Rapid SARS-CoV-2 RT-LAMP Assay Setup using Gene Construct Design Approach

Background and Aim: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still an ongoing challenge in health system worldwide. Molecular detection methods including reverse transcription loop-mediated isothermal amplification (RT-LAMP) can help cutting off the transmission chain of the virus. In this study, prompt setting up of the RT-LAMP assays were investigated using gene construct design approach. Materials and Methods: RT-LAMP assays were initially setup and optimized using gene constructs as positive controls which worked simultaneously for RT-LAMP and RT-PCR assays. Constructed genes included T7 promoter sequence and partial sequences of SARS-CoV-2 RdRp, N and E genes cloned into pUC57 multiple cloning site (MCS) via synthetic and subcloning procedures. These templates were then used for artificial RNA synthesis. Finally, the RT-LAMP assay parameters were optimized and applied to clinical RNA specimens. Results: As an initial rapid approach, RT-LAMP assays were successfully setup for SARS-CoV-2 diagnostic using in vitro RNA transcripts from gene constructs. The assays were then examined on clinical samples with reaction times of 35 and 40 min required for assay one and two respectively. The detection limit was 100 copies of the target gene per reaction for each assay. Conclusion: Here, prompt setting up of RT-LAMP assays was carried out initially on designed gene constructs containing different SARS-CoV-2 target genes and simultaneous validation by RT-PCR. This approach could be used as an efficient solution where the clinical specimens may not initially be available, with the feasibility for new target gene of interest to be inserted into the MCS position.

Rapid SARS-CoV-2 RT-LAMP Assay Setup using Gene Construct Design Approach Keywords:

SARS-CoV-2 , COVID-19 Pandemic , Molecular Diagnostic Techniques , RT-LAMP Assay , ویروس سندروم حاد و شدید تنفسی 2 ,  بیماری عالم گیر کووید-19 , روش تکثیر هم دما به واسطه حلقه با رونوشت برداری معکوس ,  روش های تشخیصی مولکولی