Flexible upscaling of laboratory PCR testing capacity at the Robert Koch Institute during the SARS-CoV-2 pandemic DOI Creative Commons
Eva Krause, Janine Michel, Andreas Puyskens

et al.

Virology Journal, Journal Year: 2023, Volume and Issue: 20(1)

Published: July 5, 2023

Abstract Background Over the course of COVID-19 pandemic, laboratories worldwide have been facing an unprecedented increase in demand for PCR testing because high importance diagnostics prevention and control virus spread. Moreover, has varying considerably over time, depending on epidemiological situation, rendering efficient resource allocation difficult. Here, we present a scalable workflow which implemented our laboratory to capacity while maintaining flexibility regarding number samples be processed. Methods We compared performance five automated extraction instruments, using dilutions SARS-CoV-2 cell culture supernatant as well clinical samples. To throughput, combined two duplex reactions previously published assay into one quadruplex reaction their limit detection low viral loads Furthermore, developed sample pooling protocol with either or four per pool, specifically adapted assay, diagnostic sensitivity pooled individual testing. Results All tested instruments yielded comparable results subsequent by PCR. While (E-Gene assay: 28.7 genome equivalents (ge)/reaction, orf1ab 32.0 ge/reaction) was slightly higher than that assays 9.8 ge/reaction, 6.6 ge/reaction), rate correctly identified positive patient both assays. Sample optimized downstream showed no loss Conclusion Specific adaptation can help overcome potential due levels multiplexing dilution Combining these different processing strategies provides simple highly adjustable resource-efficient diagnostics. The presented principles easily adopted variety settings pathogens other SARS-CoV-2, making it feasible any conducts

Language: Английский

Advances in RT-LAMP for COVID-19 testing and diagnosis DOI Creative Commons
Gihoon Choi, Taylor J. Moehling, Robert J. Meagher

et al.

Expert Review of Molecular Diagnostics, Journal Year: 2023, Volume and Issue: 23(1), P. 9 - 28

Published: Jan. 2, 2023

Introduction : The SARS-CoV-2 pandemic, and the subsequent limitations on standard diagnostics, has vastly expanded user base of Reverse Transcription Loop-mediated isothermal Amplification (RT-LAMP) in fundamental research development. RT-LAMP also penetrated commercial markets, with emergency use authorizations for clinical diagnosis.Areas covered This review discusses role within context other technologies like RT-qPCR rapid antigen tests, progress sample preparation strategies to enable simplified workflow directly from specimens, new challenges primer assay design evolving prominent detection modalities including colorimetric CRISPR-mediated methods, translational development applications.Expert opinion occupies a middle ground between tests. simplicity approaches that making it suitable point-of-care use, but sensitivity nears RT-qPCR. still lags understanding mechanism, interplay performance. Industry is now beginning address issues around scalability usability, which could finally LAMP find future widespread application as diagnostic conditions, pathogens pandemic potential.

Language: Английский

Citations

36
Conventional and Novel Diagnostic Tools for the Diagnosis of Emerging SARS-CoV-2 Variants DOI Creative Commons
Vivek P. Chavda,

Disha Valu,

Palak K. Parikh

et al.

Vaccines, Journal Year: 2023, Volume and Issue: 11(2), P. 374 - 374

Published: Feb. 6, 2023

Accurate identification at an early stage of infection is critical for effective care any infectious disease. The “coronavirus disease 2019 (COVID-19)” outbreak, caused by the virus “Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)”, corresponds to current and global pandemic, characterized several developing variants, many which are classified as variants concern (VOCs) “World Health Organization (WHO, Geneva, Switzerland)”. primary diagnosis made using either molecular technique RT-PCR, detects parts viral genome’s RNA, or immunodiagnostic procedures, identify proteins antibodies generated host. As demand RT-PCR test grew fast, inexperienced producers joined market with innovative kits, increasing number laboratories diagnostic field, rendering results increasingly prone mistakes. It difficult determine how outcomes one unnoticed result could influence decisions about patient quarantine social isolation, particularly when patients themselves health providers. development point-of-care testing helps in rapid in-field disease, such can also be used a bedside monitor mapping progression patients. In this review, we have provided readers available techniques their pitfalls detecting emerging VOCs SARS-CoV-2, lastly, discussed AI-ML- nanotechnology-based smart SARS-CoV-2 detection.

Language: Английский

Citations

30
RUNCOV: a one-pot triplex real-time RT-LAMP as a point-of-care diagnostic tool for detecting SARS-CoV-2 DOI Creative Commons
Isabelle Robène, Emmanuel Jouen,

Véronique Maillot-Lebon

et al.

Biology Methods and Protocols, Journal Year: 2025, Volume and Issue: 10(1)

Published: Jan. 1, 2025

Given the risk of zoonotic disease emergence, including new SARS-CoV-2 variants COVID-19, rapid diagnostic tools are urgently needed to improve control spread infectious diseases. A one-pot triplex real-time RT-LAMP (reverse-transcription-loop-mediated isothermal amplification) assay, based on two regions genome SARS-CoV-2-specifically Orf1ab and N genes-along with one internal control, human RNase P gene, was developed. The multiplexing relies distinct melting peaks produced during an annealing step. This tool, named RUNCOV, compared gold-standard reverse-transcription quantitative PCR (RT-qPCR) assay. simple sample preparation step designed alongside making it ready for use site, as a point-of-care tool. RUNCOV is (typically less than 40 minutes), highly sensitive specific. When tested clinical samples known status, its limit detection (LOD) ranges between 5 20 copies per reaction sensitivity (97.44%) specificity (100%) values high RT-qPCR gold standard. These results were supported extensive in silico analysis over 14 million genomes, demonstrating this tool capable detecting all variants, most recent ones KP.3.1.1 BA2.86.1. molecular assay portable, demonstrated when used successfully La Réunion different contexts outside laboratory.

Language: Английский

Citations

1
Recent progress in diagnosis and treatment of Human African Trypanosomiasis has made the elimination of this disease a realistic target by 2030 DOI Creative Commons
Andrés Álvarez-Rodríguez, Bo-kyung Jin, Magdalena Radwanska

et al.

Frontiers in Medicine, Journal Year: 2022, Volume and Issue: 9

Published: Nov. 3, 2022

Human African Trypanosomiasis (HAT) is caused by unicellular flagellated protozoan parasites of the genus Trypanosoma brucei . The subspecies T. b. gambiense mainly responsible for mostly chronic anthroponotic infections in West- and Central Africa, accounting roughly 95% all HAT cases. rhodesiense results more acute zoonotic East-Africa. Because has a two-stage pathogenesis, treatment depends on clinical assessment patients determination whether or not have crossed blood brain barrier. Today, ultimate confirmation parasitemia still done microscopy analysis. However, introduction diagnostic lateral flow devices been major contributor to recent dramatic drop HAT. Other techniques such as loop mediated isothermal amplification (LAMP) recombinant polymerase (RPA)-based tests published but are widely used field. Most recently, CRISPR-Cas technology proposed improve intrinsic characteristics molecular approaches. This will become crucial near future, preventing resurgence be priority require tools with extreme high positive negative predicted values, well excellent sensitivity specificity. As treatment, pentamidine suramin historically drugs choice blood-stage gambiense-HAT rhodesiense-HAT, respectively. For second-stage infections, that pass barrier needed, melarsoprol effectively both forms past. due occurrence post-treatment encephalopathy, drug recommended use Here, combination therapy eflornithine nifurtimox (NECT) since 2009. this requires IV perfusion eflornithine, efforts were launched 2003 neglected disease initiative (DNDi) find an oral-only solution, suitable rural sub-Saharan Africa conditions. In 2019 resulted fexinidazole, regimen non-severe infections. Experimental now initiated well.

Language: Английский

Citations

34
Promise and perils of paper-based point-of-care nucleic acid detection for endemic and pandemic pathogens DOI

Navaporn Sritong,

Marina Sala de Medeiros, Laud Anthony Basing

et al.

Lab on a Chip, Journal Year: 2023, Volume and Issue: 23(5), P. 888 - 912

Published: Jan. 1, 2023

Review work on the challenges of paper-based NAATs covering sample-to-answer procedures along with three main types clinical samples as well broader operational, scale up, and regulatory aspects device development implementation.

Language: Английский

Citations

16
Digital microfluidics with distance-based detection – a new approach for nucleic acid diagnostics DOI
Man Ho, N. Sathishkumar, Alexandros A. Sklavounos

et al.

Lab on a Chip, Journal Year: 2023, Volume and Issue: 24(1), P. 63 - 73

Published: Nov. 21, 2023

There is great enthusiasm for using loop-mediated isothermal amplification (LAMP) in point-of-care nucleic acid tests (POC NAATs), as an alternative to PCR. While techniques like LAMP eliminate the need rapid temperature cycling a portable format, these systems are still plagued by requirements dedicated optical detection apparatus analysis and manual off-chip sample processing. Here, we developed new microfluidic system LAMP-based POC NAATs address limitations. The combines digital microfluidics (DMF) with distance-based (DBD) direct signal readout. This first report of use (i) or (ii) DMF DBD - thus, describe number characterization steps taken determine optimal combinations reagents, materials, processes reliable operation. For example, was found be quite sensitive background signals from low molecular weight products; Capto™ adhere bead-based clean-up procedure isolate desirable high-molecular-weight products analysis. method validated application SARS-CoV-2 saliva. able distinguish between saliva containing no virus, viral load (10

Language: Английский

Citations

15
Loop-Mediated Isothermal Amplification: From Theory to Practice DOI Open Access
Fedor V. Shirshikov, Julia Bespyatykh

Russian Journal of Bioorganic Chemistry, Journal Year: 2022, Volume and Issue: 48(6), P. 1159 - 1174

Published: Dec. 1, 2022

Language: Английский

Citations

22
Progression of LAMP as a Result of the COVID-19 Pandemic: Is PCR Finally Rivaled? DOI Creative Commons

Cassidy Mannier,

Jeong‐Yeol Yoon

Biosensors, Journal Year: 2022, Volume and Issue: 12(7), P. 492 - 492

Published: July 6, 2022

Reflecting on the past three years and coronavirus disease 19 (COVID-19) pandemic, varying global tactics offer insights into most effective public-health responses. In US, specifically, rapid widespread testing was quickly prioritized to lower restrictions sooner. Essentially, only two types of COVID-19 diagnostic tests were publicly employed during peak pandemic: antigen test reverse transcription polymerase chain reaction (RT-PCR). However, neither ideally suited situation, as are far too inaccurate, RT-PCR require skilled personnel sophisticated equipment, leading long wait times. Loop-mediated isothermal amplification (LAMP) is another exceptionally accurate nucleic acid (NAAT) that offers quicker time results. RT-LAMP have not been embraced extensively or RT-PCR. This review will investigate performance current RT-LAMP-based summarize reasons behind hesitancy embrace instead We also look at other LAMP platforms explore possible improvements in accuracy portability LAMP, which could be applied diagnostics future outbreaks.

Language: Английский

Citations

20
Vivid COVID-19 LAMP is an ultrasensitive, quadruplexed test using LNA-modified primers and a zinc ion and 5-Br-PAPS colorimetric detection system DOI Creative Commons
Adrián Szobi, Katarína Buranovská,

Nina Vojtaššáková

et al.

Communications Biology, Journal Year: 2023, Volume and Issue: 6(1)

Published: March 2, 2023

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 minimal equipment requirements, although limitations exist regarding sensitivity methods used detect reaction products. We describe development of Vivid COVID-19 LAMP, which leverages a metallochromic detection system utilizing zinc ions sensor, 5-Br-PAPS, circumvent classic systems dependent on pH indicators or magnesium chelators. make strides improving RT-LAMP by establishing principles for using LNA-modified LAMP primers, multiplexing, conducting extensive optimizations parameters. To enable testing, we introduce sample inactivation procedure without RNA extraction that is compatible with self-collected, non-invasive gargle samples. Our quadruplexed assay (targeting E, N, ORF1a, RdRP) reliably detects 1 copy/µl (=8 copies/reaction) from extracted 2 copies/µl (=16 directly samples, making it one most sensitive tests even comparable RT-qPCR. Additionally, demonstrate self-contained, mobile version our variety high-throughput field testing scenarios nearly 9,000 crude can be asset endemic phase well preparing future pandemics.

Language: Английский

Citations

12
Detection and absolute quantification biosensing tools for food authentication: CRISPR/Cas, digital CRISPR and beyond DOI
Xiaolin Wu,

Xuanming Lou,

Hanzhang Zhou

et al.

Trends in Food Science & Technology, Journal Year: 2024, Volume and Issue: 145, P. 104349 - 104349

Published: Jan. 29, 2024

Language: Английский

Citations

4