Title : Development of Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) assay as a point-of-care diagnostics for SARS-CoV-2
Abstract:
The present study aimed to develop a reverse-transcription Loop-Mediated Isothermal Amplification (RT-LAMP) assay as a “Point-of-care” (PoC) diagnostic solution which can be useful for the detection of SARS CoV-2. To develop this assay, the complete genome sequences of SARS CoV-2 reported from different States and Union Territories of India were downloaded from GISAID (global initiative on sharing avian influenza data) database. The sequences were aligned to identify conserved regions for designing RT-LAMP primers. After the literature search, three conserved regions with no previously reported primers for detection of SARS CoV-2 were selected and the primers were designed uniquely using Primer Explorer V5. A total of six sets (A, B, C, D, E, and F) of primers, two each from the single conserved region were designed. The specificity of these primers was again checked in silico with BLAST tool in the NCBI server. The designed primers did not yield any similarity matches with the other human coronavirus except MERS-CoV and Bat SARS-CoV. On the other hand for the development of LAMP assay with these designed primers, the target conserved regions were got synthesized and analyzed for quality parameters before their use in the LAMP assay. Later, the primers (type, concentration, and ratio) and LAMP reaction temperature were optimized in the development of LAMP assay using synthetic gene constructs. Further, the performance of the developed assay was evaluated at KCGMC, Karnal, Haryana, using cDNA synthesized from RNA of SARS-CoV-2, and the developed LAMP technique was found effective in the detection of SARS-CoV-2. The developed LAMP technique was further explored for the development of colorimetric/fluorometric LAMP assay for rapid visual detection of SARS-CoV-2. The performance evaluation of developed colorimetric and fluorometric LAMP assays yielded satisfactory results in the detection of SARS-CoV-2 RNA. Hence, the developed Colorimetric and fluorometric LAMP assays can be used as PoC diagnostic tests for the detection of SARS-CoV-2. However, its performance directly with RNA or samples needs to be evaluated to make it an RT-LAMP assay and further validation is required with a larger sample size as per ICMR, India guidelines.
