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Background: Sever acute respiratory syndrome Coronavirus-2 (SARS-COV-2) spread prompted mass testing. The main method for testing is by any FDA approved kits for RNA extraction followed by One-Step RT-qPCR based on primer-probe assays. Yet, the high demand for these kits created a global bottleneck in the testing capacity. Methods: We developed a Full-In-House Method (FinHM) suitable for automated viral RNA extraction using full in-house solutions utilizing the MagMaxTM beads followed by an In-House RT-qPCR based on the CDC/WHO recommended ‘primer-probe’ assay targeting the following genes; E, RdRp2, and RdRp4. FinHM was validated by an FDA approved kit that targets S, N, and ORF1b genes made by Thermo Fisher Scientific (TF). Results: The sensitivity and specificity of the automated RNA extraction were evaluated on serial dilutions of in-laboratory propagated SARS-COV-2 with a successful detection down to 46 copies in both assays (P>0.05). Moreover, automated FinHM was successful in extraction of SARS-COV-2 RNA in 266 clinical samples, in which the test results replicated the FDA approved test results (>99% similarity, P>0.05). The In-House RT-qPCR assay had low limit of detection (5 RNA templates), with significant negative correlation between the Ct values and RNA titrations as shown by Pearson correlation (-0.8, -0.8 and -0.7 for E, RdRp2 and RdRp4, respectively). Finally, FinHM was also successful in extraction of SARS-COV-2-spiked plasma and patient plasma samples. Conclusion: We report a reliable, reproducible, specific, sensitive and low-cost platform for automated RNA extraction and detection from SARS-COV-2 and other viruses which is suitable for clinical and mass testing.

american journal of internal medicine

Full-in-House Method (FinHM) for SARS-COV-2 Automated Viral RNA Extraction, Followed by in-House ‘Primer-Probe’ Based RT-qPCR Detection; Low Cost Mass Testing