Recent advances and challenges of RT-PCR tests for the diagnosis of COVID-19

Teymouri, Manoucher; Mollazadeh, Samaneh; Mortazavi, Hamed; Ghale-Noie, Zari Naderi; Keyvani, Vahideh; Aghababaei, Farzaneh; Hamblin, Michael R.; Abbaszadeh-Goudarzi, Ghasem; Pourghadamyari, Hossein; Hashemian, Seyed Mohammad Reza; Mirzaei, Hamed

doi:10.1016/j.prp.2021.153443

Cited by 144 publications

(110 citation statements)

References 54 publications

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“…While the detection of viral nucleic acid is a definitive indication of an infection, the infrastructure and personnel requirements, along with the time, cost, and discomfort of sample collection, are prohibitive for its implementation in the surveillance of large populations. Furthermore, studies suggest that false-negative test results can occur in up to 40% of swab and sputum specimens [ 10 ]. Serology-based diagnostics offer the distinct advantage that pathogen-specific antibodies are available for detection in any sample of blood.…”

Section: Introductionmentioning

confidence: 99%

Multiepitope Proteins for the Differential Detection of IgG Antibodies against RBD of the Spike Protein and Non-RBD Regions of SARS-CoV-2

et al. 2021

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The COVID-19 pandemic has exposed the extent of global connectivity and collective vulnerability to emerging diseases. From its suspected origins in Wuhan, China, it spread to all corners of the world in a matter of months. The absence of high-performance, rapid diagnostic methods that could identify asymptomatic carriers contributed to its worldwide transmission. Serological tests offer numerous benefits compared to other assay platforms to screen large populations. First-generation assays contain targets that represent proteins from SARS-CoV-2. While they could be quickly produced, each actually has a mixture of specific and non-specific epitopes that vary in their reactivity for antibodies. To generate the next generation of the assay, epitopes were identified in three SARS-Cov-2 proteins (S, N, and Orf3a) by SPOT synthesis analysis. After their similarity to other pathogen sequences was analyzed, 11 epitopes outside of the receptor-binding domain (RBD) of the spike protein that showed high reactivity and uniqueness to the virus. These were incorporated into a ß-barrel protein core to create a highly chimeric protein. Another de novo protein was designed that contained only epitopes in the RBD. In-house ELISAs suggest that both multiepitope proteins can serve as targets for high-performance diagnostic tests. Our approach to bioengineer chimeric proteins is highly amenable to other pathogens and immunological uses.

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Section: Introductionmentioning

confidence: 99%

Multiepitope Proteins for the Differential Detection of IgG Antibodies against RBD of the Spike Protein and Non-RBD Regions of SARS-CoV-2

et al. 2021

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“…Here, the benefit of decoupling analysis automation from peripheral software functions such as dashboarding, monitoring, and reporting allowed new tools to be deployed to automatically identify samples that potentially were affected due to proximity to highly positive wells and needed to be flagged for retest, maintaining TaT for unaffected samples. Now, well into the pandemic, decentralized testing initiatives, reinforcement of regional networks of clinical laboratories, installation of mobile laboratories [14] and point-ofcare testing [15,16], and the adoption of different laboratory technologies [15,[17][18][19] will allow to further reduce TaT, and recent initiatives will soon lead to turnaround times below the hour.…”

Section: Maintaining Tat With Growing Volumesmentioning

confidence: 99%

Reliable and Scalable SARS-CoV-2 qPCR Testing at a High Sample Throughput: Lessons Learned from the Belgian Initiative

Vooren¹,

Grayson²,

Ranst

et al. 2022

Life

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We present our approach to rapidly establishing a standardized, multi-site, nation-wide COVID-19 screening program in Belgium. Under auspices of a federal government Task Force responsible for upscaling the country’s testing capacity, we were able to set up a national testing initiative with readily available resources, putting in place a robust, validated, high-throughput, and decentralized qPCR molecular testing platform with embedded proficiency testing. We demonstrate how during an acute scarcity of equipment, kits, reagents, personnel, protective equipment, and sterile plastic supplies, we introduced an approach to rapidly build a reliable, validated, high-volume, high-confidence workflow based on heterogeneous instrumentation and diverse assays, assay components, and protocols. The workflow was set up with continuous quality control monitoring, tied together through a clinical-grade information management platform for automated data analysis, real-time result reporting across different participating sites, qc monitoring, and making result data available to the requesting physician and the patient. In this overview, we address challenges in optimizing high-throughput cross-laboratory workflows with minimal manual intervention through software, instrument and assay validation and standardization, and a process for harmonized result reporting and nation-level infection statistics monitoring across the disparate testing methodologies and workflows, necessitated by a rapid scale-up as a response to the pandemic.

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“…Polymerase chain reaction (PCR) is the most common technique for amplifying nucleic acids and has been proven to be an accurate method, with high specificity and sensitivity [ 3 , 4 , 5 ]. Once again, during the coronavirus disease 2019 (COVID-19) pandemic, PCR has proved to be the most reliable method for the early, rapid detection of COVID-19 [ 6 , 7 , 8 ]. However, PCR usually requires several hours for the amplification process using a thermal cycler, which makes it difficult to utilize its applications in resource-limited settings where the pandemic and burden of many infectious diseases are the most severe [ 9 ].…”

Section: Introductionmentioning

confidence: 99%

Fabrication of Wearable PDMS Device for Rapid Detection of Nucleic Acids via Recombinase Polymerase Amplification Operated by Human Body Heat

Trinh

Lee

2022

Biosensors

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Pathogen detection by nucleic acid amplification proved its significance during the current coronavirus disease 2019 (COVID-19) pandemic. The emergence of recombinase polymerase amplification (RPA) has enabled nucleic acid amplification in limited-resource conditions owing to the low operating temperatures around the human body. In this study, we fabricated a wearable RPA microdevice using poly(dimethylsiloxane) (PDMS), which can form soft—but tight—contact with human skin without external support during the body-heat-based reaction process. In particular, the curing agent ratio of PDMS was tuned to improve the flexibility and adhesion of the device for better contact with human skin, as well as to temporally bond the microdevice without requiring further surface modification steps. For PDMS characterization, water contact angle measurements and tests for flexibility, stretchability, bond strength, comfortability, and bendability were conducted to confirm the surface properties of the different mixing ratios of PDMS. By using human body heat, the wearable RPA microdevices were successfully applied to amplify 210 bp from Escherichia coli O157:H7 (E. coli O157:H7) and 203 bp from the DNA plasmid SARS-CoV-2 within 23 min. The limit of detection (LOD) was approximately 500 pg/reaction for genomic DNA template (E. coli O157:H7), and 600 fg/reaction for plasmid DNA template (SARS-CoV-2), based on gel electrophoresis. The wearable RPA microdevice could have a high impact on DNA amplification in instrument-free and resource-limited settings.

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Recent advances and challenges of RT-PCR tests for the diagnosis of COVID-19

Cited by 144 publications

References 54 publications

Multiepitope Proteins for the Differential Detection of IgG Antibodies against RBD of the Spike Protein and Non-RBD Regions of SARS-CoV-2

Multiepitope Proteins for the Differential Detection of IgG Antibodies against RBD of the Spike Protein and Non-RBD Regions of SARS-CoV-2

Reliable and Scalable SARS-CoV-2 qPCR Testing at a High Sample Throughput: Lessons Learned from the Belgian Initiative

Fabrication of Wearable PDMS Device for Rapid Detection of Nucleic Acids via Recombinase Polymerase Amplification Operated by Human Body Heat

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