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Nanopore Targeted Sequencing for the Accurate and Comprehensive Detection of SARS-CoV-2 and Other Respiratory Viruses.

ABSTRACT: The ongoing global novel coronavirus pneumonia COVID-19 outbreak has engendered numerous cases of infection and death. COVID-19 diagnosis relies upon nucleic acid detection; however, currently recommended methods exhibit high false-negative rates and are unable to identify other respiratory virus infections, thereby resulting in patient misdiagnosis and impeding epidemic containment. Combining the advantages of targeted amplification and long-read, real-time nanopore sequencing, herein, nanopore targeted sequencing (NTS) is developed to detect SARS-CoV-2 and other respiratory viruses simultaneously within 6-10 h, with a limit of detection of ten standard plasmid copies per reaction. Compared with its specificity for five common respiratory viruses, the specificity of NTS for SARS-CoV-2 reaches 100%. Parallel testing with approved real-time reverse transcription-polymerase chain reaction kits for SARS-CoV-2 and NTS using 61 nucleic acid samples from suspected COVID-19 cases show that NTS identifies more infected patients (22/61) as positive, while also effectively monitoring for mutated nucleic acid sequences, categorizing types of SARS-CoV-2, and detecting other respiratory viruses in the test sample. NTS is thus suitable for COVID-19 diagnosis; moreover, this platform can be further extended for diagnosing other viruses and pathogens.

SUBMITTER: Wang M

PROVIDER: S-EPMC7361204 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundNosocomially acquired severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection has become the most significant pandemic of our lifetime. Though its transmission was essentially attributed to droplets from an infected person, with recent advancements in knowledge, aerosol transmission seems to be a viable pathway, as well. Because of the lower biological load in ambient aerosol, detection of SARS-CoV-2 is challenging. A few recent attempts of sampling large aerosol volumes and using next-generation sequencing (NGS) to detect the presence of SARS-CoV-2 in the air at very low levels gave positive results. These results suggest the potential of using this technique to detect the presence of SARS-CoV-2 and use it as an early warning signal for possible outbreak or recurrence of coronavirus disease 2019 (COVID-19).AimTo assess efficacy of comprehensive respiratory viral panel (CRVP) sequencing and RT-PCR for low-level identification of SARS-CoV-2 and other respiratory viruses in indoor air.MethodsA large volume of indoor aerosol samples from three major hospitals involved in COVID-19 care in Kuwait was collected. Viral RNA was isolated and subjected to comprehensive respiratory viral panel sequencing (CRVP) as per the standard protocol to detect the SARS-CoV-2 and other respiratory viruses in the hospital aerosol and monitor variations within the sequences. RT-PCR was also employed to estimate the viral load of SARS-CoV-2.Findings13 of 15 (86.7%) samples exhibited SARS-CoV-2 with a relative abundance of 0.2-33.3%. The co-occurrence of human adenoviruses (type C1, C2, C5, C4), respiratory syncytial virus (RSV), influenza B, and non-SARS-CoV-229E were also recorded. Alignment of SARS-CoV-2 sequences against the reference strain of Wuhan China revealed variations in the form of single nucleotide polymorphisms (SNPs-17), insertions and deletions (indels-1). These variations were predicted to create missense (16), synonymous (15), frameshift (1) and stop-gained (1) mutations with a high (2), low (15), and moderate (16) impact.ConclusionsOur results suggest that using CRVP on a large volume aerosol sample was a valuable tool for detecting SARS-CoV-2 in indoor aerosols of health care settings. Owing to its higher sensitivity, it can be employed as a surveillance strategy in the post COVID times to act as an early warning system to possibly control future outbreaks.

Dataset Information

Nanopore Targeted Sequencing for the Accurate and Comprehensive Detection of SARS-CoV-2 and Other Respiratory Viruses.

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