1008-LBP: Comparison of Single Nucleotide Polymorphism Genotyping of CYP2C19∗2 and CYP2C19∗3 alleles by Loop-mediated Isothermal Amplification and Real-time PCR Melting Curve Analysis

Abstract

To validate the effectiveness of CYP2C19 -LAMP assay for the detection of CYP2C19 ∗ 2 and CYPC19 ∗ 3 in comparison with real time PCR melting curve analysis. CYP2C19 -LAMP primers were essentially adapted from Iwasaki M et al. [1] . The LAMP reaction was optimized at a temperature range from 58 °C to 62 °C. DNA were extracted from whole blood samples collected from forty patients suffering from acute coronary syndrome in a single center of Hong Kong. With the addition of SYBR Green, LAMP results were examined visually in ambient light and also under UV illumination at 365 nm. Real-time PCR melting curve analysis was done by LightMix Kit human CYP2C19 ∗ 2 and CYP2C19 ∗ 3 (TIB Molecular Biology). The two variants exhibit Tm 48.1[o]C and 59.2[o]C and were detected by LightCycler 480 II. CYP2C19 -LAMP assay successfully detected CYP2C19 ∗ 2 and CYPC19 ∗ 3 mutants. Our study found that the assay time of CYP2C19 -LAMP assay was only 30 min whereas real time PCR melting curve analysis was 60 min. We observed that the temperature requirement was very stringent and any temperature outside 60 °C might result in non-specific amplifications. In the LAMP assay, non-specific amplifications were observed with an error rate of 5% (8/160) and resulted in 15% (6/40) being mistyped. These non-specific amplifications, however, could be resolved with duplicated runs. A trial of one heat treated heparinized blood sample was found to be sustainable in LAMP reactions without prior DNA extraction. We showed that CYP2C19 -LAMP assay was a simple, rapid and cost- effective method for CYP2C19 genotyping. Owing to the stringent temperature requirement in LAMP assay, the test is recommended to be run in duplicate with better quality control in obtaining a more reliable result by a thermal cycler.