Objectives: According to OECD guidelines methods implemented in a diagnostic laboratory should be properly validated prior their implementation. For this purpose we selected genotyping by High Resolution Melting (HRM) of small amplicons using common variants in MTHFR as a model.
Design And Methods: We selected previously typed samples on which selected analytical validation-related parameters relevant to DNA diagnostics - specificity, sensitivity, precision, robustness and ability to perform reliable calls were evaluated.
Results: Correct genotype was assigned in 375/381 (98.4%) for c.677 C>T (rs1801133: C>T; p.A222 V) and in 102/104 (98.1%) for c.1298 A>C (rs1801131: A>C; p.E429A) of all cases. Low analytical failure rate and very high specificity/sensitivity were achieved. Similarly, precision and robustness were consistent.
Conclusions: We have successfully validated HRM of small amplicons using common MTHFR variants as a model. We proved that this technique is highly reliable for routine diagnostics and our diagnostic validation strategy can serve as a model for other applications.
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