Quantitative description of SARS-CoV-2 RT-PCR, a cohort of 76 COVID-19 older hospitalized adults.
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ABSTRACT:
SUBMITTER: Meyer M
PROVIDER: S-EPMC8250917 | biostudies-literature | 2021 May
REPOSITORIES: biostudies-literature
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Quantitative description of SARS-CoV-2 RT-PCR, a cohort of 76 COVID-19 older hospitalized adults.
Meyer Maxence M Meyer Anita A Calabrese Lidia L Constancias Florentin F Porter Louise F LF Muller Marion M Leitner Manon M Leitner Amandine A Michaud Antonin A Boussuge Alexandre A Kaltenbach Georges G Schmitt Elise E Karcher Patrick P Sauleau Erik E Chayer Saïd S Blanc Frédéric F Fafi-Kremer Samira S Velay Aurélie A Vogel Thomas T
Journal of the American Geriatrics Society 20210401 5
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Project description:BackgroundThe outbreak of novel coronavirus disease 2019 (COVID-19) has become a public health emergency of international concern. Quantitative testing of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) virus is demanded in evaluating the efficacy of antiviral drugs and vaccines and RT-PCR can be widely deployed in the clinical assay of viral loads. Here, we developed a quantitative RT-PCR method for SARS-CoV-2 virus detection in this study.MethodsRT-PCR kits targeting E (envelope) gene, N (nucleocapsid) gene and RdRP (RNA-dependent RNA polymerase) gene of SARS-CoV-2 from Roche Diagnostics were evaluated and E gene kit was employed for quantitative detection of COVID-19 virus using Cobas Z480. Viral load was calculated according to the standard curve established by series dilution of an E-gene RNA standard provided by Tib-Molbiol (a division of Roche Diagnostics). Assay performance was evaluated.ResultsThe performance of the assay is acceptable with limit of detection (LOD) below 10E1 copies/μL and lower limit of quantification (LLOQ) as 10E2 copies/μL.ConclusionA quantitative detection of the COVID-19 virus based on RT-PCR was established.
| S-EPMC8677905 | biostudies-literature
Project description:The world is currently facing a novel viral pandemic (SARS-CoV-2), and large-scale testing is central to decision-making for the design of effective policies and control strategies to minimize its impact on the global population. However, testing for the presence of the virus is a major bottleneck in tracking the spreading of the disease. Given its adaptability regarding the nucleotide sequence of target regions, RT-qPCR is a strong ally to reveal the rapid geographical spreading of novel viruses. We assessed PCR variations in the SARS-CoV-2 diagnosis taking into account public genome sequences and diagnosis kits used by different countries. We analyzed 226 SARS-CoV-2 genome sequences from samples collected by March 22, 2020. Our work utilizes a phylogenetic approach that reveals the early evolution of the virus sequence as it spreads around the globe and informs the design of RT-qPCR primers and probes. The quick expansion of testing capabilities of a country during a pandemic is largely impaired by the availability of adequately trained personnel on RNA isolation and PCR analysis, as well as the availability of hardware (thermocyclers). We propose that rapid capacity development can circumvent these bottlenecks by training medical and non-medical personnel with some laboratory experience, such as biology-related graduate students. Furthermore, the use of thermocyclers available in academic and commercial labs can be promptly calibrated and certified to properly conduct testing during a pandemic. A decentralized, fast-acting training and testing certification pipeline will better prepare us to manage future pandemics.
| S-EPMC7567654 | biostudies-literature
Project description: Not available
| S-EPMC7151314 | biostudies-literature
Project description:Most individuals acutely infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exhibit mild symptoms. However, 10 to 20% of those infected develop long-term symptoms, referred to as post-coronavirus disease 2019 (COVID-19) condition (PCC). One hypothesis is that PCC might be exacerbated by viral persistence in tissue sanctuaries. Therefore, the accurate detection and quantification of SARS-CoV-2 are not only necessary for viral load monitoring but also crucial for detecting long-term viral persistence and determining whether viral replication is occurring in tissue reservoirs. In this study, the sensitivity and robustness of reverse transcription (RT)-droplet digital PCR (ddPCR) and RT-quantitative PCR (qPCR) techniques have been compared for the detection and quantification of SARS-CoV-2 genomic and subgenomic RNAs from oropharyngeal swabs taken from confirmed SARS-CoV-2-positive, SARS-CoV-2-exposed, and nonexposed individuals as well as from samples from mice infected with SARS-CoV-2. Our data demonstrated that both techniques presented equivalent results in the mid- and high-viral-load ranges. Additionally, RT-ddPCR was more sensitive than RT-qPCR in the low-viral-load range, allowing the accurate detection of positive results in individuals exposed to the virus. Overall, these data suggest that RT-ddPCR might be an alternative to RT-qPCR for detecting low viral loads in samples and for assessing viral persistence in samples from individuals with PCC. IMPORTANCE We developed one-step reverse transcription (RT)-droplet digital PCR (ddPCR) protocols to detect SARS-CoV-2 RNA and compared them to the gold-standard RT-quantitative PCR (RT-qPCR) method. RT-ddPCR was more sensitive than RT-qPCR in the low-viral-load range, while both techniques were equivalent in the mid- and high-viral-load ranges. Overall, these results suggest that RT-ddPCR might be a viable alternative to RT-qPCR when it comes to detecting low viral loads in samples, which is a highly relevant issue for determining viral persistence in as-yet-unknown tissue reservoirs in individuals suffering from post-COVID conditions or long COVID.
| S-EPMC10100669 | biostudies-literature
Project description:Widespread testing for the presence of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise, and/or instrumentation necessary to detect the virus by quantitative RT-PCR (RT-qPCR), the most robust, sensitive, and specific assay currently available. Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can replicate commercially available SARS-CoV-2 RT-qPCR test kits and backfill pipeline shortages. The Georgia Tech COVID-19 Test Kit Support Group, composed of faculty, staff, and trainees across the biotechnology quad at Georgia Institute of Technology, synthesized multiplexed primers and probes and formulated a master mix composed of enzymes and proteins produced in-house. Our in-house kit compares favorably with a commercial product used for diagnostic testing. We also developed an environmental testing protocol to readily monitor surfaces for the presence of SARS-CoV-2. Our blueprint should be readily reproducible by research teams at other institutions, and our protocols may be modified and adapted to enable SARS-CoV-2 detection in more resource-limited settings.
| S-EPMC7667971 | biostudies-literature
Project description:Coronavirus disease 2019 (COVID-19) has become pandemic since March 11, 2020. Thus, development and integration in clinics of fast and sensitive diagnostic tools are essential. The aim of the study is a development and evaluation of a one-step quantitative reverse transcription-polymerase chain reaction (RT-qPCR) assay (COVID-19 Amp) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection with an armored positive control and internal controls constructed from synthetic MS2-phage-based RNA particles. The COVID-19 Amp assay limit of detection was 103 copies/ml, the analytical specificity was 100%. A total of 109 biological samples were examined using COVID-19 Amp and World Health Organization (WHO)-based assay. Discordance in nine samples was observed (negative by the WHO-based assay) and discordant samples were retested as positive according to the results obtained from the Vector-PCRrv-2019-nCoV-RG assay. The developed COVID-19 Amp assay has high sensitivity and specificity, includes virus particles-based controls, provides the direct definition of the SARS-CoV-2 RdRp gene partial sequence, and is suitable for any hospital and laboratory equipped for RT-qPCR.
| S-EPMC7537076 | biostudies-literature
Project description:ObjectiveTo evaluate the efficacy of sample pooling compared to the individual analysis for the diagnosis of coronavirus disease 2019 (COVID-19) by using different commercial platforms for nucleic acid extraction and amplification.MethodsA total of 3519 nasopharyngeal samples received at nine Spanish clinical microbiology laboratories were processed individually and in pools (342 pools of ten samples and 11 pools of nine samples) according to the existing methodology in place at each centre.ResultsWe found that 253 pools (2519 samples) were negative and 99 pools (990 samples) were positive; with 241 positive samples (6.85%), our pooling strategy would have saved 2167 PCR tests. For 29 pools (made out of 290 samples), we found discordant results when compared to their correspondent individual samples, as follows: in 22 of 29 pools (28 samples), minor discordances were found; for seven pools (7 samples), we found major discordances. Sensitivity, specificity and positive and negative predictive values for pooling were 97.10% (95% confidence interval (CI), 94.11-98.82), 100%, 100% and 99.79% (95% CI, 99.56-99.90) respectively; accuracy was 99.80% (95% CI, 99.59-99.92), and the kappa concordant coefficient was 0.984. The dilution of samples in our pooling strategy resulted in a median loss of 2.87 (95% CI, 2.46-3.28) cycle threshold (Ct) for E gene, 3.36 (95% CI, 2.89-3.85) Ct for the RdRP gene and 2.99 (95% CI, 2.56-3.43) Ct for the N gene.ConclusionsWe found a high efficiency of pooling strategies for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA testing across different RNA extraction and amplification platforms, with excellent performance in terms of sensitivity, specificity and positive and negative predictive values.
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Project description:Bivalve shellfish are readily contaminated by human pathogens present in waters impacted by municipal sewage, and the detection of SARS-CoV-2 in feces of infected patients and in wastewater has drawn attention to the possible presence of the virus in bivalves. The aim of this study was to collect data on SARS-CoV-2 prevalence in bivalve mollusks from harvesting areas of Campania region. A total of 179 samples were collected between September 2019 and April 2021 and were tested using droplet digital RT-PCR (dd RT-PCR) and real-time RT-PCR. Combining results obtained with different assays, SARS-CoV-2 presence was detected in 27/179 (15.1%) of samples. A median viral concentration of 1.1 × 102 and 1.4 × 102 g.c./g was obtained using either Orf1b nsp14 or RdRp/gene E, respectively. Positive results were unevenly distributed among harvesting areas and over time, positive samples being more frequent after January 2021. Partial sequencing of the spike region was achieved for five samples, one of which displaying mutations characteristic of the Alpha variant (lineage B.1.1.7). This study confirms that bivalve mollusks may bioaccumulate SARS-CoV-2 to detectable levels and that they may represent a valuable approach to track SARS-CoV-2 in water bodies and to monitor outbreak trends and viral diversity.
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Project description:Rising demands for repetitive SARS-CoV-2 screens and mass testing necessitate additional test strategies. Saliva may serve as an alternative to nasopharyngeal swab (NPS) as its collection is simple, non-invasive and amenable for mass- and home testing, but its rigorous validation, particularly in children, is missing. We conducted a large-scale head-to-head comparison of SARS-CoV-2 detection by RT-PCR in saliva and NPS of 1270 adults and children reporting to outpatient test centers and an emergency unit. In total, 273 individuals were tested positive for SARS-CoV-2 in either NPS or saliva. SARS-CoV-2 RT-PCR results in the two specimens showed a high agreement (overall percent agreement = 97.8%). Despite lower viral loads in the saliva of both adults and children, detection of SARS-CoV-2 in saliva fared well compared to NPS (positive percent agreement = 92.5%). Importantly, in children, SARS-CoV-2 infections were more often detected in saliva than NPS (positive predictive value = 84.8%), underlining that NPS sampling in children can be challenging. The comprehensive parallel analysis reported here establishes saliva as a generally reliable specimen for the detection of SARS-CoV-2, with particular advantages for testing children, that is readily applicable to increase and facilitate repetitive and mass testing in adults and children.
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Project description:Quantitative viral load assays have transformed our understanding of viral diseases. They hold similar potential to advance COVID-19 control and prevention, but SARS-CoV-2 viral load tests are not yet widely available. SARS-CoV-2 molecular diagnostic tests, which typically employ real-time RT-PCR, yield semiquantitative results only. Droplet digital RT-PCR (RT-ddPCR) offers an attractive platform for SARS-CoV-2 RNA quantification. Eight primer/probe sets originally developed for real-time RT-PCR-based SARS-CoV-2 diagnostic tests were evaluated for use in RT-ddPCR; three were identified as the most efficient, precise, and sensitive for RT-ddPCR-based SARS-CoV-2 RNA quantification. For example, the analytical efficiency for the E-Sarbeco primer/probe set was approximately 83%, whereas assay precision, measured as the coefficient of variation, was approximately 2% at 1000 input copies/reaction. Lower limits of quantification and detection for this primer/probe set were 18.6 and 4.4 input SARS-CoV-2 RNA copies/reaction, respectively. SARS-CoV-2 RNA viral loads in a convenience panel of 48 COVID-19-positive diagnostic specimens spanned a 6.2log10 range, confirming substantial viral load variation in vivo. RT-ddPCR-derived SARS-CoV-2 E gene copy numbers were further calibrated against cycle threshold values from a commercial real-time RT-PCR diagnostic platform. This log-linear relationship can be used to mathematically derive SARS-CoV-2 RNA copy numbers from cycle threshold values, allowing the wealth of available diagnostic test data to be harnessed to address foundational questions in SARS-CoV-2 biology.
| S-EPMC8164350 | biostudies-literature