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.
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.
Authors: Allison B Chambliss; Molly Resnick; Athena K Petrides; William A Clarke; Mark A Marzinke Journal: Clin Chem Lab Med Date: 2017-03-01 Impact factor: 3.694
Authors: Dafni-Maria Kagkli; Silvia Folloni; Elodie Barbau-Piednoir; Guy Van den Eede; Marc Van den Bulcke Journal: PLoS One Date: 2012-06-25 Impact factor: 3.240
Authors: Johanna Teräsjärvi; Antti Hakanen; Matti Korppi; Kirsi Nuolivirta; Kirsi Gröndahl-Yli-Hannuksela; Jussi Mertsola; Ville Peltola; Qiushui He Journal: Sci Rep Date: 2017-02-02 Impact factor: 4.379