Rapid and Sensitive Diagnosis of COVID-19 Using an Electricity-Free Self-Testing System.

Rapid and sensitive detection of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for early diagnosis and effective treatment. Nucleic acid testing has been considered the gold standard method for the diagnosis of COVID-19 for its high sensitivity and specificity. However, the polymerase chain reaction (PCR)-based method in the central lab requires expensive equipment and well-trained personnel, which makes it difficult to be used in resource-limited settings. It highlights the need for a sensitive and simple assay that allows potential patients to detect SARS-CoV-2 by themselves. Here, we developed an electricity-free self-testing system based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) that allows for rapid and accurate detection of SARS-CoV-2. Our system employs a heating bag as the heat source, and a 3D-printed box filled with phase change material (PCM) that successfully regulates the temperature for the RT-LAMP. The colorimetric method could be completed in 40 min and the results could be read out by the naked eye. A ratiometric measurement for exact readout was also incorporated to improve the detection accuracy of the system. This self-testing system is a promising tool for point-of-care testing (POCT) that enables rapid and sensitive diagnosis of SARS-CoV-2 in the real world and will improve the current COVID-19 screening efforts for control and mitigation of the pandemic.

CoLAMP: CRISPR-based one-pot loop-mediated isothermal amplification enables at-home diagnosis of SARS-CoV-2 RNA with nearly eliminated contamination utilizing amplicons depletion strategy.

Rapid point-of-care diagnostics, essential in settings such as airport on-site testing and home-based screening, displayed important implications for infectious disease control during the SARS-CoV-2 outbreak. However, the deployment of simple and sensitive assays in real-life scenarios still faces the concern of aerosol contamination. Here, we report an amplicon-depleting CRISPR-based one-pot loop-mediated isothermal amplification (CoLAMP) assay for point-of-care diagnosis of SARS-CoV-2 RNA. In this work, AapCas12b sgRNA is designed to recognize the activator sequence sited in the loop region of the LAMP product, which is crucial for exponential amplification. By destroying the aerosol-prone amplifiable products at the end of each amplification reaction, our design can significantly reduce the amplicons contamination that causes false positive results in point-of-care diagnostics. For at-home self-testing, we designed a low-cost sample-to-result device for fluorescence-based visual interpretation. As well, a commercial portable electrochemical platform was deployed as a proof-of-concept of ready-to-use point-of-care diagnostic systems. The field deployable CoLAMP assay can detect as low as 0.5 copies/µL of SARS-CoV-2 RNA in clinical nasopharyngeal swab samples within 40 min without the need for specialists for its operation.

Current regulatory landscape for viral point-of-care testing in the United States.

Historically, the diagnosis of viral infections has been accomplished using a combination of laboratory-based methods, including culture, serology, antigen-based tests, and molecular (e.g., real-time PCR) assays. Although these methods provide an accurate way to detect viral pathogens, testing in a centralized laboratory may delay results, which could impact patient diagnosis and management. Point-of-care tests, including antigen- and molecular-based assays, have been developed to assist with the timely diagnosis of several viral infections, such as influenza, respiratory syncytial virus, and COVID-19. Despite the ability of point-of-care tests to provide rapid results (i.e., <30 min), there are issues to consider prior to their routine use, including test performance and specific regulatory requirements. This review will provide a summary of the regulatory landscape of point-of-care tests for viral infections in the United States, and address important considerations such as site certification, training and inspection readiness.

Field-deployable multiplex detection method of SARS-CoV-2 and influenza virus using loop-mediated isothermal amplification and DNA chromatography.

A novel multiplex loop-mediated isothermal amplification (LAMP) method combined with DNA chromatography was developed for the simultaneous detection of three important respiratory disease-causing viruses: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, and influenza B virus. Amplification was performed at a constant temperature, and a positive result was confirmed by a visible colored band. An in-house drying protocol with trehalose was used to prepare the dried format multiplex LAMP test. Using this dried multiplex LAMP test, the analytical sensitivity was determined to be 100 copies for each viral target and 100-1000 copies for the simultaneous detection of mixed targets. The multiplex LAMP system was validated using clinical COVID-19 specimens and compared with the real-time qRT-PCR method as a reference test. The determined sensitivity of the multiplex LAMP system for SARS-CoV-2 was 71% (95% CI: 0.62-0.79) for cycle threshold (Ct) ≤ 35 samples and 61% (95% CI: 0.53-0.69) for Ct ≤40 samples. The specificity was 99% (95%CI: 0.92-1.00) for Ct ≤35 samples and 100% (95%CI: 0.92-1.00) for the Ct ≤40 samples. The developed simple, rapid, low-cost, and laboratory-free multiplex LAMP system for the two major important respiratory viral diseases, COVID-19 and influenza, is a promising field-deployable diagnosis tool for the possible future 'twindemic, ' especially in resource-limited settings.

Rapid production and free distribution of a synthetic RNA material to support SARS-CoV-2 molecular diagnostic testing.

In response to the COVID-19 pandemic, the National Institute of Standards and Technology released a synthetic RNA material for SARS-CoV-2 in June 2020. The goal was to rapidly produce a material to support molecular diagnostic testing applications. This material, referred to as Research Grade Test Material 10169, was shipped free of charge to laboratories across the globe to provide a non-hazardous material for assay development and assay calibration. The material consisted of two unique regions of the SARS-CoV-2 genome approximately 4 kb nucleotides in length. The concentration of each synthetic fragment was measured using RT-dPCR methods and confirmed to be compatible with RT-qPCR methods. In this report, the preparation, stability, and limitations of this material are described.

Integrating tuberculosis and COVID-19 molecular testing in Lima, Peru: a cross-sectional, diagnostic accuracy study.

BACKGROUND: Integrated molecular testing could be an opportunity to detect and provide care for both tuberculosis and COVID-19. Many high tuberculosis burden countries, such as Peru, have existing GeneXpert systems for tuberculosis testing with GeneXpert Xpert MTB/RIF Ultra (Xpert Ultra), and a GeneXpert SARS-CoV-2 assay, GeneXpert Xpert Xpress SARS-CoV-2 (Xpert Xpress), is also available. We aimed to assess the feasibility of integrating tuberculosis and COVID-19 testing using one sputum specimen with Xpert Ultra and Xpert Xpress in Lima, Peru. METHODS: In this cross-sectional, diagnostic accuracy study, we recruited adults presenting with clinical symptoms or suggestive history of tuberculosis or COVID-19, or both. Participants were recruited from a total of 35 primary health facilities in Lima, Peru. Participants provided one nasopharyngeal swab and one sputum sample. For COVID-19, we tested nasopharyngeal swabs and sputum using Xpert Xpress; for tuberculosis, we tested sputum using culture and Xpert Ultra. We compared diagnostic accuracy of sputum testing using Xpert Xpress with nasopharyngeal swab testing using Xpert Xpress. Individuals with positive Xpert Xpress nasopharyngeal swab results were considered COVID-19 positive, and a positive culture indicated tuberculosis. To assess testing integration, the proportion of cases identified in sputum by Xpert Xpress was compared with Xpert Xpress on nasopharyngeal swabs, and sputum by Xpert Ultra was compared with culture. FINDINGS: Between Jan 11, 2021, and April 26, 2022, we recruited 600 participants (312 [52%] women and 288 [48%] men). In-study prevalence of tuberculosis was 13% (80 participants, 95% CI 11-16) and of SARS-CoV-2 was 35% (212 participants, 32-39). Among tuberculosis cases, 13 (2·2%, 1·2-3·7) participants were concurrently positive for SARS-CoV-2. Regarding the diagnostic yield of integrated testing, Xpert Ultra detected 96% (89-99) of culture-confirmed tuberculosis cases (n=77), and Xpert Xpress-sputum detected 67% (60-73) of COVID-19 cases (n=134). All five study staff reported that integrated molecular testing was easy and acceptable. INTERPRETATION: The diagnostic yield of Xpert Xpress on sputum was moderate, but integrated testing for tuberculosis and COVID-19 with GeneXpert was feasible. However, systematic testing for both diseases might not be the ideal approach for everyone presenting with presumptive tuberculosis or COVID-19, as concurrent positive cases were rare during the study period. Further research might help to identify when integrated testing is most worthwhile and its optimal implementation. FUNDING: Canadian Institutes of Health Research and International Development Research Centre. TRANSLATION: For the Spanish translation of the abstract see Supplementary Materials section.

Real-Time, Multiplexed SHERLOCK for in Vitro Diagnostics.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the need for simple, low-cost, and scalable diagnostics that can be widely deployed for rapid testing. Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics have emerged as a promising technology, but its implementation in clinical laboratories has been limited by the requirement of a separate amplification step prior to CRISPR-associated (Cas) enzyme-based detection. This article reports the discovery of two novel Cas12 enzymes (SLK9 and SLK5-2) that exhibit enzymatic activity at 60°C, which, when combined with loop-mediated isothermal amplification (LAMP), enable a real-time, single-step nucleic acid detection method [real-time SHERLOCK (real-time SLK)]. Real-time SLK was demonstrated to provide accurate results comparable to those from real-time quantitative RT-PCR in clinical samples, with 100% positive and 100% negative percent agreement. The method is further demonstrated to be compatible with direct testing (real-time SLK Direct) of samples from anterior nasal swabs, without the need for standard nucleic acid extraction. Lastly, SLK9 was combined with either Alicyclobacillus acidoterrestris AacCas12b or with SLK5-2 to generate a real-time, multiplexed CRISPR-based diagnostic assay for the simultaneous detection of SARS-CoV-2 and a human-based control in a single reaction, with sensitivity down to 5 copies/µL and a time to result of under 30 minutes.

A Portable RT-LAMP/CRISPR Machine for Rapid COVID-19 Screening.

The COVID-19 pandemic has changed people's lives and has brought society to a sudden standstill, with lockdowns and social distancing as the preferred preventative measures. To lift these measurements and reduce society's burden, developing an easy-to-use, rapid, and portable system to detect SARS-CoV-2 is mandatory. To this end, we developed a portable and semi-automated device for SARS-CoV-2 detection based on reverse transcription loop-mediated isothermal amplification followed by a CRISPR/Cas12a reaction. The device contains a heater element mounted on a printed circuit board, a cooler fan, a proportional integral derivative controller to control the temperature, and designated areas for 0.2 mL Eppendorf® PCR tubes. Our system has a limit of detection of 35 copies of the virus per microliter, which is significant and has the capability of being used in crisis centers, mobile laboratories, remote locations, or airports to diagnose individuals infected with SARS-CoV-2. We believe the current methodology that we have implemented in this article is beneficial for the early screening of infectious diseases, in which fast screening with high accuracy is necessary.

Current methods for diagnosis of human coronaviruses: pros and cons.

The current global fight against coronavirus disease (COVID-19) to flatten the transmission curve is put forth by the World Health Organization (WHO) as there is no immediate diagnosis or cure for COVID-19 so far. In order to stop the spread, researchers worldwide are working around the clock aiming to develop reliable tools for early diagnosis of severe acute respiratory syndrome (SARS-CoV-2) understanding the infection path and mechanisms. Currently, nucleic acid-based molecular diagnosis (real-time reverse transcription polymerase chain reaction (RT-PCR) test) is considered the gold standard for early diagnosis of SARS-CoV-2. Antibody-based serology detection is ineffective for the purpose of early diagnosis, but a potential tool for serosurveys, providing people with immune certificates for clearance from COVID-19 infection. Meanwhile, there are various blooming methods developed these days. In this review, we summarise different types of coronavirus discovered which can be transmitted between human beings. Methods used for diagnosis of the discovered human coronavirus (SARS, MERS, COVID-19) including nucleic acid detection, gene sequencing, antibody detection, antigen detection, and clinical diagnosis are presented. Their merits, demerits and prospects are discussed which can help the researchers to develop new generation of advanced diagnostic tools for accurate and effective control of human coronavirus transmission in the communities and hospitals.

Real-time detection of SARS-CoV-2 in clinical samples via ultrafast ligation-dependent RNA transcription amplification.

RNA has been recognized as an important biomarker of many infectious pathogens; thus, sensitive, simple and rapid detection of RNA is urgently required for the control of epidemics. Herein, we report an ultrafast ligation-dependent RNA transcription amplification assay with high sensitivity and specificity for real-time detection of SARS-CoV-2 in real clinical samples, termed splint-based cascade transcription amplification (SCAN). Target RNA is first recognized by two DNA probes, which are then ligated together by SplintR, followed by the binding of the T7 promotor and T7 RNA polymerase to the ligated probe and the start of the transcription process. By introducing a vesicular stomatitis virus (VSV) terminator in the ligated probe, large amounts of RNA transcripts are rapidly produced within 10 min, which then directly hybridize with molecular beacons (MBs) and trigger the conformational switch of the MBs to generate a fluorescence signal that can be monitored in real time. The SCAN assay, which can be completed within 30-50 min, has a limit of detection of 104 copies per mL, while exhibiting high specificity to distinguish the target pathogen from those causing similar syndromes. More importantly, the results of SCAN for SARS-CoV-2 detection in clinical samples display great agreement with the most used qRT-PCR and qRT-LAMP, indicating great potential in the diagnosis of pathogens in clinical practice.

Carrageenan from Gigartina skottsbergii: A Novel Molecular Probe to Detect SARS-CoV-2.

The COVID-19 pandemic has caused an unprecedented health and economic crisis, highlighting the importance of developing new molecular tools to monitor and detect SARS-CoV-2. Hence, this study proposed to employ the carrageenan extracted from Gigartina skottsbergii algae as a probe for SARS-CoV-2 virus binding capacity and potential use in molecular methods. G. skottsbergii specimens were collected in the Chilean subantarctic ecoregion, and the carrageenan was extracted -using a modified version of Webber's method-, characterized, and quantified. After 24 h of incubation with an inactivated viral suspension, the carrageenan's capacity to bind SARS-CoV-2 was tested. The probe-bound viral RNA was quantified using the reverse transcription and reverse transcription loop-mediated isothermal amplification (RT-LAMP) methods. Our findings showed that carrageenan extraction from seaweed has a similar spectrum to commercial carrageenan, achieving an excellent proportion of binding to SARS-CoV-2, with a yield of 8.3%. Viral RNA was also detected in the RT-LAMP assay. This study shows, for the first time, the binding capacity of carrageenan extracted from G. skottsbergii, which proved to be a low-cost and highly efficient method of binding to SARS-CoV-2 viral particles.

A novel, ultrafast, ultrasensitive diagnosis platform for the detection of SARS-COV-2 using restriction endonuclease-mediated reverse transcription multiple cross displacement amplification.

Coronavirus disease 2019 (COVID-19) is a highly infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-COV-2). Though many methods have been used for detecting SARS-COV-2, development of an ultrafast and highly sensitive detection strategy to screen and/or diagnose suspected cases in the population, especially early-stage patients with low viral load, is significant for the prevention and treatment of COVID-19. In this study, a novel restriction endonuclease-mediated reverse transcription multiple cross displacement amplification (MCDA) combined with real-time fluorescence analysis (rRT-MCDA) was successfully established and performed to diagnose COVID-19 infection (COVID-19 rRT-MCDA). Two sets of specific SARS-COV-2 rRT-MCDA primers targeting opening reading frame 1a/b (ORF1ab) and nucleoprotein (NP) genes were designed and modified according to the reaction mechanism. The SARS-COV-2 rRT-MCDA test was optimized and evaluated using various pathogens and clinical samples. The optimal reaction condition of SARS-COV-2 rRT-MCDA assay was 65°C for 36 min. The SARS-COV-2 rRT-MCDA limit of detection (LoD) was 6.8 copies per reaction. Meanwhile, the specificity of SARS-COV-2 rRT-MCDA assay was 100%, and there was no cross-reaction with nucleic acids of other pathogens. In addition, the whole detection process of SARS-COV-2 rRT-MCDA, containing the RNA template processing (15 min) and real-time amplification (36 min), can be accomplished within 1 h. The SARS-COV-2 rRT-MCDA test established in the current report is a novel, ultrafast, ultrasensitive, and highly specific detection method, which can be performed as a valuable screening and/or diagnostic tool for COVID-19 in clinical application.

A loop-mediated isothermal amplification-enabled analytical assay for the detection of SARS-CoV-2: A review.

The number of words: 4645, the number of figures: 4, the number of tables: 1The outbreak of COVID-19 in December 2019 caused a global pandemic of acute respiratory disease, and with the increasing virulence of mutant strains and the number of confirmed cases, this has resulted in a tremendous threat to global public health. Therefore, an accurate diagnosis of COVID-19 is urgently needed for rapid control of SARS-CoV-2 transmission. As a new molecular biology technology, loop-mediated isothermal amplification (LAMP) has the advantages of convenient operation, speed, low cost and high sensitivity and specificity. In the past two years, rampant COVID-19 and the continuous variation in the virus strains have demanded higher requirements for the rapid detection of pathogens. Compared with conventional RT-PCR and real-time RT-PCR methods, genotyping RT-LAMP method and LAMP plus peptide nucleic acid (PNA) probe detection methods have been developed to correctly identified SARS-CoV-2 variants, which is also why LAMP technology has attracted much attention. LAMP detection technology combined with lateral flow assay, microfluidic technology and other sensing technologies can effectively enhance signals by nucleic acid amplification and help to give the resulting output in a faster, more convenient and user-friendly way. At present, LAMP plays an important role in the detection of SARS-CoV-2.

Ultrafast Plasmonic Nucleic Acid Amplification and Real-Time Quantification for Decentralized Molecular Diagnostics.

Point-of-care real-time reverse-transcription polymerase chain reaction (RT-PCR) facilitates the widespread use of rapid, accurate, and cost-effective near-patient testing that is available to the public. Here, we report ultrafast plasmonic nucleic acid amplification and real-time quantification for decentralized molecular diagnostics. The plasmonic real-time RT-PCR system features an ultrafast plasmonic thermocycler (PTC), a disposable plastic-on-metal (PoM) cartridge, and an ultrathin microlens array fluorescence (MAF) microscope. The PTC provides ultrafast photothermal cycling under white-light-emitting diode illumination and precise temperature monitoring with an integrated resistance temperature detector. The PoM thin film cartridge allows rapid heat transfer as well as complete light blocking from the photothermal excitation source, resulting in real-time and highly efficient PCR quantification. Besides, the MAF microscope exhibits close-up and high-contrast fluorescence microscopic imaging. All of the systems were fully packaged in a palm size for point-of-care testing. The real-time RT-PCR system demonstrates the rapid diagnosis of coronavirus disease-19 RNA virus within 10 min and yields 95.6% of amplification efficiency, 96.6% of classification accuracy for preoperational test, and 91% of total percent agreement for clinical diagnostic test. The ultrafast and compact PCR system can decentralize point-of-care molecular diagnostic testing in primary care and developing countries.

Assessment of the clinical and analytical performance of the Aptima SARS-CoV-2 assay using the VALCOR protocol.

BACKGROUND: The COVID-19 pandemic highlighted the importance of diagnostic testing against curbing the spread of SARS-CoV-2. The urgent need and scale for diagnostic tools resulted in manufacturers of SARS-CoV-2 assays receiving emergency authorization that lacked robust analytical or clinical evaluation. As it is highly likely that testing for SARS-CoV-2 will continue to play a central role in public health, the performance characteristics of assays should be evaluated to ensure reliable diagnostic outcomes are achieved. METHODS: VALCOR or "VALidation of SARS-CORona Virus-2 assays" is a study protocol designed to set up a framework for test validation of SARS-CoV-2 virus assays. Using clinical samples collated from VALCOR, the performance of Aptima SARS-CoV-2 assay was assessed against a standard comparator assay. Diagnostic test parameters such as sensitivity, specificity and overall per cent agreement were calculated for the clinical performance of Aptima SARS-CoV-2 assay. RESULTS: A total of 180 clinical samples were tested with an addition of 40 diluted clinical specimens to determine the limit of detection. When compared to the standard comparator assay Aptima had a sensitivity of 100.0% [95% CI 95.9-100.0] and specificity of 96.7% [95% CI 90.8-99.3]. The overall percent agreement was 98.3% with an excellent Cohen's coefficient of κ = 0.967 [95% CI 0.929-1.000]. For the limit of detection, Aptima was able to detect all of the diluted clinical samples. CONCLUSION: In conclusion. validation of Aptima SARS-CoV-2 assay using clinical samples collated through the VALCOR protocol showed excellent test performance. Additionally, Aptima demonstrated high analytical sensitivity by detecting all diluted clinical samples corresponding to a low limit of detection.

Development of loop-mediated isothermal amplification (LAMP) assays using five primers reduces the false-positive rate in COVID-19 diagnosis.

The reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is a cheaper and faster testing alternative for detecting SARS-CoV-2. However, a high false-positive rate due to misamplification is one of the major limitations. To overcome misamplifications, we developed colorimetric and fluorometric RT-LAMP assays using five LAMP primers, instead of six. The gold-standard RT-PCR technique verified the assays' performance. Compared to other primer sets with six primers (N, S, and RdRp), the E-ID1 primer set, including five primers, performed superbly on both colorimetric and fluorometric assays. The sensitivity of colorimetric and fluorometric assays was 89.5% and 92.2%, respectively, with a limit of detection of 20 copies/µL. The colorimetric RT-LAMP had a specificity of 97.2% and an accuracy of 94.5%, while the fluorometric RT-LAMP obtained 99% and 96.7%, respectively. No misamplification was evident even after 120 min, which is crucial for the success of this technique. These findings are important to support the use of RT-LAMP in the healthcare systems in fighting COVID-19.

Forty Years of Molecular Diagnostics for Infectious Diseases.

Nearly 40 years have elapsed since the invention of the PCR, with its extremely sensitive and specific ability to detect nucleic acids via in vitro enzyme-mediated amplification. In turn, more than 2 years have passed since the onset of the coronavirus disease 2019 (COVID-19) pandemic, during which time molecular diagnostics for infectious diseases have assumed a larger global role than ever before. In this context, we review broadly the progression of molecular techniques in clinical microbiology, to their current prominence. Notably, these methods now entail both the detection and quantification of microbial nucleic acids, along with their sequence-based characterization. Overall, we seek to provide a combined perspective on the techniques themselves, as well as how they have come to shape health care at the intersection of technologic innovation, pathophysiologic knowledge, clinical/laboratory logistics, and even financial/regulatory factors.

Recent advances in point of care testing for COVID-19 detection.

The World Health Organizations declaration of the COVID-19 pandemic was a milestone for the scientific community. The high transmission rate and the huge number of deaths, along with the lack of knowledge about the virus and the evolution of the disease, stimulated a relentless search for diagnostic tests, treatments, and vaccines. The main challenges were the differential diagnosis of COVID-19 and the development of specific, rapid, and sensitive tests that could reach all people. RT-PCR remains the gold standard for diagnosing COVID-19. However, new methods, such as other molecular techniques and immunoassays emerged. Also, the need for accessible tests with quick results boosted the development of point of care tests (POCT) that are fast, and automated, with high precision and accuracy. This assay reduces the dependence on laboratory conditions and mass testing of the population, dispersing the pressure regarding screening and detection. This review summarizes the advances in the diagnostic field since the pandemic started, emphasizing various laboratory techniques for detecting COVID-19. We reviewed the main existing diagnostic methods, as well as POCT under development, starting with RT-PCR detection, but also exploring other nucleic acid techniques, such as digital PCR, loop-mediated isothermal amplification-based assay (RT-LAMP), clustered regularly interspaced short palindromic repeats (CRISPR), and next-generation sequencing (NGS), and immunoassay tests, and nanoparticle-based biosensors, developed as portable instruments for the rapid standard diagnosis of COVID-19.

Rapid differential diagnosis of SARS-CoV-2, influenza A/B and respiratory syncytial viruses: Validation of a novel RT-PCR assay.

BACKGROUND: Influenza and respiratory syncytial (RSV) viruses are expected to co-circulate with SARS-CoV-2 in the upcoming seasons and clinical differential diagnosis between them is difficult. Laboratory-based RT-PCR is a gold standard diagnostic method for influenza, RSV and SARS-CoV-2. The objective of this study was to estimate the diagnostic performance of a novel point-of-care RT-PCR assay STANDARD M10 Flu/RSV/SARS-CoV-2 (SD Biosensor) in a large number of clinical specimens with diversified (co)-infection patterns and viral loads. METHODS: This was a retrospective study, in which all samples were tested in both STANDARD M10 Flu/RSV/SARS-CoV-2 index and Allplex SARS-CoV-2/Respiratory Panel 1 (Seegene) reference kits. Samples with discordant results were further processed in a third resolver test (Resp-4-Plex, Abbott). RESULTS: A total of 1,019 naso-/oropharyngeal samples (50.3% positive for at least one virus) were processed in both STANDARD M10 Flu/RSV/SARS-CoV-2 and Allplex assays and the overall between-assay agreement was as high as 94.6%. Positive percent agreement of the STANDARD M10 Flu/RSV/SARS-CoV-2 was 100%, 96.6%, 97.3% and 99.4% for influenza A, B, RSV and SARS-CoV-2, respectively. The corresponding negative percent agreement was 99.7%. 100%, 100% and 98.4%, respectively. The expected positive and negative predictive values for all viruses were constantly above 96% in a reasonable range of disease prevalence. CONCLUSIONS: STANDARD M10 Flu/RSV/SARS-CoV-2 is a reliable RT-PCR assay able to detect influenza A, influenza B, RSV and SARS-CoV-2 in one hour or less, fostering a rapid differential diagnosis of common respiratory viruses.

Advances in RT-LAMP for COVID-19 testing and diagnosis.

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