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Diagnosing the novel SARS-CoV-2 by quantitative RT-PCR: variations and opportunities.

ABSTRACT: 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.

SUBMITTER: Barreto HG

PROVIDER: S-EPMC7567654 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Establishment of a quantitative RT-PCR detection of SARS-CoV-2 virus.

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

Seven discharged patients turning positive again for SARS-CoV-2 on quantitative RT-PCR.

Project description: Not available

| S-EPMC7151314 | biostudies-literature

A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits.

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

One-step quantitative RT-PCR assay with armored RNA controls for detection of SARS-CoV-2.

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

Sample pooling for SARS-CoV-2 RT-PCR screening.

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.

| S-EPMC7481316 | biostudies-literature

A novel ORF1a-based SARS-CoV-2 RT-PCR assay to resolve inconclusive samples.

Project description:BackgroundIndia bears the second largest burden of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. A multitude of reverse transcription polymerase chain reaction (RT-PCR) detection assays with disparate gene targets, including automated high-throughput platforms, are available. Varying concordance and interpretation of diagnostic results in this setting can result in significant reporting delays, leading to suboptimal disease management. This article reports the development of a novel ORF1a-based SARS-CoV-2 RT-PCR assay - Viroselect - that shows high concordance with conventional assays and the ability to resolve inconclusive results generated during the peak of the epidemic in Mumbai, India.MethodsA unique target region within SARS-CoV-2 ORF1a - the non-structural protein 3 (nsp3) region - was used to design and develop the assay. This hypervariable region (1923-3956) between SARS-CoV-2, SARS-CoV-1 and Middle East respiratory syndrome coronavirus was utilized to design the primers and probes for the RT-PCR assay. The concordance of this assay with commonly used emergency use authorization (US Food and Drug Administration) manual kits and an automated high-throughput testing platform was evaluated. Further, a retrospective analysis was carried out using Viroselect on samples reported as 'inconclusive' between April and October 2020.ResultsIn total, 701 samples were tested. Concordance analysis of 477 samples demonstrated high overall agreement of Viroselect with both manual (87.6%) and automated (84.7%) assays. Also, in the retrospective analysis of 224 additional samples reported as 'inconclusive', Viroselect was able to resolve 100% (19/19) and 93.7% (192/205) of samples which had inconclusive results on manual and automated high-throughput platforms, respectively.ConclusionViroselect had high concordance with conventional assays, both manual and automated, and has potential to resolve inconclusive samples.

| S-EPMC8036167 | biostudies-literature

Evaluation of Intra- and Interlaboratory Variations in SARS-CoV-2 Real-Time RT-PCR Through Nationwide Proficiency Testing.

Project description:ObjectiveThis study aimed to examine the intra- and interlaboratory variations of cycle threshold (Ct) values using the nationwide proficiency testing for SARS-CoV-2.MethodsTriplicated strong-positive contrived samples duplicated weak-positive contrived samples, and 2 negative samples were transported to participating laboratories in October 2021.ResultsA total of 232 laboratories responded. All except 4 laboratories correctly answered. Six false-negative results, including 2 false-negatives with Ct values beyond the threshold and 1 clerical error, were noted from weak-positive samples. Intralaboratory variations of Ct values of weak-positive and strong-positive samples were not acceptable (Ct > 1.66) in 17 and 7 laboratories, respectively. High interlaboratory variations of Ct values (up to 7 cycles) for the 2 commonly used polymerase chain reaction (PCR) reagents were observed.ConclusionThe overall qualitative performance was acceptable; intralaboratory variation was acceptable. However, interlaboratory variations of Ct values were remarkable even when the same PCR reagents were used.

| S-EPMC9214160 | biostudies-literature

Quantitative description of SARS-CoV-2 RT-PCR, a cohort of 76 COVID-19 older hospitalized adults.

Project description: Not available

| S-EPMC8250917 | biostudies-literature

Detection of SARS-CoV-2 RNA in Bivalve Mollusks by Droplet Digital RT-PCR (dd RT-PCR).

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.

| S-EPMC8776039 | biostudies-literature

Coronavirus antibody profiling in convalescent sera of RT-PCR SARS-CoV-2 confirmed hospitalised patients

Project description:Convalescent sera of RT-PCR SARS-CoV-2 confirmed hospitalised patients were tested on the protein array to profile IgG, IgM, and IgA antibody levels against human coronaviruses.

2022-03-08 | E-MTAB-11523 | biostudies-arrayexpress

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