scholarly journals Commercial stocks of SARS-CoV-2 RNA may report low concentration values, leading to artificially increased apparent sensitivity of diagnostic assays

2020 ◽  
Author(s):  
Erik Jue ◽  
Rustem F. Ismagilov
Keyword(s):  
Diagnostic Test ◽  
Single Molecule ◽  
Reverse Transcription ◽  
Plasmid Dna ◽  
Limit Of Detection ◽  
Analytical Sensitivity ◽  
Genomic Rna ◽  
Actual Concentration ◽  
Test Kit

AbstractIn response to the rapidly evolving COVID-19 pandemic, the U.S. Food and Drug Administration (FDA) has rapidly issued 49 emergency use authorizations (EUAs) for SARS-CoV-2 in vitro diagnostic test-kits. A critical metric in the performance evaluation for a diagnostic test kit is the analytical sensitivity, which is measured by the limit of detection (LOD). Commercial RNA stocks with known titers are used to determine LOD. We identified a problem with the titer reported for the commercial stocks when examining the analytical sensitivity of the reverse transcription quantitative PCR (RT-qPCR) protocol that is recommended by the Centers for Disease Control and Prevention (CDC) using plasmid DNA from Integrated DNA Technologies (IDT), synthetic RNA from BEI Resources (BEI), and extracted genomic RNA from BEI. We detected 3/3 positives for reactions containing synthetic RNA at a concentration of 0.1 copies/reaction (based on the supplier’s label concentration). The apparent better-than-single-molecule performance is a statistically highly unlikely event, indicating a potential inaccuracy in the supplier’s quantification of the stock material. Using an ultrasensitive and precise assay, reverse transcription digital PCR (RT-dPCR), we independently quantified concentrations of commercial SARS-CoV-2 plasmid DNA and SARS-CoV-2 RNA stocks. For plasmid DNA, the actual concentration measured by RT-dPCR was 11% of the nominal label concentration. For synthetic RNA, the actual concentration measured by RT-dPCR for one lot was 770% of the label concentration and for a different lot was 57% of the label concentration. For genomic RNA, the concentration measured by RT-dPCR for one lot was 240% of the label concentration and for a different lot it was 300% of the label concentration. This SARS-CoV-2 genomic RNA from BEI Resources has been used in at least 11 approved FDA Emergency Use Authorizations as of April 27, 2020. Such deviations of reported RNA or DNA stock concentrations from true concentrations can result in inaccurate quantification and calculation of LOD. Precise and accurate reporting of DNA and RNA stock concentrations by commercial suppliers will enable accurate quantification of assay performance, which is urgently needed to improve evaluation of different assays by diagnostic developers and regulatory bodies.

scholarly journals High resolution biosensor to test the capping level and integrity of mRNAs

2020 ◽  
Vol 48 (22) ◽  
pp. e129-e129
Author(s):  
Ignacio Moya-Ramírez ◽  
Clement Bouton ◽  
Cleo Kontoravdi ◽  
Karen Polizzi
Keyword(s):  
Single Molecule ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Chimeric Protein ◽  
Single Step ◽  
Quality Analysis ◽  
Eukaryotic Mrnas ◽  
In Vitro Capping

Abstract 5′ Cap structures are ubiquitous on eukaryotic mRNAs, essential for post-transcriptional processing, translation initiation and stability. Here we describe a biosensor designed to detect the presence of cap structures on mRNAs that is also sensitive to mRNA degradation, so uncapped or degraded mRNAs can be detected in a single step. The biosensor is based on a chimeric protein that combines the recognition and transduction roles in a single molecule. The main feature of this sensor is its simplicity, enabling semi-quantitative analyses of capping levels with minimal instrumentation. The biosensor was demonstrated to detect the capping level on several in vitro transcribed mRNAs. Its sensitivity and dynamic range remained constant with RNAs ranging in size from 250 nt to approximately 2700 nt and the biosensor was able to detect variations in the capping level in increments of at least 20%, with a limit of detection of 2.4 pmol. Remarkably, it also can be applied to more complex analytes, such mRNA vaccines and mRNAs transcribed in vivo. This biosensor is an innovative example of a technology able to detect analytically challenging structures such as mRNA caps. It could find application in a variety of scenarios, from quality analysis of mRNA-based products such as vaccines to optimization of in vitro capping reactions.

scholarly journals Fullerene Derivatives Prevent Packaging of Viral Genomic RNA into HIV-1 Particles by Binding Nucleocapsid Protein

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2451
Author(s):  
Ivana Křížová ◽  
Alžběta Dostálková ◽  
Edison Castro ◽  
Jan Prchal ◽  
Romana Hadravová ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Biological Activities ◽  
Reverse Transcriptase Activity ◽  
Genomic Rna ◽  
Rna Packaging ◽  
Fullerene Derivatives ◽  
Viral Genomic Rna ◽  
Anti Hiv ◽  
Hiv 1

Fullerene derivatives with hydrophilic substituents have been shown to exhibit a range of biological activities, including antiviral ones. For a long time, the anti-HIV activity of fullerene derivatives was believed to be due to their binding into the hydrophobic pocket of HIV-1 protease, thereby blocking its activity. Recent work, however, brought new evidence of a novel, protease-independent mechanism of fullerene derivatives’ action. We studied in more detail the mechanism of the anti-HIV-1 activity of N,N-dimethyl[70]fulleropyrrolidinium iodide fullerene derivatives. By using a combination of in vitro and cell-based approaches, we showed that these C70 derivatives inhibited neither HIV-1 protease nor HIV-1 maturation. Instead, our data indicate effects of fullerene C70 derivatives on viral genomic RNA packaging and HIV-1 cDNA synthesis during reverse transcription—without impairing reverse transcriptase activity though. Molecularly, this could be explained by a strong binding affinity of these fullerene derivatives to HIV-1 nucleocapsid domain, preventing its proper interaction with viral genomic RNA, thereby blocking reverse transcription and HIV-1 infectivity. Moreover, the fullerene derivatives’ oxidative activity and fluorescence quenching, which could be one of the reasons for the inconsistency among reported anti-HIV-1 mechanisms, are discussed herein.

scholarly journals Building-Level Wastewater Monitoring for COVID-19 Using Tampon Swabs and RT-LAMP for Rapid SARS-Cov-2 RNA Detection

2021 ◽  
Author(s):  
Aaron Bivins ◽  
Megan Lott ◽  
Marlee Shaffer ◽  
Zhenyu Wu ◽  
Devin North ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Predictive Value ◽  
Limit Of Detection ◽  
Cost Effective ◽  
Digital Pcr ◽  
Analytical Sensitivity ◽  
Clinical Testing ◽  
Predictive Capacity ◽  
Gene Copies ◽  
Building Level

Community-level wastewater monitoring for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA has demonstrated useful correlation with both coronavirus disease 2019 (COVID-19) case numbers and clinical testing positivity. Wastewater monitoring on college campuses has demonstrated promising predictive capacity for the presence and absence of COVID-19 cases. However, to date, such monitoring has largely relied upon composite or grab samples and reverse transcription quantitative PCR (RT-qPCR) techniques, which limits the accessibility and scalability of wastewater monitoring. In this study, we piloted a workflow that uses tampons as passive swabs for collection and reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect SARS-CoV-2 RNA in wastewater. Results for the developed workflow were available same day, with a time to result following tampon swab collection of approximately three hours. The RT-LAMP 95% limit of detection (76 gene copies reaction-1) was greater than RT-droplet digital PCR (ddPCR; 3.3 gene copies reaction-1). Nonetheless, during a building-level wastewater monitoring campaign conducted in the midst of weekly clinical testing of all students, the workflow demonstrated a same-day positive predictive value (PPV) of 33% and negative predictive value (NPV) of 80% for incident COVID-19 cases. The NPV is comparable to that reported by wastewater monitoring using RT-qPCR. These observations suggest that even with lower analytical sensitivity the tampon swab and RT-LAMP workflow offers a cost-effective and rapid approach that could be leveraged for scalable same-day building-level wastewater monitoring for COVID-19.

scholarly journals Approbation of RT-qPCR test kit for differential diagnosis of African and Classical swine fever

2018 ◽  
Vol 20 (83) ◽  
pp. 221-225
Author(s):  
S.S. Mandyhra ◽  
L.M. Muzykina ◽  
L.M. Ishchenko ◽  
G.A. Kovalenko ◽  
I.V. Halka ◽  
...  
Keyword(s):  
Differential Diagnosis ◽  
Veterinary Medicine ◽  
Internal Control ◽  
Limit Of Detection ◽  
Control Sample ◽  
Classical Swine Fever ◽  
Analytical Sensitivity ◽  
Test Kit ◽  
Field Samples ◽  
Sensitivity Specificity

The first Ukrainian real-time reverse-transcriptase polymerase chain reaction (RT-qPCR) based test kit for the differential diagnosis of African (AFS) and Classical swine fever (CSF) has been developed in the Institute of Veterinary Medicine of NAAS. The proposed test kit allows simultaneous detection of three targets: ASFV DNA, CSFV cDNA and an internal control sample. The goal of this work was to provide an expert evaluation of the RT-qPCR kit for differential diagnosis of ASF and CSF according to appearance, analytical sensitivity, specificity and repeatability. Interdepartmental evaluation of the kit was conducted in the State Scientific and Research Institute of Laboratory Diagnostics and Veterinary and Sanitary Expertise (DNDILDEVSE) in accordance with the approved methodology. The RT-qPCR kit sensitivity was determined by testing 10-fold serial dilutions of the ASFV DNA and CSFV cDNA (concentration range was 103–100 copies/μl). For specificity determination reference samples of ASFV DNA different genotypes, ASF and CSF positive and negative field samples, as well as pathogens which cause similar to ASF and CSF clinical syndromes were used. Sample preparation and amplification were performed according to the test kit instructions. The amplification was accomplished on QuantStudio™ 5 System (Applied Biosystems). As a result of accomplished interdepartmental evaluation high sensitivity, specificity and repeatability of RT-qPCR kit were confirmed. In particular, it was determined that the limit of detection of the RT-qPCR kit was 5 copies of the ASFV and CSFV genomes per one reaction. The high specificity of the assay to ASFV (I, II, V, VIII, IX and X genotypes) and CSFV was confirmed. It was showп no cross-reactions with closely related pigs viruses (porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and virus of Aujeszky's disease). The high enough repeatability of results was also confirmed. In conclusion, the obtained results are in compliance with the requirements of the RT-qPCR kit normative and technical documentation. This RT-qPCR kit will be recommended for use in veterinary medicine laboratories after its registration would be done.

scholarly journals Clinical Performance and Analytical Sensitivity of Three SARS-CoV-2 Nucleic Acid Diagnostic Tests

2021 ◽  
Vol 104 (4) ◽  
pp. 1516-1518
Author(s):  
Byron Freire-Paspuel ◽  
Miguel Angel Garcia-Bereguiain
Keyword(s):  
Nucleic Acid ◽  
Gold Standard ◽  
Limit Of Detection ◽  
Clinical Performance ◽  
Analytical Sensitivity ◽  
Nucleic Acid Test ◽  
Test Kit ◽  
Food Drug Administration ◽  
Made In China ◽  
Acid Test

ABSTRACTHundreds of RT-qPCR kits are available in the market for SARS-CoV-2 diagnosis, some of them with emergency use authorization (EUA) by the Food Drug Administration (FDA) or their country of origin agency, but also many of them without any independent clinical performance evaluation. We performed a clinical evaluation for two Chinese SARS-CoV-2 RT-PCR kits available in South America, COVID-19 Nucleic Acid Test Kit (eDiagnosis Biomedicine, Wuhan, China) and 2019-nCoV Nucleic Acid Diagnostic Kit (Sansure Biotech, Changsha, China), for RT-qPCR SARS-CoV-2 diagnosis using the FDA EUA 2019-nCoV CDC kit (IDT, Coralville, IA) as gold standard. We found an excellent clinical performance and analytical sensitivity for both kits with sensitivity values of 100% and 95.3% and estimated limit of detection of 500 copies/mL and 1,000 copies/mL, for eDiagnosis and Sansure Biotech kits, respectively. COVID-19 Nucleic Acid Test Kit (eDiagnosis) and 2019-nCoV Nucleic Acid Diagnostic Kit (Sansure Biotech) are both made in China and hold EUA by the Chinese CDC. Also, Sansure Biotech kit has EUA by the FDA. In conclusion, our results endorse the use of these two commercially available kits imported to Ecuador for SARS-CoV-2 diagnosis, as they had the similar clinical performance as the gold standard from the CDC.

scholarly journals Analytical Sensitivity of the Abbott BinaxNOW COVID-19 Ag Card

2020 ◽  
Vol 59 (3) ◽  
Author(s):  
Garrett A. Perchetti ◽  
Meei-Li Huang ◽  
Margaret G. Mills ◽  
Keith R. Jerome ◽  
Alexander L. Greninger
Keyword(s):  
Reverse Transcription ◽  
Limit Of Detection ◽  
Digital Pcr ◽  
Detection Methods ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Qrt Pcr ◽  
Viral Transport Medium ◽  
Antigen Tests ◽  
Antigen Testing

ABSTRACT Multiple rapid antigen (Ag) tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have recently received emergency-use authorization (EUA) from the U.S. Food and Drug Administration (FDA). Although less sensitive than molecular detection methods, rapid antigen testing offers the potential for inexpensive, quick, decentralized testing. Robust analytical sensitivity data in comparison to reverse transcription-quantitative PCR (qRT-PCR) are currently lacking for many rapid antigen tests. Here, we evaluated the analytical sensitivity of the Abbott BinaxNOW COVID-19 Ag card using SARS-CoV-2-positive clinical specimens quantified by reverse transcription-droplet digital PCR (RT-ddPCR) and multiple FDA EUA qRT-PCR platforms using RNA standards. Initial and confirmatory limits of detection for the BinaxNOW COVID-19 Ag card were determined to be equivalent to 4.04 × 104 to 8.06 × 104 copies/swab. We further confirmed this limit of detection with 72 additional clinical samples positive for SARS-CoV-2 in either phosphate-buffered saline or viral transport medium. One hundred percent of samples with viral loads of >40,000 copies/swab were detected by rapid antigen testing. These data indicate that the BinaxNOW COVID-19 Ag card has an analytical sensitivity approximately equivalent to a generic qRT-PCR cycle threshold (CT) value of 29 to 30.

Evaluation of biocompatibility and efficiency of plasmid DNA delivery by gene-activated hydrogels in vitro

2019 ◽  
Vol XIV (2) ◽  
Author(s):  
I.Y. Bozo ◽  
A.A. Titova ◽  
M.N. Zhuravleva ◽  
A.I. Bilyalov ◽  
M.O. Mavlikeev ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Dna Delivery

Detectability of Plasma Proteins in SRM Measurements

2018 ◽  
Vol 16 (1) ◽  
pp. 74-81 ◽  
Author(s):  
Olga I. Kiseleva ◽  
Elena A. Ponomarenko ◽  
Yulia A. Romashova ◽  
Ekaterina V. Poverennaya ◽  
Andrey V. Lisitsa
Keyword(s):  
Blood Plasma ◽  
Human Plasma ◽  
Limit Of Detection ◽  
Human Blood Plasma ◽  
Analytical Sensitivity ◽  
Plasma Proteome ◽  
Protein Targets ◽  
Blood Plasma Sample ◽  
Specificity And Sensitivity ◽  
Human Plasma Proteome

Background: Liquid chromatography coupled with targeted mass spectrometry underwent rapid technical evolution during last years and has become widely used technology in clinical laboratories. It offers confident specificity and sensitivity superior to those of traditional immunoassays. However, due to controversial reports on reproducibility of SRM measurements, the prospects of clinical appliance of the method are worth discussing. </P><P> Objective: The study was aimed at assessment of capabilities of SRM to achieve a thorough assembly of the human plasma proteome. </P><P> Method: We examined set of 19 human blood plasma samples to measure 100 proteins, including FDA-approved biomarkers, via SRM-assay. </P><P> Results: Out of 100 target proteins 43 proteins were confidently detected in at least two blood plasma sample runs, 36 and 21 proteins were either not detected in any run or inconsistently detected, respectively. Empiric dependences on protein detectability were derived to predict the number of biological samples required to detect with certainty a diagnostically relevant quantum of the human plasma proteome. </P><P> Conclusion: The number of samples exponentially increases with an increase in the number of protein targets, while proportionally decreasing to the logarithm of the limit of detection. Analytical sensitivity and enormous proteome heterogeneity are major bottlenecks of the human proteome exploration.

Sign in / Sign up

Export Citation Format

Share Document