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

Wang, Ming; Fu, Aisi; Hu, Ben; Tong, Yongqing; Liu, Ran; Liu, Zhen; Gu, Jiashuang; Xiang, Bin; Liu, Jianghao; Jiang, Wen; Shen, Gaigai; Zhao, Wenhui; Men, Dong; Deng, Zixin; Yu, Lilei; Wu, Wei; Li, Yan; Liu, Tiangang

doi:10.1002/smll.202002169

Cited by 177 publications

(215 citation statements)

References 48 publications

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“…We observed that the RT-LAMP assay was less sensitive and informative than multiplex PCR-based NGS in our study and other literature (20)(21)(22)(23)(24). NGS is a robust tool for obtaining extensive genetic information, allowing LOD values as low as 10 copies/ml for SARS-CoV-2 and serving as a reference test for COVID-19, especially for those challenging samples with a low viral content (2,(22)(23)(24).…”

Section: Discussionmentioning

confidence: 47%

Development and Clinical Application of a Rapid and Sensitive Loop-Mediated Isothermal Amplification Test for SARS-CoV-2 Infection

Deng

et al. 2020

mSphere

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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak urgently necessitates sensitive and convenient COVID-19 diagnostics for the containment and timely treatment of patients. We aimed to develop and validate a novel reverse transcription–loop-mediated isothermal amplification (RT-LAMP) assay to detect SARS-CoV-2. Patients with suspected COVID-19 and close contacts were recruited from two hospitals between 26 January and 8 April 2020. Respiratory samples were collected and tested using RT-LAMP, and the results were compared with those obtained by reverse transcription-quantitative PCR (RT-qPCR). Samples yielding inconsistent results between these two methods were subjected to next-generation sequencing for confirmation. RT-LAMP was also applied to an asymptomatic COVID-19 carrier and patients with other respiratory viral infections. Samples were collected from a cohort of 129 cases (329 nasopharyngeal swabs) and an independent cohort of 76 patients (152 nasopharyngeal swabs and sputum samples). The RT-LAMP assay was validated to be accurate (overall sensitivity and specificity of 88.89% and 99.00%, respectively) and diagnostically useful (positive and negative likelihood ratios of 88.89 and 0.11, respectively). RT-LAMP showed increased sensitivity (88.89% versus 81.48%) and high consistency (kappa, 0.92) compared to those of RT-qPCR for SARS-CoV-2 screening while requiring only constant-temperature heating and visual inspection. The time required for RT-LAMP was less than 1 h from sample preparation to the result. In addition, RT-LAMP was feasible for use with asymptomatic patients and did not cross-react with other respiratory pathogens. The developed RT-LAMP assay offers rapid, sensitive, and straightforward detection of SARS-CoV-2 infection and may aid the expansion of COVID-19 testing in the public domain and hospitals. IMPORTANCE We developed a visual and rapid reverse transcription–loop-mediated isothermal amplification (RT-LAMP) assay targeting the S gene for SARS-CoV-2 infection. The strength of our study was that we validated the RT-LAMP assay using 481 clinical respiratory samples from two prospective cohorts of suspected COVID-19 patients and on the serial samples from an asymptomatic carrier. The developed RT-LAMP approach showed an increased sensitivity (88.89%) and high consistency (kappa, 0.92) compared with those of reverse transcription-quantitative PCR (RT-qPCR) for SARS-CoV-2 screening while requiring only constant-temperature heating and visual inspection, facilitating SARS-CoV-2 screening in well-equipped labs as well as in the field. The time required for RT-LAMP was less than 1 h from sample preparation to the result (more than 2 h for RT-qPCR). This study showed that the RT-LAMP assay was a simple, rapid, and sensitive approach for SARS-CoV-2 infection and can facilitate COVID-19 diagnosis, especially in resource-poor settings.

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Section: Discussionmentioning

confidence: 47%

Development and Clinical Application of a Rapid and Sensitive Loop-Mediated Isothermal Amplification Test for SARS-CoV-2 Infection

Deng

et al. 2020

mSphere

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“…The CRISPR/Cas technology is characterized by high specificity, high precision, high efficiency, and simple and fast operation. 38,59 0.5-1 h 29,111,112 0.5-1 h 38,40,113 Sensitivity N. A. † 73%-86% 32,33,114 81% -100% 38,40,115 Specificity 100% 116 86% -100% 32,33,114 99% -100% 38,40,115 Advantages Unknown virus identification, viral mutation and evolution screening.…”

Section: Crisprmentioning

confidence: 99%

COVID‐19 diagnostic testing: Technology perspective

Wang

et al. 2020

Clinical & Translational Med

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The corona virus disease 2019 (COVID‐19) is a highly contagious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). More than 18 million people were infected with a total of 0.7 million deaths in ∼188 countries. Controlling the spread of SARS‐CoV‐2 is therefore inherently dependent on identifying and isolating infected individuals, especially since COVID‐19 can result in little to no symptoms. Here, we provide a comprehensive review of the different primary technologies used to test for COVID‐19 infection, discuss the advantages and disadvantages of each technology, and highlight the studies that have employed them. We also describe technologies that have the potential to accelerate SARS‐CoV‐2 detection in the future, including digital PCR, CRISPR, and microarray. Finally, remaining challenges in COVID‐19 diagnostic testing are discussed, including (a) the lack of universal standards for diagnostic testing; (b) the identification of appropriate sample collection site(s); (c) the difficulty in performing large population screening; and (d) the limited understanding of SARS‐COV‐2 viral invasion, replication, and transmission.

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“…Wang et al, developed a nanopore targeted sequencing (NTS) method, combining the advantages of target amplication and long-read, real-time nanopore sequencing, to be used for diagnosing COVID-19 and simultaneously detecting other common respiratory viruses and mutated nucleic acid sequences, within 6-10 h. Parallel testing of 61 nucleic acid samples from suspected COVID-19 cases with commercial RT-PCR kits and the proposed NTS method, showed the NTS method was more sensitive for SARS-CoV-2 detection and identied more positive cases. 21 Although the accuracy of gene sequencing to detect SARS-CoV-2 is high, there are limitations in its clinical application due to testing duration, high cost, and complex equipment. Finally, with the ongoing spread of COVID-19, genome sequencing has shied from initial virus identication to investigation of the origin and evolution of the SARS-CoV-2 virus.…”

Section: Nucleic Acid-based Detection Methodsmentioning

confidence: 99%