Limit of detection evaluation of a synthetic SARS-CoV-۲ RNAby RT-LAMP and Real-time PCR

Background and Aim : Molecular techniques have advantages over antigen-antibody basedtechniques in detection of microorganism including viral diseases such as COVID-۱۹ caused bySARS-CoV-۲. Recently, two molecular methods including reverse transcriptase-loop-mediatedisothermal amplification (RT-LAMP) and Real-time PCR are mainly used in detection of SARSCoV-۲. The former one uses ۶ primers targeting eight regions of the gene and to detect theinfectious agent in less than one hour without any needs for complicated equipment and highlytrained staff. The second method which is a gold standard is routinely used in diagnosticlaboratories. These techniques are both very sensitive to detect as little as ۱ copy number of RNAmolecules. RT-LAMP can also be performed for large sample scales so it can be a supportivechoice during a pandemic when the capacities of the diagnostic laboratories are out of theircapacities.Methods : Of around ۳۰Kb reference genome of SARS-CoV-۲, bioinformatics analysis wereperformed using different softwares. Then a short fragment of the genome was synthesizedartificially as RNA. Specific primers then designed for the target region. After determining theRNA concentration with nanodrop, a serial dilution was prepared to be applied for detection viaRT-LAMP and Real-time PCR in triplicates. The results were read by naked eye and machinerespectively.Results : The results showed that RT-LAMP and Real-time PCR can detect as little as ۲۵ and ۱۰copies of synthetic SARS-CoV-۲ RNA number in the samples respectively. The reaction for RTLAMPwas very shorter than RT-PCR completed in less than one hour. The results were read usingdye and also gel electrophoresis.Conclusion : Here, we showed that both RT-LAMP and Real-time PCR are very sensitive methodsfor detection of SARS-CoV-۲ RNA. The former one is simpler, quicker and easier for a field workapplication required minimum equipment and trained staff. RT-LAMP is also under the Food andDrug Administration's Emergency Use Authorization and can support the society health by higherdiagnostic and detection tests for cutting off the transmission chains of virus.

Keywords:

SARS-CoV-۲ , Loop-mediated isothermal amplification , Real-time PCR , Limit ofdetection