We combined RT-LAMP with deep sequencing to detect as few as 5–10 virions of SARS-CoV-2 in unprocessed human saliva. Based on a multi-dimensional barcoding strategy, COV-ID can be used to test thousands of samples overnight in a single sequencing run with limited labor and laboratory equipment. The sequencing-based readout allows COV-ID to detect multiple amplicons simultaneously, including key controls such as host transcripts and artificial spike-ins, as well as multiple pathogens. Here we demonstrate this flexibility by simultaneous detection of 4 amplicons in contrived saliva samples: SARS-CoV-2, influenza A, human STATHERIN, and an artificial SARS spike-in. The approach was validated on clinical saliva samples, where it showed 100% agreement with RT-qPCR. COV-ID can also be performed directly on saliva adsorbed on filter paper, simplifying collection logistics and sample handling.
Overall design
Water or human saliva was spiked with synthetic SARS-CoV-2 RNA or influenza A RNA at various concentrations. Modified set of RT-LAMP primers including sample-specific barcodes as well as landing sites for universal Illumina adapters were used to perform various RT-LAMP reactions in multiplex. Different barcoded and amplified samples were pooled and Illumina adapter sequences introduced by PCR. The resulting pools were sequenced and analyzed.
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