.

(3) Dilute all restriction products with distilled water (e.g. 1:5). Some reaction buffers can inhibit PCR in high concentrations and make amplification impossible. Dilution can solve this problem.

(4) Run all diluted restriction products in qPCR with parasite 7-1 primers (and with reference gene if used) to measure parasite load in the sample. Check melting curves to make sure that the PCR-product is specific. If unspecific products are found in the sample, it is uninfected.

(5) Use Cq's of the infected samples to calculate parasite load in the sample. See Lipponen et al. (submitted) for full details of calculating parasite loads.

A workflow of the working nested qPCR