A new SYBR Green real-time PCR to detect SARS-CoV-2

Marinowic, Daniel Rodrigo; Zanirati, Gabriele; Rodrigues, Fernanda Silva; Grahl, Matheus V. Coste; Alcará, Allan Marinho; Costa, Jaderson da

doi:10.1038/s41598-021-81245-0

Cited by 27 publications

(32 citation statements)

References 13 publications

(5 reference statements)

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“…The key advantages of intercalating dyes (particularly SYBR Green) are their wide availability, low cost, simplicity of use, wide range of thermocyclers capable of detecting them and the easy ordering of oligonucleotides without the requirement for labelled probes. While the use of SYBR Green-based alternatives has been previously described [ 10 , 15 , 16 ], here we show that this strategy could significantly expand the PCR testing capacities by applying it to a range of protocols and reagents that makes this a flexible and cheap alternative to current commercial kits. In addition, the chosen pair of primers used here anneal to sequences of the viral genome that present no mutations in the current variants of concern ( Fig.…”

Section: Discussionmentioning

confidence: 77%

A DNA intercalating dye-based RT-qPCR alternative to diagnose SARS-CoV-2

et al. 2021

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Early detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been proven crucial during the efforts to mitigate the effects of the COVID-19 pandemic. Several diagnostic methods have emerged in the past few months, each with different shortcomings and limitations. The current gold standard, RT-qPCR using fluorescent probes, relies on demanding equipment requirements plus the high costs of the probes and specific reaction mixes. To broaden the possibilities of reagents and thermocyclers that could be allocated towards this task, we have optimized an alternative strategy for RT-qPCR diagnosis. This is based on a widely used DNA-intercalating dye and can be implemented with several different qPCR reagents and instruments. Remarkably, the proposed qPCR method performs similarly to the broadly used TaqMan-based detection, in terms of specificity and sensitivity, thus representing a reliable tool. We think that, through enabling the use of vast range of thermocycler models and laboratory facilities for SARS-CoV-2 diagnosis, the alternative proposed here can increase dramatically the testing capability, especially in countries with limited access to costly technology and reagents.

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Section: Discussionmentioning

confidence: 77%

A DNA intercalating dye-based RT-qPCR alternative to diagnose SARS-CoV-2

et al. 2021

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“…Kit development and optimisation are ongoing processes all over the world and are ever more urgent as we face the rise and spread of new virus variants. Many different, upgraded commercial kits are available for the diagnosis of patients with SARS-CoV-2 [ 47 , 48 , 49 , 50 , 51 ]. As they significantly differ in the above-mentioned parameters, reporting row Ct values is very questionable, and Ct values cannot be directly compared between assays.…”

Section: Discussionmentioning

confidence: 99%

Comparison of SARS-CoV-2 Detection by Rapid Antigen and by Three Commercial RT-qPCR Tests: A Study from Martin University Hospital in Slovakia

Daňková

Nováková

Škereňová

et al. 2021

IJERPH

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The global pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is having a tremendous impact on the global economy, health care systems and the lives of almost all people in the world. The Central European country of Slovakia reached one of the highest daily mortality rates per 100,000 inhabitants in the first 3 months of 2021, despite implementing strong prophylactic measures, lockdowns and repeated nationwide antigen testing. The present study reports a comparison of the performance of the Standard Q COVID-19 antigen test (SD Biosensor) with three commercial RT-qPCR kits (vDetect COVID-19-MultiplexDX, gb SARS-CoV-2 Multiplex-GENERI BIOTECH Ltd. and Genvinset COVID-19 [E]-BDR Diagnostics) in the detection of infected individuals among employees of the Martin University Hospital in Slovakia. Health care providers, such as doctors and nurses, are classified as “critical infrastructure”, and there is no doubt about the huge impact that incorrect results could have on patients. Out of 1231 samples, 14 were evaluated as positive for SARS-CoV-2 antigen presence, and all of them were confirmed by RT-qPCR kit 1 and kit 2. As another 26 samples had a signal in the E gene, these 40 samples were re-isolated and subsequently re-analysed using the three kits, which detected the virus in 22, 23 and 12 cases, respectively. The results point to a divergence not only between antigen and RT-qPCR tests, but also within the “gold standard” RT-qPCR testing. Performance analysis of the diagnostic antigen test showed the positive predictive value (PPV) to be 100% and negative predictive value (NPV) to be 98.10%, indicating that 1.90% of individuals with a negative result were, in fact, positive. If these data are extrapolated to the national level, where the mean daily number of antigen tests was 250,000 in April 2021, it points to over 4700 people per day being misinterpreted and posing a risk of virus shedding. While mean Ct values of the samples that were both antigen and RT-qPCR positive were about 20 (kit 1: 20.47 and 20.16 for Sarbeco E and RdRP, kit 2: 19.37 and 19.99 for Sarbeco E and RdRP and kit 3: 17.47 for ORF1b/RdRP), mean Ct values of the samples that were antigen-negative but RT-qPCR-positive were about 30 (kit 1: 30.67 and 30.00 for Sarbeco E and RdRP, kit 2: 29.86 and 31.01 for Sarbeco E and RdRP and kit 3: 27.47 for ORF1b/RdRP). It confirms the advantage of antigen test in detecting the most infectious individuals with a higher viral load. However, the reporting of Ct values is still a matter of ongoing debates and should not be conducted without normalisation to standardised controls of known concentration.

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“…in May of 2020, there were 81 kits and systems approved by the US FDA. Several low-cost intercalating dye-based methods for SARS-CoV2 diagnosis were published to overcome the financial struggle and elevate the throughput of virus diagnostics [9,10]. In the case of SARS-CoV-2 PCR assays targeting ORF1a, ORF1b, S and N genes can detect less, than 10 genome equivalents [11].…”

Section: Introductionmentioning

confidence: 99%

A Practical Approach for Quantitative Polymerase Chain Reaction, the Gold Standard in Microbiological Diagnosis

Mosolygó¹,

Laczi²,

Spengler³

et al. 2021

Preprint

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From gene expression studies to identifying microbes quantitative polymerase chain reaction (qPCR) is widely used in research and medical diagnostics. In transmittable diseases like the Ebola outbreak in West Africa (2014-2016), or the present SARS-CoV2 pandemic qPCR plays a key role in the detection of infected patients. Although the technique itself is decades old with reliable approaches (eg. TaqMan essay) in the diagnosis of pathogens many people showed distrust in it during the SARS-CoV2 outbreak. This came mainly from not understanding or misunderstanding the principles of qPCR. This situation motivated us to design a simple laboratory practical class, in which students have opportunities to understand the underlying principles of qPCR and its advantages in microbiological diagnosis. Moreover, during the exercise, students can develop skills such as handling experimental assays, and the ability to solve problems, discuss their observations. Finally, this activity brings them closer to the clinical practice and they can see the impact of the science on real life. The class is addressed to undergraduate students of biological sciences.

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A new SYBR Green real-time PCR to detect SARS-CoV-2

Cited by 27 publications

References 13 publications

A DNA intercalating dye-based RT-qPCR alternative to diagnose SARS-CoV-2

A DNA intercalating dye-based RT-qPCR alternative to diagnose SARS-CoV-2

Comparison of SARS-CoV-2 Detection by Rapid Antigen and by Three Commercial RT-qPCR Tests: A Study from Martin University Hospital in Slovakia

A Practical Approach for Quantitative Polymerase Chain Reaction, the Gold Standard in Microbiological Diagnosis

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