Sensitivity Evaluation of SYBR Green I, SYBR Safe and Calcein Dyes for Detection of Human Papillomavirus 16 by Loop-Mediated Isothermal Amplification

Abstract

ABSTRAK Amplikasi isoterma berpengantara gelung (LAMP) merupakan teknik amplifikasi gen yang menghasilkan produk akhir iaitu mendapan keruh magnesium pirofosfat yang boleh dianalisis dengan hanya menggunakan mata kasar. Penggunaan pewarna interkalat yang sesuai adalah penting kerana ia boleh meningkatkan sensitiviti dan ABSTRACTLoop-mediated isothermal amplification (LAMP) is a gene amplification technique whereby the amplification products are commonly visualized as turbidity by naked eye in the presence of magnesium pyrophosphate precipitation. An appropriate intercalating dye is important as it could increase the sensitivity and reduce the false positive and false negative results for the detection. The study aimed to compare the performance of three different intercalating dyes; SYBR Green I, SYBR Safe and calcein-based dyes in HPV-16 LAMP assay by naked-eye visualization, gel electrophoresis and real-time monitoring. The LAMP assay was carried out using a Loopamp DNA amplification kit in 25 μl volumes. The reaction mixture was incubated at 60�C for 60 mins and terminated at 80�C for 5 mins in a real-time turbidimeter. For naked eye detection, SYBR Green I and SYBR Safe were diluted at 1:10 of DMSO and was added to the solution after the reaction was completed while calcein was added before the amplification process. The sensitivity of the LAMP assay was investigated ranging from 10 1 copies/μl to 10 8 copies/μl of the HPV 16 DNA template. All three dyes exhibited similar results in term of sensitivity with the detection limit of 10 3 copies/μl. Addition of calcein dye showed decrease in detection time by 10 mins by real-time turbidimeter. The performance all three dyes for naked-eye detection are comparable and can be used for endpoint screening applications in HPV 16 assay, whereas in real-time evaluation, addition of calcein delay the detection time by 10 mins.