A comparison between a colorimetric and a fluorescent dye ina SARS-CoV-۲ reverse transcriptase-loop-mediated isothermalamplification (RT-LAMP) assay

Background and Aim : Loop-mediated isothermal amplification (LAMP) as a new moleculartechnique in the third millennium which has a broad application in detection of infectious diseasesincluding emerging contagious disease such as viruses including influenza, Ebola, HIV, Zika. Thismethod also has been wildly used in detection of severe acute respiratory syndrome coronavirus ۲(SARS-CoV-۲). This virus causes COVID-۱۹ disease and after more than ۲.۵ years it is still anongoing pandemic when it was first reported in December ۲۰۱۹. Reverse transcriptase-loopmediatedisothermal amplification (RT-LAMP) is able to amplify the viral gene with ۶ primerstargeting eight regions of the gene and to detect the infectious agent in less than one hour withoutany needs for complicated equipment and highly trained staff. This technique uses different waysfor reading the results and data interpretation. One of the approaches is using different dyesincluding colorful and fluorescent dyes. The aim of this study is to compare the efficiency andsimplicity of a a pH sensitive dye with a fluorescent dye in an RT-LAMP assay.Methods : In this study, hydroxy naphthol blue (HNB) as a pH sensitive coulometric dye wascompared with SYBR Gold fluorescent intercalating dye in an RT-LAMP reaction. Aftervalidation of an optimum dye concentration, the time for reaction end point as well as ease of theirapplications were evaluated.Results : The results show that HNB is simpler and more quick dye of choice for RT-LAMPresults read when compared with SYBR Gold. From RT-LAMP reaction to the result being read,the tubes with HNB have no need for the tubes to be opened while for SYBR Gold application, itis needed that the fluorescent dye to be applied after the reaction was finished by opening the tubesand observing the fluorescent color in a separate tube which caused high cross contamination.Conclusion : Here, we showed that in a comparison between HNB as a pH-sensitive dye and theSYBR Gold fluorescent dye in an RT-LAMP assay, the former one not only gave an easier wayfor reading the results to be recorded with naked eyes but also prevents the pre-amplificationsample cross contamination.