LAMP-Seq enables sensitive, multiplexed COVID-19 diagnostics using molecular barcoding

Ludwig, Kerstin U.; Schmithausen, Ricarda Maria; Li, David; Jacobs, Max L.; Hollstein, Ronja; Blumenstock, Katja; Liebing, Jana; Słabicki, Mikołaj; Ben-Shmuel, Amir; Israeli, Ofir; Weiss, Shay; Ebert, Thomas S.; Paran, Nir; Rüdiger, Wibke; Wilbring, Gero; Feldman, David; Lippke, Bärbel; Ishorst, Nina; Hochfeld, Lara M.; Beins, Eva C.; Kaltheuner, Ines H.; Schmitz, Maximilian; Wöhler, Aliona; Döhla, Manuel; Sib, Esther; Jentzsch, Marius; Moench, Eva-Maria C.; Borrajo, Jacob; Strecker, Jonathan; Reinhardt, Julia; Cleary, Brian; Geyer, Matthias; Hölzel, Michael; Macrae, Rhiannon K.; Nöthen, Markus M.; Hoffmann, Per; Exner, Martin; Regev, Aviv; Zhang, Feng; Schmid-Burgk, Jonathan L.

doi:10.1038/s41587-021-00966-9

Cited by 53 publications

(56 citation statements)

References 24 publications

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“…However, microfluidic devices are expensive to fabricate, complex to use, and require trained personnel and stable operation conditions, which makes this technology not feasible for field‐deployable real‐time diagnosis (Almassian et al, 2013, de Oliveira et al, 2021, El‐Tholoth et al, 2021, Ganguli et al, 2020, Kaymaz & Elitas, 2021, Ma et al, 2018, Mori et al, 2001, Wan et al, 2017, Wan et al, 2019). Therefore, portable, rapid, cost‐effective, automated, and remotely controllable LAMP platforms with colorimetric readouts will be the future of field‐ready modern nucleic‐acid‐based detection platforms (Kaygusuz et al, 2019, Ludwing et al, 2021, Puri et al, 2021, Xun et al, 2021).…”

Section: Resultsmentioning

confidence: 99%

A low‐cost, portable, and practical LAMP device for point‐of‐diagnosis in the field

Kaymaz

Ergenç

Aytekin

et al. 2022

Biotech & Bioengineering

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Transition of rapid, ready‐to‐use, and low‐cost nucleic acid‐based detection technologies from laboratories to points of sample collection has drastically accelerated. However, most of these approaches are still incapable of diagnosis starting from sampling through nucleic acid isolation and detection in the field. Here we developed a simple, portable, low‐cost, colorimetric, and remotely controllable platform for reliable, high‐throughput, and rapid diagnosis using loop‐mediated isothermal amplification (LAMP) assays. It consists of a thermally isolated cup, low‐cost electronic components, a polydimethylsiloxane sample well, and a fast prototyped case that covers electronic components. The steady‐state temperature error of the system is <1%. We performed LAMP, Colony‐LAMP, and Colony polymerase chain reactions (PCRs) using the yaiO2 primer set for Escherichia coli and Pseudomonas aeruginosa samples at 65°C and 30 min. We detected the end‐point colorimetric readouts by the naked eye under day light. We confirmed the specificity and sensitivity of our approach using pure genomic DNA and crude bacterial colonies. We benchmarked our Colony‐LAMP detection against Colony PCR. The number of samples tested can easily be modified for higher throughput in our system. We strongly believe that our platform can greatly contribute rapid and reliable diagnosis in versatile operational environments.

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Section: Resultsmentioning

confidence: 99%

A low‐cost, portable, and practical LAMP device for point‐of‐diagnosis in the field

Kaymaz

Ergenç

Aytekin

et al. 2022

Biotech & Bioengineering

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“…Multiple differences exist between recently published NGS-based diagnostic assays (8)(9)(10)(11)(12)(13)(14)(15)(16), including sample type (e.g., saliva, nasal swab) and reaction type (e.g., RT-PCR, LAMP). Early hybridization and pooling as implemented in ApharSeq are compatible with various extraction methods, enzymatic reactions, and protocols.…”

Section: Discussionmentioning

confidence: 99%

“…In the last decade, next generation sequencing (NGS) has replaced RT-qPCR and microarrays as the assay of choice for quantifying RNA molecules in research. During the COVID-19 pandemic, different NGS-based assays have been proposed to measure the presence and abundance of the viral genome in samples (8)(9)(10)(11)(12)(13)(14)(15)(16). In addition to detection and quantification, these assays can provide near real-time sequence information and provide epidemiologists with data on the emergence of new variants (17,18).…”

Section: Introductionmentioning

confidence: 99%

Early sample tagging and pooling enables simultaneous SARS-CoV-2 detection and variant sequencing

et al. 2021

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Most SARS-CoV-2 diagnostic tests have relied on RNA extraction followed by quantitative reversetranscription PCR assays (RT-qPCR). Whereas automation improved logistics and different pooling strategies increased testing capacity, highly multiplexed next generation sequencing (NGS) diagnostics remain a largely untapped resource. NGS tests have the potential to dramatically increase throughput while providing crucial SARS-CoV-2 variant information. Current NGS-based detection and genotyping assays for SARS-CoV-2 are costly, mostly due to parallel sample processing through multiple steps. Here, we have established ApharSeq, in which samples are barcoded in the lysis buffer and pooled prior to reverse transcription. We validated this assay by applying ApharSeq to more than 500 clinical samples from the Clinical Virology Lab at Hadassah hospital in a robotic workflow. The assay was linear across five orders of magnitude, and the limit of detection was Ct 33 (~1000 copies/ml, 95% sensitivity) with >99.5% specificity. ApharSeq provided targeted high-confidence genotype information due to unique molecular identifiers incorporated into this method. Due to early pooling, we were able to estimate a 10-fold to a 100fold reduction in labor, automated liquid handling, and reagent requirements in high throughput settings compared to current testing methods. The protocol can be tailored to assay other host or pathogen RNA targets simultaneously. These results suggest that ApharSeq can be a promising tool for current and future mass diagnostic challenges.

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“…NP ( Torres et al., 2020 ) have also been extensively accessed. The approach has even been tested with other non-PCR NAATs, with relative success ( Ludwig et al., 2021 ).…”

Section: Sample Collectionmentioning

confidence: 99%

SARS-CoV-2 Diagnostics Based on Nucleic Acids Amplification: From Fundamental Concepts to Applications and Beyond

Vindeirinho

Pinho

Azevedo

et al. 2022

Front. Cell. Infect. Microbiol.

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COVID-19 pandemic ignited the development of countless molecular methods for the diagnosis of SARS-CoV-2 based either on nucleic acid, or protein analysis, with the first establishing as the most used for routine diagnosis. The methods trusted for day to day analysis of nucleic acids rely on amplification, in order to enable specific SARS-CoV-2 RNA detection. This review aims to compile the state-of-the-art in the field of nucleic acid amplification tests (NAATs) used for SARS-CoV-2 detection, either at the clinic level, or at the Point-Of-Care (POC), thus focusing on isothermal and non-isothermal amplification-based diagnostics, while looking carefully at the concerning virology aspects, steps and instruments a test can involve. Following a theme contextualization in introduction, topics about fundamental knowledge on underlying virology aspects, collection and processing of clinical samples pave the way for a detailed assessment of the amplification and detection technologies. In order to address such themes, nucleic acid amplification methods, the different types of molecular reactions used for DNA detection, as well as the instruments requested for executing such routes of analysis are discussed in the subsequent sections. The benchmark of paradigmatic commercial tests further contributes toward discussion, building on technical aspects addressed in the previous sections and other additional information supplied in that part. The last lines are reserved for looking ahead to the future of NAATs and its importance in tackling this pandemic and other identical upcoming challenges.

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LAMP-Seq enables sensitive, multiplexed COVID-19 diagnostics using molecular barcoding

Cited by 53 publications

References 24 publications

A low‐cost, portable, and practical LAMP device for point‐of‐diagnosis in the field

A low‐cost, portable, and practical LAMP device for point‐of‐diagnosis in the field

Early sample tagging and pooling enables simultaneous SARS-CoV-2 detection and variant sequencing

SARS-CoV-2 Diagnostics Based on Nucleic Acids Amplification: From Fundamental Concepts to Applications and Beyond

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