Strategies That Facilitate Extraction-Free SARS-CoV-2 Nucleic Acid Amplification Tests

Delgado-Diaz, David Jose; Sakthivel, Dhanasekaran; Nguyen, Hanh Thi Ngoc; Farrokzhad, Khashayar; Hopper, William; Narh, Charles A.; Richards, James L.

doi:10.3390/v14061311

Cited by 9 publications

(8 citation statements)

References 41 publications

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“…Regardless of the chosen diagnostic method, the establishment of reliable protocols to produce enzymes with high yield and activity that are not covered by patents is essential to avoid the worldwide reagent shortage problems that happened during the COVID-19 pandemic. [30][31][32] This learned lesson may be crucial to deal with future threats of new pandemics, such as other respiratory and tropical diseases caused by infectious agents. 33,34 Another important lesson learned from the pandemic is the need to decentralize test performance, allowing for both faster diagnosis and access to remotely populated areas.…”

Section: Impact Statementmentioning

confidence: 99%

Improved protocol for Bst polymerase and reverse transcriptase production and application to a point-of-care diagnostics system

de Souza,

Silva,

Celis-Silva

et al. 2023

Exp Biol Med (Maywood)

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The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has raised awareness in the scientific community about the importance of being prepared for sanitary emergencies. Many measures implemented during the COVID pandemic are now being expanded to other applications. In the field of molecular and immunological diagnostics, the need to massively test the population worldwide resulted in the application of a variety of methods to detect viral infection. Besides gold standard reverse transcription quantitative polymerase chain reaction (RT-qPCR), the use of reverse transcription loop-mediated isothermal amplification (RT-LAMP) arose as an alternative and sensitive method to amplify and detect viral genetic material. We have used openly available protocols and have improved the protein production of RT-LAMP enzymes Bst polymerase and HIV-reverse transcriptase. To optimize enzyme production, we tested different protein tags, and we shortened the protein purification protocol, resulting in reduced processing time and handling of the enzymes and, thus, preserved the protein activity with high purity. The enzymes showed significant stability at 4 °C and 25 °C, over 60 days, and were highly reliable when used as a one-step RT-LAMP reaction in a portable point-of-care device with clinical samples. The enzymes and the reaction setup can be further expanded to detect other infectious diseases agents.

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Section: Impact Statementmentioning

confidence: 99%

Improved protocol for Bst polymerase and reverse transcriptase production and application to a point-of-care diagnostics system

de Souza,

Silva,

Celis-Silva

et al. 2023

Exp Biol Med (Maywood)

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“…This happened all around the world [ 15 ] but specially affected developing countries and areas with less infrastructure adaptation and capacity planning [ 16 ]. Therefore, alternatives were required to surmount supply chain limitations [ 17 , 18 ], as well as the shortage of healthcare professionals [ 19 ]. Despite huge efforts, alternatives are still required to replace commercial kits, lowering their cost if necessary, and allowing the scientific community to support high-throughput screenings.…”

Section: Introductionmentioning

confidence: 99%

A Highly Sensitive Immunoassay for Determination of Immune Response to SARS-CoV-2 in Capillary Blood Samples

et al. 2022

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Throughout the pandemic, serological assays have been revealed as crucial for detecting previous exposures to the virus and determining the timing of antibody maintenance after vaccination or natural infection. This study aimed to develop an optimized enzyme-linked immunosorbent assay (ELISA)-based serology, which could be used in case of reagent shortages, such as that occurred in the beginning of this health emergency. As a result, we present a high-sensitive immunoassay for the determination of IgG levels in venous serum samples, using 2 μg/mL antigen (receptor-binding domain of the spike protein S1) for coating the plate and utilizing human samples at a dilution 1:1000. This method showed non-inferiority features versus a commercial kit, is less expensive, and has a higher spectrophotometric range that allows for a better quantification of the antibody titers. The optical density values before and after heating venous serum samples at 56 °C during 30 min was quite similar, showing that heat inactivation can be used to reduce the biohazardous risks while handling samples. Furthermore, we show that finger-stick capillary blood samples can also serve as a suitable source for IgG detection, bypassing the need for serum isolation and being suitable for point-of-care application (Pearson’s coefficient correlation with capillary serum was 0.95, being statistically significant).

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“…To date, more than 200 SARS-CoV-2 molecular assays have been granted emergency use authorization by the US Food and Drug Administration, the vast majority of which require viral RNA extraction from clinical specimens prior to amplification [6]. RNA extraction is crucial to remove potential inhibitors that may interfere with viral target gene amplification; however, conventional RNA extraction processes consisting of lysis and purification steps are time-consuming and costly [3,4]. Recently, streamlined RNA extraction methods, such as treatment with proteinase K or heat treatment, have been suggested as alternatives to conventional RNA extraction methods [3,[5][6][7][8][9][10][11][12][13][14][15][16][17][18].…”

mentioning

confidence: 99%

“…Rapid and accurate diagnosis is necessary to contain the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and molecular assays based on real-time reverse transcription (rRT)-PCR are currently considered the reference standard for the diagnosis of SARS-CoV-2 infection [ 1 , 2 ]. These assays generally require a time-consuming and costly RNA extraction step, which remains a major bottleneck in the diagnosis of SARS-CoV-2 infection [ 3 , 4 ]. Furthermore, the unprecedented demand for SARS-CoV-2 molecular assays has led to a global shortage of RNA extraction kits [ 5 ].…”

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confidence: 99%

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Evaluation of the AdvanSure One-Stop COVID-19 Plus Kit for SARS-CoV-2 Detection Using a Streamlined RNA Extraction Method

et al. 2023

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Real-time reverse transcription (rRT)-PCR, which is the reference standard for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, generally involves a time-consuming and costly RNA extraction step prior to amplification. We evaluated the performance of the AdvanSure One-Stop COVID-19 Plus Kit (LG Chem, Seoul, Korea), a novel rRT-PCR assay that can detect SARS-CoV-2 within 90 minutes using a streamlined RNA extraction method. In total, 509 nasopharyngeal swab (NPS) specimens (SARS-CoV-2 positive: N = 205; SARS-CoV-2 negative: N = 304) previously tested using the PowerChek SARS-CoV-2 Real-time PCR Kit (Kogene Biotech, Seoul, Korea) were tested using the AdvanSure assay. The limit of detection (LOD) of the AdvanSure assay was determined using serially diluted inactivated SARS-CoV-2. The positive and negative percent agreements between the AdvanSure and PowerChek assays were 99.5% (204/205) and 99.3% (302/304), respectively. The LODs of the AdvanSure assay for SARS-CoV-2 nucleocapsid and spike/RNA-dependent RNA polymerase genes were 672 and 846 copies/mL, respectively. The results show that the performance of the AdvanSure assay is comparable to that of the PowerChek assay used for routine SARS-CoV-2 testing, suggesting that the AdvanSure assay is a useful diagnostic tool for rapid and accurate detection of SARS-CoV-2 infection.

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Strategies That Facilitate Extraction-Free SARS-CoV-2 Nucleic Acid Amplification Tests

Cited by 9 publications

References 41 publications

Improved protocol for Bst polymerase and reverse transcriptase production and application to a point-of-care diagnostics system

Improved protocol for Bst polymerase and reverse transcriptase production and application to a point-of-care diagnostics system

A Highly Sensitive Immunoassay for Determination of Immune Response to SARS-CoV-2 in Capillary Blood Samples

Evaluation of the AdvanSure One-Stop COVID-19 Plus Kit for SARS-CoV-2 Detection Using a Streamlined RNA Extraction Method

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