Effect of Reaction Parameters in the Real-Time PCR Detection of Grape Chitinase

Grapevine is a well-known and popular cultural plant that is grown on thousands of hectares worldwide. It possesses a lot of nutritional benefits but, on the other side, some of the people may suffer an allergic reaction when eating grapes or even in the case of consumption of the processed forms. Although grape allergy is not a common allergic reaction, the prevalence of this type of allergy is increasing mainly in southern European countries. One of the main proteins that has the potential to cause grapes allergy is chitinase. Here, a real-time PCR protocol was optimized to analyse the expression of chitinase in the mature grape berries. Different parameter such as analytic kit, primer annealing temperatures or cDNA dilution effects were tested. The final optimized protocol was proved for the chitinase unique isoform amplification by post melting analysis and testing of the protocol was performed for selected grape varieties