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  3. Vivid COVID-19 LAMP is an ultrasensitive, quadruplexed test using LNA-modified primers and a zinc ion and 5-Br-PAPS colorimetric detection system

Vivid COVID-19 LAMP is an ultrasensitive, quadruplexed test using LNA-modified primers and a zinc ion and 5-Br-PAPS colorimetric detection system

  • Commun Biol. 2023 Mar 2;6(1):233. doi: 10.1038/s42003-023-04612-9.
Adrián Szobi 1 2,  Katarína Buranovská # 1 2,  Nina Vojtaššáková # 1 2,  Daniel Lovíšek 1 2,  Halil Önder Özbaşak 1 2,  Sandra Szeibeczederová 1 2,  Liudmyla Kapustian 1 2,  Zuzana Hudáčová 1 2 3,  Viera Kováčová 1 2 4,  Diana Drobná 1 2,  Piotr Putaj 1 2,  Stanislava Bírová 1 2,  Ivana Čirková 1 2,  Martin Čarnecký 1 2,  Peter Kilián 1 2,  Peter Jurkáček 5,  Viktória Čabanová 6,  Kristína Boršová 6,  Monika Sláviková 6,  Veronika Vaňová 6 7,  Boris Klempa 6 7,  Pavol Čekan 8 9,  Evan D Paul 10 11
Affiliations

Affiliations

  • 1 MultiplexDX, s.r.o., Comenius University Science Park, Ilkovičova 8, 841 04, Bratislava, Slovakia.
  • 2 MultiplexDX, Inc., One Research Court, Suite 450, Rockville, MD, 20850, USA.
  • 3 Stanford University, 730 Escondido Rd., Stanford, CA, 94305, USA.
  • 4 University of Cologne, Institute for Biological Physics, Zülpicher Str. 77, 50937, Köln, Germany.
  • 5 AstonITM s.r.o., Račianska 153, 831 54, Bratislava, Slovakia.
  • 6 Biomedical Research Center, Slovak Academy of Sciences, Institute of Virology, Dúbravská cesta 9, 845 05, Bratislava, Slovakia.
  • 7 Department of Microbiology and Virology, Faculty of Natural Sciences, Comenius University, Ilkovičova 6, 842 15, Bratislava, Slovakia.
  • 8 MultiplexDX, s.r.o., Comenius University Science Park, Ilkovičova 8, 841 04, Bratislava, Slovakia. pavol@multiplexdx.com.
  • 9 MultiplexDX, Inc., One Research Court, Suite 450, Rockville, MD, 20850, USA. pavol@multiplexdx.com.
  • 10 MultiplexDX, s.r.o., Comenius University Science Park, Ilkovičova 8, 841 04, Bratislava, Slovakia. paul@multiplexdx.com.
  • 11 MultiplexDX, Inc., One Research Court, Suite 450, Rockville, MD, 20850, USA. paul@multiplexdx.com.
  • # Contributed equally.
PMID: 36864129 DOI: 10.1038/s42003-023-04612-9
Abstract

Sensitive and rapid point-of-care assays have been crucial in the global response to SARS-CoV-2. Loop-mediated isothermal amplification (LAMP) has emerged as an important diagnostic tool given its simplicity and minimal equipment requirements, although limitations exist regarding sensitivity and the methods used to detect reaction products. We describe the development of Vivid COVID-19 LAMP, which leverages a metallochromic detection system utilizing zinc ions and a zinc sensor, 5-Br-PAPS, to circumvent the limitations of classic detection systems dependent on PH Indicators or magnesium Chelators. We make important strides in improving RT-LAMP sensitivity by establishing principles for using LNA-modified LAMP primers, multiplexing, and conducting extensive optimizations of reaction parameters. To enable point-of-care testing, we introduce a rapid sample inactivation procedure without RNA extraction that is compatible with self-collected, non-invasive gargle samples. Our quadruplexed assay (targeting E, N, ORF1a, and RdRP) reliably detects 1 RNA copy/µl of sample (=8 copies/reaction) from extracted RNA and 2 RNA copies/µl of sample (=16 copies/reaction) directly from gargle samples, making it one of the most sensitive RT-LAMP tests and even comparable to RT-qPCR. Additionally, we demonstrate a self-contained, mobile version of our assay in a variety of high-throughput field testing scenarios on nearly 9,000 crude gargle samples. Vivid COVID-19 LAMP can be an important asset for the endemic phase of COVID-19 as well as preparing for future pandemics.

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