Optimization and clinical validation of dual-target RT-LAMP for SARS-CoV-2

Abu Naser Mohon; Lisa Oberding; Jana Hundt; Guido van Marle; Kanti Pabbaraju; Byron M Berenger; Luiz Lisboa; Thomas Griener; Markus Czub; Cody Doolan; Venice Servellita; Charles Y Chiu; Alexander L Greninger; Keith R Jerome; Dylan R Pillai

doi:10.1016/j.jviromet.2020.113972

Optimization and clinical validation of dual-target RT-LAMP for SARS-CoV-2

J Virol Methods. 2020 Dec:286:113972. doi: 10.1016/j.jviromet.2020.113972. Epub 2020 Sep 15.

Authors

Affiliations

¹ Department of Microbiology, Immunology, and Infectious Diseases, University of Calgary, AB, Canada.
² Department of Microbiology, Immunology, and Infectious Diseases, University of Calgary, AB, Canada; Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada.
³ Alberta Public Health Laboratory, Alberta Precision Laboratory, Calgary, AB, Canada.
⁴ Alberta Public Health Laboratory, Alberta Precision Laboratory, Calgary, AB, Canada; Clinical Section of Microbiology, Alberta Precision Laboratories, Calgary, AB, Canada.
⁵ Clinical Section of Microbiology, Alberta Precision Laboratories, Calgary, AB, Canada; Department Pathology and Laboratory Medicine, University of Calgary, Calgary, AB, Canada.
⁶ Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada.
⁷ Illucidx Inc., Calgary, AB, Canada.
⁸ Department Pathology and Laboratory Medicine, University of California, San Francisco, USA.
⁹ Department of Laboratory Medicine, University of Washington, Seattle, WA, USA.
¹⁰ Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
¹¹ Department of Microbiology, Immunology, and Infectious Diseases, University of Calgary, AB, Canada; Clinical Section of Microbiology, Alberta Precision Laboratories, Calgary, AB, Canada; Department Pathology and Laboratory Medicine, University of Calgary, Calgary, AB, Canada; Department of Medicine, University of Calgary, Calgary, AB, Canada. Electronic address: drpillai@ucalgary.ca.

Abstract

A novel reverse-transcriptase loop mediated amplification (RT-LAMP) method targeting genes encoding the Spike (S) protein and RNA-dependent RNA polymerase (RdRP) of SARS-CoV-2 has been developed. The LAMP assay achieves a comparable limit of detection (25-50 copies per reaction) to commonly used RT-PCR protocols using clinical samples quantified by digital droplet PCR. Precision, cross-reactivity, inclusivity, and limit of detection studies were performed according to regulatory standards. Clinical validation of dual-target RT-LAMP (S and RdRP gene) achieved a PPA of 98.48 % (95 % CI 91.84%-99.96%) and NPA 100.00 % (95 % CI 93.84%-100.00%) based on the E gene and N2 gene reference RT-PCR methods. The method has implications for development of point of care technology using isothermal amplification.

Keywords: Assay; Covid; Diagnostics; LAMP; Molecular; PCR; SARS-CoV-2; Validation.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Betacoronavirus / genetics*
Betacoronavirus / isolation & purification*
COVID-19
COVID-19 Testing
Clinical Laboratory Techniques / methods*
Computer Simulation
Coronavirus Envelope Proteins
Coronavirus Infections / diagnosis*
Coronavirus Infections / virology*
Humans
Molecular Diagnostic Techniques / methods*
Nucleic Acid Amplification Techniques / methods*
Pandemics
Pneumonia, Viral / diagnosis*
Pneumonia, Viral / virology*
Point-of-Care Systems
RNA, Viral / genetics
RNA, Viral / isolation & purification*
RNA-Directed DNA Polymerase
SARS-CoV-2
Sensitivity and Specificity
Spike Glycoprotein, Coronavirus / genetics
Viral Envelope Proteins / genetics

Substances

Coronavirus Envelope Proteins
RNA, Viral
Spike Glycoprotein, Coronavirus
Viral Envelope Proteins
envelope protein, SARS-CoV-2
spike protein, SARS-CoV-2
RNA-Directed DNA Polymerase

Supplementary concepts

LAMP assay

Grants and funding

Canadian Institutes for Health Research/International