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Developing RT-LAMP Assays for Detection of SARS-CoV-2 in Saliva

ABSTRACT: Developing RT-LAMP Assays for Detection of SARS-CoV-2 in Saliva

PROVIDER: PRJNA723769 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR14299253.fastq.gz	Fastqsanger.gz
	SRR14299254.fastq.gz	Fastqsanger.gz

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Developing RT-LAMP Assays for Detection of SARS-CoV-2 in Saliva

Project description:Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a fast and convenient method to amplify and identify the transcripts of a targeted pathogen. We combined bioinformatic and experimental analyses to improve the RT-LAMP assay performance for COVID-19 diagnosis. First, we developed an improved algorithm to design LAMP primers targeting the nucleocapsid (N), membrane (M), and spike (S) genes of SARS-CoV-2. Next, we rigorously validated these new assays for their efficacy and specificity. Further, we demonstrated that multiplexed RT-LAMP assays could directly detect as low as a few copies of SARS-CoV-2 RNA in saliva, without the need of RNA isolation. Importantly, further testing using saliva samples from COVID-19 patients indicated that the new RT-LAMP assays were in total agreement in sensitivity and specificity with standard RT-qPCR. In summary, our new LAMP primer design algorithm along with the validated assays provide a fast and reliable method for the diagnosis of COVID-19 cases.

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COV-ID: A LAMP sequencing approach for high-throughput co-detection of SARS-CoV-2 and influenza virus in human saliva

Project description: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.

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