Method validation of a high resolution melting analysis of a candidate genetic marker of hypertension / Journal of the ASEAN Federation of Endocrine Societies

ABSTRACT

Objectives@#The study aimed to validate a high resolution melting (HRM) – polymerase chain reaction (PCR) based method for analysis of ATP2B1 rs2681472, a candidate genetic marker for hypertension. Specifically, the study aimed to establish method accuracy, inter- and intra-day precision, instrument detection limits (IDL) and linearity. @*Methodology@#DNA samples from whole blood of selected respondents of the 2008 National Nutrition Survey (NNS) were analyzed for ATP2B1 rs2681472, following the HRM-PCR method. Analytical validation parameters such as method accuracy, inter- and intra-day precision, IDL and assay linearity were evaluated. @*Results@#The data obtained using HRM conformed to data obtained from the current gold standard, which is direct DNA sequence analysis. Precision of the method is shown in the low intra-day and inter-day coefficients of variation of 0.11% and 0.14% respectively. The IDL was found at 1x10-2 ng DNA. Correlation coefficient using the method has met the acceptance criteria for linearity that is equal to 0.98. @*Conclusion@#HRM-PCR was successfully applied for analysis of ATP2B1 rs2681472. HRM is a highly commendable tool that can identify existence of polymorphisms in a genome which may be related to or which may cause hypertension. The method was found to be accurate and precise.