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Development and Validation of a Rapid, Single-Step Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) System Potentially to Be Used for Reliable and High-Throughput Screening of COVID-19.
Jiang, Minghua; Pan, Weihua; Arasthfer, Amir; Fang, Wenjie; Ling, Liyan; Fang, Hua; Daneshnia, Farnaz; Yu, Jian; Liao, Wanqing; Pei, Hao; Li, Xiaojing; Lass-Flörl, Cornelia.
  • Jiang M; Department of Laboratory Medicine, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China.
  • Pan W; Shanghai Key Laboratory of Molecular Medical Mycology, Shanghai Institute of Mycology, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, China.
  • Arasthfer A; Center for Discovery and Innovation, Hackensack Meridian Health, Nutley, NJ, United States.
  • Fang W; Shanghai Key Laboratory of Molecular Medical Mycology, Shanghai Institute of Mycology, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, China.
  • Ling L; Department of Laboratory Medicine, Pinghu Second People's Hospital, Jiaxing, China.
  • Fang H; Department of Laboratory Medicine, Pudong New Area People's Hospital, Shanghai, China.
  • Daneshnia F; Center for Discovery and Innovation, Hackensack Meridian Health, Nutley, NJ, United States.
  • Yu J; Department of Laboratory Medicine, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China.
  • Liao W; Shanghai Key Laboratory of Molecular Medical Mycology, Shanghai Institute of Mycology, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, China.
  • Pei H; Department of Laboratory Medicine, Wuxi No. 5 People's Hospital, Wuxi, China.
  • Li X; Department of Dermatology, Affiliated Hospital of Hebei University of Engineering, Handan, China.
  • Lass-Flörl C; Institute of Hygiene and Medical Microbiology, Medical University of Innsbruck, Innsbruck, Austria.
Front Cell Infect Microbiol ; 10: 331, 2020.
Article in English | MEDLINE | ID: covidwho-634362
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ABSTRACT

Objectives:

Development and validation of a single-step and accurate reverse transcriptase loop-mediated isothermal amplification technique (RT-LAMP) for rapid identification of SARS-CoV-2 relative to commercial quantitative reverse transcriptase real-time PCR (qRT-PCR) assays to allow prompt initiation of proper medical care and containment of virus spread.

Methods:

Primers showing optimal in-silico features were subjected to analytical sensitivity and specificity to assess the limit of detection (LOD) and cross-reaction with closely- and distantly-related viral species, and clinically prominent bacterial and fungal species. In order to evaluate the clinical utility, our RT-LAMP was subjected to a large number of clinical samples, including 213 negative and 47 positive patients, relative to two commercial quantitative RT-PCR assays.

Results:

The analytical specificity and sensitivity of our assay was 100% and 500 copies/ml when serial dilution was performed in both water and sputum. Subjecting our RT-LAMP assay to clinical samples showed a high degree of specificity (99.5%), sensitivity (91.4%), positive predictive value (97.7%), and negative predictive value (98.1%) when used relative to qRT-PCR. Our RT-LAMP assay was two times faster than qRT-PCR and is storable at room temperature. A suspected case that later became positive tested positive using both our RT-LAMP and the two qRT-PCR assays, which shows the capability of our assay for screening purposes.

Conclusions:

We present a rapid RT-LAMP assay that could extend the capacity of laboratories to process two times more clinical samples relative to qRT-PCR and potentially could be used for high-throughput screening purposes when demand is increasing at critical situations.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: Pneumonia, Viral / Mass Screening / Coronavirus Infections / Nucleic Acid Amplification Techniques / Betacoronavirus Type of study: Diagnostic study / Prognostic study / Randomized controlled trials / Screening study Limits: Humans Language: English Journal: Front Cell Infect Microbiol Year: 2020 Document Type: Article Affiliation country: Fcimb.2020.00331

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Full text: Available Collection: International databases Database: MEDLINE Main subject: Pneumonia, Viral / Mass Screening / Coronavirus Infections / Nucleic Acid Amplification Techniques / Betacoronavirus Type of study: Diagnostic study / Prognostic study / Randomized controlled trials / Screening study Limits: Humans Language: English Journal: Front Cell Infect Microbiol Year: 2020 Document Type: Article Affiliation country: Fcimb.2020.00331