Comparison of commercial RT-PCR diagnostic kits for COVID-19

ABSTRACTThe final months of 2019 witnessed the emergence of a novel coronavirus in the human population. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has since spread across the globe and is posing a major burden on society. Measures taken to reduce its spread critically depend on timely and accurate identification of virus-infected individuals by the most sensitive and specific method available, i.e. real-time reverse transcriptase PCR (RT-PCR). Many commercial kits have recently become available, but their performance has not yet been independently assessed.The aim of this study was to compare basic analytical and clinical performance of selected RT-PCR kits from seven different manufacturers (Altona Diagnostics, BGI, CerTest Biotec, KH Medical, PrimerDesign, R-Biopharm AG, and Seegene).We used serial dilutions of viral RNA to establish PCR efficiency and estimate the 95% limit of detection (LOD95%). Furthermore, we ran a panel of SARS-CoV-2-positive clinical samples (n=16) for a preliminary evaluation of clinical sensitivity. Finally, we used clinical samples positive for non-coronavirus respiratory viral infections (n=6) and a panel of RNA from related human coronaviruses to evaluate assay specificity.PCR efficiency was ≥96% for all assays and the estimated LOD95% varied within a 6-fold range. Using clinical samples, we observed some variations in detection rate between kits. Importantly, none of the assays showed cross-reactivity with other respiratory (corona)viruses, except as expected for the SARS-CoV-1 E-gene.We conclude that all RT-PCR kits assessed in this study may be used for routine diagnostics of COVID-19 in patients by experienced molecular diagnostic laboratories.

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Evaluation of accuracy, exclusivity, limit-of-detection and ease-of-use of LumiraDx™ - Antigen-detecting point-of-care device for SARS-CoV-2

10.1101/2021.03.02.21252430 ◽

2021 ◽

Author(s):

Lisa Johanna Krüger ◽

Julian A.F. Klein ◽

Frank Tobian ◽

Mary Gaeddert ◽

Federica Lainati ◽

...

Keyword(s):

Viral Load ◽

Limit Of Detection ◽

Point Of Care ◽

Scale Up ◽

Cross Reactivity ◽

Ease Of Use ◽

Analytical Sensitivity ◽

Rt Pcr ◽

Clinical Sensitivity ◽

Lateral Flow Assays

Background: Rapid antigen-detecting tests (Ag-RDTs) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transform pandemic control. Thus far, sensitivity (≤85%) of lateral-flow assays has limited scale-up. Conceivably, microfluidic immunofluorescence Ag-RDTs could increase sensitivity for SARS-CoV-2 detection. Materials and Methods: This multi-centre diagnostic accuracy study investigated performance of the microfluidic immunofluorescence LumiraDx™ assay, enrolling symptomatic and asymptomatic participants with suspected SARS-CoV-2 infection. Participants collected a supervised nasal mid-turbinate (NMT) self-swab for Ag-RDT testing, in addition to a professionally-collected nasopharyngeal (NP) swab for routine testing with reverse transcriptase polymerase chain reaction (RT-PCR). Results were compared to calculate sensitivity and specificity. Sub-analyses investigated the results by viral load, symptom presence and duration. An analytical study assessed exclusivity and limit-of-detection (LOD). In addition, we evaluated ease-of-use. Results: Study conduct was between November 2nd 2020 and January 21st 2021. 761 participants were enrolled, with 486 participants reporting symptoms on testing day. 120 out of 146 RT-PCR positive cases were detected positive by LumiraDx™, resulting in a sensitivity of 82.2% (95% CI: 75.2%-87.5%). Specificity was 99.3% (CI: 98.3-99.7%). Sensitivity was increased in individuals with viral load ≥ 7 log10 SARS-CoV2 RNA copies/ml (93.8%; CI: 86.2%-97.3%). Testing against common respiratory commensals and pathogens showed no cross-reactivity and LOD was estimated to be 2-56 PFU/mL. The ease-of-use-assessment was favourable for lower throughput settings. Conclusion: The LumiraDx™ assay showed excellent analytical sensitivity, exclusivity and clinical specificity with good clinical sensitivity using supervised NMT self-sampling.

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Evaluation of accuracy, exclusivity, limit-of-detection and ease-of-use of LumiraDx™: An antigen-detecting point-of-care device for SARS-CoV-2

Infection ◽

10.1007/s15010-021-01681-y ◽

2021 ◽

Author(s):

Lisa J. Krüger ◽

Julian A. F. Klein ◽

Frank Tobian ◽

Mary Gaeddert ◽

Federica Lainati ◽

...

Keyword(s):

Viral Load ◽

Limit Of Detection ◽

Point Of Care ◽

Scale Up ◽

Cross Reactivity ◽

Ease Of Use ◽

Analytical Sensitivity ◽

Rt Pcr ◽

Clinical Sensitivity ◽

Lateral Flow Assays

Abstract Purpose Rapid antigen-detecting tests (Ag-RDTs) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transform pandemic control. Thus far, sensitivity (≤ 85%) of lateral-flow assays has limited scale-up. Conceivably, microfluidic immunofluorescence Ag-RDTs could increase sensitivity for SARS-CoV-2 detection. Methods This multi-centre diagnostic accuracy study investigated performance of the microfluidic immunofluorescence LumiraDx™ assay, enrolling symptomatic and asymptomatic participants with suspected SARS-CoV-2 infection. Participants collected a supervised nasal mid-turbinate (NMT) self-swab for Ag-RDT testing, in addition to a professionally collected nasopharyngeal (NP) swab for routine testing with reverse transcriptase polymerase chain reaction (RT-PCR). Results were compared to calculate sensitivity and specificity. Sub-analyses investigated the results by viral load, symptom presence and duration. An analytical study assessed exclusivity and limit-of-detection (LOD). In addition, we evaluated ease-of-use. Results The study was conducted between November 2nd 2020 and 4th of December 2020. 761 participants were enrolled, with 486 participants reporting symptoms on testing day. 120 out of 146 RT-PCR positive cases were detected positive by LumiraDx™, resulting in a sensitivity of 82.2% (95% CI 75.2–87.5%). Specificity was 99.3% (CI 98.3–99.7%). Sensitivity was increased in individuals with viral load ≥ 7 log10 SARS-CoV2 RNA copies/ml (93.8%; CI 86.2–97.3%). Testing against common respiratory commensals and pathogens showed no cross-reactivity and LOD was estimated to be 2–56 PFU/mL. The ease-of-use-assessment was favourable for lower throughput settings. Conclusion The LumiraDx™ assay showed excellent analytical sensitivity, exclusivity and clinical specificity with good clinical sensitivity using supervised NMT self-sampling. Trial registration number and registration date DRKS00021220 and 01.04.2020

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Isothermal Recombinase Polymerase Amplification Assay Applied to the Detection of Group B Streptococci in Vaginal/Anal Samples

Clinical Chemistry ◽

10.1373/clinchem.2013.213504 ◽

2014 ◽

Vol 60 (4) ◽

pp. 660-666 ◽

Cited By ~ 30

Author(s):

Rana K Daher ◽

Gale Stewart ◽

Maurice Boissinot ◽

Michel G Bergeron

Keyword(s):

Limit Of Detection ◽

Positive Sample ◽

Recombinase Polymerase Amplification ◽

Molecular Diagnostic ◽

Rt Pcr ◽

Group B Streptococci ◽

Clinical Sensitivity ◽

Amplification Assay ◽

Group B ◽

Recombinase Polymerase Amplification Assay

Abstract BACKGROUND Group B streptococcal infections are the leading cause of sepsis and meningitis in newborns. A rapid and reliable method for the detection of this pathogen at the time of delivery is needed for the early treatment of neonates. Isothermal amplification techniques such as recombinase polymerase amplification have advantages relative to PCR in terms of the speed of reaction and simplicity. METHODS We studied the clinical performance of recombinase polymerase amplification for the screening of group B streptococci in vaginal/anal samples from 50 pregnant women. We also compared the limit of detection and the analytical specificity of this isothermal assay to real-time PCR (RT-PCR). RESULTS Compared to RT-PCR, the recombinase polymerase amplification assay showed a clinical sensitivity of 96% and a clinical specificity of 100%. The limit of detection was 98 genome copies and the analytical specificity was 100% for a panel of 15 bacterial and/or fungal strains naturally found in the vaginal/anal flora. Time-to-result for the recombinase polymerase amplification assay was <20 min compared to 45 min for the RT-PCR assay; a positive sample could be detected as early as 8 min. CONCLUSIONS We demonstrate the potential of isothermal recombinase polymerase amplification assay as a clinically useful molecular diagnostic tool that is simple and faster than PCR/RT-PCR. Recombinase polymerase amplification offers great potential for nucleic acid–based diagnostics at the point of care.

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Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays

PLoS ONE ◽

10.1371/journal.pone.0252687 ◽

2021 ◽

Vol 16 (6) ◽

pp. e0252687

Author(s):

Sukalyani Banik ◽

Kaheerman Saibire ◽

Shraddha Suryavanshi ◽

Glenn Johns ◽

Soumitesh Chakravorty ◽

...

Keyword(s):

Limit Of Detection ◽

Viral Rna ◽

Clinical Samples ◽

Sample Collection ◽

Rt Pcr ◽

Diagnostic Assays ◽

Guanidinium Thiocyanate ◽

Upper Respiratory ◽

Polymerase Chain ◽

The Stability

Background Upper respiratory samples used to test for SARS-CoV-2 virus may be infectious and present a hazard during transport and testing. A buffer with the ability to inactivate SARS-CoV-2 at the time of sample collection could simplify and expand testing for COVID-19 to non-conventional settings. Methods We evaluated a guanidium thiocyanate-based buffer, eNAT™ (Copan) as a possible transport and inactivation medium for downstream Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) testing to detect SARS-CoV-2. Inactivation of SARS-CoV-2 USA-WA1/2020 in eNAT and in diluted saliva was studied at different incubation times. The stability of viral RNA in eNAT was also evaluated for up to 7 days at room temperature (28°C), refrigerated conditions (4°C) and at 35°C. Results SARS-COV-2 virus spiked directly in eNAT could be inactivated at >5.6 log10 PFU/ml within a minute of incubation. When saliva was diluted 1:1 in eNAT, no cytopathic effect (CPE) on VeroE6 cells was observed, although SARS-CoV-2 RNA could be detected even after 30 min incubation and after two cell culture passages. A 1:2 (saliva:eNAT) dilution abrogated both CPE and detectable viral RNA after as little as 5 min incubation in eNAT. SARS-CoV-2 RNA from virus spiked at 5X the limit of detection remained positive up to 7 days of incubation in all tested conditions. Conclusion eNAT and similar guanidinium thiocyanate-based media may be of value for transport, stabilization, and processing of clinical samples for RT-PCR based SARS-CoV-2 detection.

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Microfluidic Nano-Scale qPCR Enables Ultra-Sensitive and Quantitative Detection of SARS-CoV-2

Processes ◽

10.3390/pr8111425 ◽

2020 ◽

Vol 8 (11) ◽

pp. 1425

Author(s):

Xin Xie ◽

Tamara Gjorgjieva ◽

Zaynoun Attieh ◽

Mame Massar Dieng ◽

Marc Arnoux ◽

...

Keyword(s):

Viral Infections ◽

Limit Of Detection ◽

False Negative ◽

False Negative Rate ◽

Clinical Samples ◽

Quantitative Detection ◽

Rt Pcr ◽

Viral Loads ◽

Nano Scale ◽

Negative Rate

A major challenge in controlling the COVID-19 pandemic is the high false-negative rate of the commonly used RT-PCR methods for SARS-CoV-2 detection in clinical samples. Accurate detection is particularly challenging in samples with low viral loads that are below the limit of detection (LoD) of standard one- or two-step RT-PCR methods. In this study, we implemented a three-step approach for SARS-CoV-2 detection and quantification that employs reverse transcription, targeted cDNA preamplification, and nano-scale qPCR based on a commercially available microfluidic chip. Using SARS-CoV-2 synthetic RNA and plasmid controls, we demonstrate that the addition of a preamplification step enhances the LoD of this microfluidic RT-qPCR by 1000-fold, enabling detection below 1 copy/µL. We applied this method to analyze 182 clinical NP swab samples previously diagnosed using a standard RT-qPCR protocol (91 positive, 91 negative) and demonstrate reproducible and quantitative detection of SARS-CoV-2 over five orders of magnitude (<1 to 106 viral copies/µL). Crucially, we detect SARS-CoV-2 with relatively low viral load estimates (<1 to 40 viral copies/µL) in 17 samples with negative clinical diagnosis, indicating a potential false-negative rate of 18.7% by clinical diagnostic procedures. In summary, this three-step nano-scale RT-qPCR method can robustly detect SARS-CoV-2 in samples with relatively low viral loads (<1 viral copy/µL) and has the potential to reduce the false-negative rate of standard RT-PCR-based diagnostic tests for SARS-CoV-2 and other viral infections.

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OC 8173 RAPID DETECTION OF MYCOBACTERIUM ULCERANS BY RECOMBINASE POLYMERASE AMPLIFICATION

BMJ Global Health ◽

10.1136/bmjgh-2019-edc.2 ◽

2019 ◽

Vol 4 (Suppl 3) ◽

pp. A2.1-A2

Author(s):

Michael Frimpong ◽

Hubert Ahor ◽

Francisca Sarpong ◽

Ken Laing ◽

Mark Wansbrough-Jones ◽

...

Keyword(s):

Sensitivity And Specificity ◽

Point Of Care ◽

Clinical Signs ◽

Buruli Ulcer ◽

Cross Reactivity ◽

Current Control ◽

Mycobacterium Ulcerans ◽

Recombinase Polymerase Amplification ◽

Clinical Samples ◽

Clinical Sensitivity

BackgroundThere are no primary measures to prevent people from contracting Buruli ulcer, mainly due to poor understanding of its epidemiology. The current control strategy emphasises early diagnosis and prompt treatment, with the goal of avoiding the complications associated with advanced stages of the disease. There is no diagnostic test for the disease appropriate for use at the primary health care level where most cases are detected and treated. Diagnosis based on clinical signs is unreliable in inexperienced hands and complicated by infections that have similar presentations. This study was to develop and evaluate the use of recombinase polymerase amplification (RPA) assay for the detection of Mycobacterium ulcerans at the point of patient care.MethodsA specific fragment of IS2404 of M. ulcerans was amplified in 15 min at a constant temperature of 42°C, using the RPA assay and analysed on a portable fluorometre. Themethod was tested for sensitivity and specificity with molecular standard of IS2404 DNA fragment, various M.ulcerans strains, other mycobacteria and environmentally associated bacteria. Additionally, the assay performance as a diagnostic tool was tested with archived DNA from symptomatic patients. All results were compared with that of a highly sensitive IS2404 PCR.ResultsThe detection limit was 50 copies of IS2404 in 15 min using plasmid standard and 125 fg with genomic Mu DNA equivalent 25 genomic copies. The assay was highly specific in detecting all strains of M. ulcerans with no observed cross reactivity with other mycobacteria and common skin colonising bacteria. The clinical sensitivity and specificity of the BU-RPA assay using clinical samples was 86% and 100% respectively.ConclusionWe have developed a real-time isothermal RPA assay for the detection of M. ulcerans as a cheaper alternative to PCR. Combining this assay with a simple extraction protocol will maximise its use as point-of-care test for Buruli ulcer.

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Rapid isothermal amplification and portable detection system for SARS-CoV-2

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2014739117 ◽

2020 ◽

Vol 117 (37) ◽

pp. 22727-22735 ◽

Cited By ~ 11

Author(s):

Anurup Ganguli ◽

Ariana Mostafa ◽

Jacob Berger ◽

Mehmet Y. Aydin ◽

Fu Sun ◽

...

Keyword(s):

Limit Of Detection ◽

Detection System ◽

Alternative Pathway ◽

Rna Extraction ◽

Lamp Assay ◽

Isothermal Amplification ◽

Clinical Samples ◽

Complete Agreement ◽

Rt Pcr ◽

Viral Transport Medium

The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay protocols and primer sequences become widely known, many laboratories perform diagnostic tests using methods such as RT-PCR or reverse transcription loop mediated isothermal amplification (RT-LAMP). Here, we report an RT-LAMP isothermal assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and demonstrate the assay on clinical samples using a simple and accessible point-of-care (POC) instrument. We characterized the assay by dipping swabs into synthetic nasal fluid spiked with the virus, moving the swab to viral transport medium (VTM), and sampling a volume of the VTM to perform the RT-LAMP assay without an RNA extraction kit. The assay has a limit of detection (LOD) of 50 RNA copies per μL in the VTM solution within 30 min. We further demonstrate our assay by detecting SARS-CoV-2 viruses from 20 clinical samples. Finally, we demonstrate a portable and real-time POC device to detect SARS-CoV-2 from VTM samples using an additively manufactured three-dimensional cartridge and a smartphone-based reader. The POC system was tested using 10 clinical samples, and was able to detect SARS-CoV-2 from these clinical samples by distinguishing positive samples from negative samples after 30 min. The POC tests are in complete agreement with RT-PCR controls. This work demonstrates an alternative pathway for SARS-CoV-2 diagnostics that does not require conventional laboratory infrastructure, in settings where diagnosis is required at the point of sample collection.

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Defining the analytical and clinical sensitivity of the ARTIC method for the detection of SARS-CoV-2

10.1101/2021.10.09.21264695 ◽

2021 ◽

Author(s):

Nabil-Fareed Alikhan ◽

Joshua Quick ◽

Alexander J. Trotter ◽

Samuel C. Robson ◽

Matthew Bashton ◽

...

Keyword(s):

Performance Metrics ◽

Limit Of Detection ◽

Diagnostic Testing ◽

Virus Detection ◽

Detection Methods ◽

Clinical Samples ◽

Sequencing Data ◽

Clinical Sensitivity ◽

Diagnostic Assays ◽

Sequencing Method

The SARS-CoV-2 ARTIC amplicon protocol is the most widely used genome sequencing method for SARS-CoV-2, accounting for over 43% of publicly-available genome sequences. The protocol utilises 98 primers to amplify ~400bp fragments of the SARS-CoV-2 genome covering all 30,000 bases. Understanding the analytical performance metrics of this protocol will improve how the data is used and interpreted. Different concentrations of SARS-CoV-2 control material were used to establish the limit of detection (LoD) of the ARTIC protocol. Results demonstrated the LoD was a minimum of 25-50 virus particles per mL. The sensitivity of ARTIC was comparable to the published sensitivities of commercial diagnostics assays and could therefore be used to confirm diagnostic testing results. A set of over 3,600 clinical samples from three UK regions were then evaluated to compare the protocols performance to clinical diagnostic assays (Roche Lightcycler 480 II, AusDiagnostics, Roche Cobas, Hologic Panther, Corman RdRp, Roche Flow, ABI QuantStudio 5, Seegene Nimbus, Qiagen Rotorgene, Abbott M2000, Thermo TaqPath, Xpert). We developed a Python tool, RonaLDO, to perform this validation (available under the GNU GPL3 open-source licence from https://github.com/quadram-institute-bioscience/ronaldo). Positives detected by diagnostic platforms were generally supported by sequencing data; platforms that used RT-qPCR were the best predictors of whether the sample would subsequently sequence successfully. To maximise success of sample sequencing for phylogenetic analysis, samples with Ct <31 should be chosen. For diagnostic tests that do not provide a quantifiable Ct value, adding a quantification step is recommended. The ARTIC SARS-CoV-2 sequencing protocol is highly sensitive, capable of detecting SARS-CoV-2 in samples with Cts in the high 30s. However, to routinely obtain whole genome coverage, samples with Ct <31 are recommended. Comparing different virus detection methods close to their LoD was challenging and significant discordance was observed.

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Single Tube qPCR detection and quantitation of hotspot mutations down to 0.01% VAF

10.1101/2021.08.12.456178 ◽

2021 ◽

Author(s):

Kerou Zhang ◽

Luis Rodriguez ◽

Lauren Yuxuan Cheng ◽

David Yu Zhang

Keyword(s):

Rare Variants ◽

Limit Of Detection ◽

Digital Pcr ◽

Clinical Samples ◽

Peripheral Blood Mononuclear ◽

Clinical Sensitivity ◽

Sequencing Technologies ◽

Median Error ◽

Allele Specific ◽

Dna Sequence Variants

Clinically and biologically, rare DNA sequence variants are significant and informative. However, existing detection technologies are either complex in workflow, or restricted in the limit of detection (LoD), or do not allow for multiplexing. Blocker displacement amplification (BDA) method can stably and effectively detect and enrich multiple rare variants with LoD around 0.1% variant allele fraction (VAF). Nonetheless, the detailed mutation information has to be identified by additional sequencing technologies. Here, we present allele-specific BDA (As-BDA), a method combining BDA with allele-specific TaqMan (As-TaqMan) probes for effective variant enrichment and simultaneous SNV profiling. We demonstrated that As-BDA could detect mutations down to 0.01% VAF. Further, As-BDA could detect up to four mutations with low to 0.1% VAF per reaction using only 15 ng DNA input. The median error of As-BDA in VAF determination is approximately 9.1%. Comparison experiments using As-BDA and droplet digital PCR (ddPCR) on peripheral blood mononuclear cell (PBMC) clinical samples showed 100% concordance for samples with mutations at 3 0.1% VAF. Hence, we have shown that As-BDA can achieve simultaneous enrichment and identification of multiple targeted mutations within the same reaction with high clinical sensitivity and specificity, thus helpful for clinical diagnosis.

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Performance evaluation of the point-of-care SAMBA II SARS-CoV-2 Test for detection of SARS-CoV-2

10.1101/2020.05.24.20100990 ◽

2020 ◽

Cited By ~ 4

Author(s):

Sonny M Assennato ◽

Allyson V Ritchie ◽

Cesar Nadala ◽

Neha Goel ◽

Hongyi Zhang ◽

...

Keyword(s):

Public Health ◽

Infection Control ◽

Sensitivity And Specificity ◽

Patient Management ◽

Limit Of Detection ◽

Point Of Care ◽

Clinical Samples ◽

Analytical Sensitivity ◽

Point Of Care Testing ◽

Clinical Sensitivity

AbstractNucleic acid amplification for the detection of SARS-CoV-2 RNA in respiratory samples is the standard method for diagnosis. These tests are centralised and therefore turnaround times can be 2-5 days. Point-of-care testing with rapid turnaround times would allow more effective triage in settings where patient management and infection control decisions need to be made rapidly.Inclusivity and specificity of the SAMBA II SARS-CoV-2 assay was determined by in silico analyses of the primers and probes. Analytical and clinical sensitivity and specificity of the SAMBA II SARS-CoV-2 Test was evaluated for analytical sensitivity and specificity. Clinical performance was evaluated in residual clinical samples compared to the Public Health England reference tests.The limit of detection of the SAMBA II SARS-CoV-2 Test is 250 cp/mL and is specific for detection of 2 regions of the SARS-CoV-2 genome. The clinical sensitivity was evaluated in 172 clinical samples provided by the Clinical Microbiology and Public Health Laboratory, Addenbrooke’s Hospital, Cambridge (CMPHL), which showed a sensitivity of 98.9% (95% CI 94.03-99.97%), specificity of 100% (95% CI 95.55-100%), PPV of 100% and NPV of 98.78% (92.02-99.82%) compared to testing by CMPHLSAMBA detected 3 positive samples that were initially negative by PHE Test. The data shows that the SAMBA II SARS-CoV-2 Test performs equivalently to the centralised testing methods with a much quicker turnaround time. Point of care testing, such as SAMBA, should enable rapid patient management and effective implementation of infection control measures.

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