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ABSTRACT: Genomic surveillance of SARS-CoV-2 using long-range PCR primers

PROVIDER: PRJNA1189558 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR31458670_1.fastq.gz	Fastqsanger.gz
	SRR31458671_1.fastq.gz	Fastqsanger.gz
	SRR31458672_1.fastq.gz	Fastqsanger.gz
	SRR31458673_1.fastq.gz	Fastqsanger.gz

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Project description:There is an urgent need for robust and high-throughput methods for SARS-CoV-2 detection in suspected pa-tient samples to facilitate disease management, surveillance, and control. Although nucleic acid detection methods such as RT-PCR are the gold standard, during the current pandemic the deployment of RT-PCR tests has been extremely slow, and key reagents such as PCR primers, and RNA extraction kits are at critical shortages. Rapid point-of-care viral antigen detec-tion methods have been previously employed for the diagnosis of respiratory viruses such as influenza and respiratory syn-cytial viruses. Therefore, the direct detection of SARS-CoV-2 viral antigens in patient samples could also be used for diagno-sis of active infection and alternative methodologies for specific and sensitive viral protein detection should be explored. Targeted mass spectrometry techniques have enabled the identification and quantitation of a defined subset of pro-teins/peptides at single amino acid resolution with attomole level sensitivity and high reproducibility. Herein we report a tar-geted mass spectrometry assay for the detection of SARS- CoV-2 spike protein and nucleoprotein in a relevant biologi-cal matrix. Recombinant full-length spike protein and nucleoprotein were digested and prototypic peptides were selected for parallel reaction monitoring (PRM) quantitation using a high resolution Orbitrap instrument. A spectral library, which con-tained 7 proteotypic peptides (4 from spike protein and 3 from nucleoprotein) and the top 3 to 4 transitionsMS2 spectra, was generated and evaluated. From the original spectral library, we selected 2 best performing peptides for the final PRM assay. The assay was evaluated using mock test samples containing inactivated SARS-CoV-2 virions, added to in-vitro de-rived mucus. The PRM assay provided a limit of detection (LOD) of ~200 attomoles and a limit of quantitation (LOQ) of ~ 390 attomoles. Extrapolating from the test samples, the projected titer of virus particles necessary for detection of SARS-CoV-2 spike and nucleoprotein detection was approximately 2E5 viral particles/mL, making it an attractive alternative to RT-PCR assays. Potentially mass spectrometry-based methods for viral antigen detection may deliver higher throughput and could serve as a complementary diagnostic tool to RT-PCR. Furthermore, this assay could be used to evaluate the pres-ence of SARS-CoV-2 in archived or recently collected biological fluids, in-vitro derived research materials, and wastewater samples

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