Results for the substitution analysis, which is also significantly lower than the permitted error rate of 10%. 1. Elaboration of the method 2.2. Robustness of the method. To assess the robustness of The amplicons obtained from all 13 Ephydatia fluviatilis the method, first the concentrations of either primer in the PCR specimens analysed contained the ITS region, including the 39 mixture were doubled (Table 1). This had no influence on the end of the 18S gene, the full length ITS1, 5.8S gene, ITS2, and the results: the absolute deviation from the mean value of 100 ng 59 end of the 28S gene
2000). point of the reaction, especially when only qualitative data is required. There are several Real-Time PCR types. The principal characteristic of the RTi-PCR is that it allows the real-time monitorization Nested PCR of the synthesis of new amplicons throughout As conventional PCR based on the agarose the PCR via the fluorescence emitted, which gel electrophoresis is generally less sensitive, is proportional to the amount of new PCR a modification, the nested PCR, which dra- products generated (Heid et al. 1996). matically increases the sensitivity and speci- Therefore, the RTi-PCR results consist of ficity of DNA amplification, can be used. It amplification curves that can be used to
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