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

Loop-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 101   copies/μl  to 108   copies/μl  of the HPV 16 DNA template.  All three dyes exhibited  similar results in term of sensitivity with the detection limit of 103  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.