Fast SARS-CoV-2 detection protocol based on RNA precipitation and RT-qPCR in nasopharyngeal swab samples

Guruceaga, X; Sierra, Alejandro Serrano; Santín, Izortze; Nieto-Garai, Jon Ander; Bilbao, José Ramón; Lorizate, Maier; Aspichueta, Patricia; Gaminde-Blasco, A; Bozal-Leorri, A; Marín-Peña, A; Castellanos–Rubio, Ainara; Iglesias-Ara, A; Rementeria, Alexandra de; Bernal-Chico, A; Ramírez-García, Almudena; Olazagoitia-Garmendia, Ane; Benito-Vicente, A; Martín, Claudia Sánchez San; Eb, Castellanos; Rueda-Alaña, E; Cavaliere, Franco; Gp, de Nanclares; Aurrekoetxea, Igor; García-Santisteban, Iraia; Bernales, Irantzu; Gonzalez-Moro, I; Tønnesen, J; Baleriola, J; Vallejo-Rodríguez, J; Aparicio-Fernandez, L; Martin-Souto, L; Lm, Mendoza; Areitio, M; Ortiz-Zarragoitia, M; Serrano‐Regal, Mari Paz; Basaras, Miren; Fernandez-Jimenez, N; Pampliega, O; Sa, Alegre; Marcos, Saioa Zalba; Fuertes-Mendizabal, T; Galicia-Garcia, U; Perez-Cuesta, U; Torrano, Verónica; Sierra-Torre,; Buqué, X; U, Mayor

doi:10.1101/2020.04.26.20081307

Cited by 9 publications

(11 citation statements)

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“…A shortage of commercial RNA extraction kits has motivated many laboratories to find in-house extraction methods: such as Trizol-based purification [18] and RNA precipitation with isopropanol. [19] At least four groups have shown that the RNA extraction step can be omitted, yielding results with minimal change in sensitivity. [20] Reverse transcription and amplification: Reverse transcription is a standard step that uses the enyzme reverse transcriptase to convert RNA into complementary DNA.…”

Section: Overview Of the Genetic Testing Protocols And Innovationsmentioning

confidence: 99%

Trends and Innovations in Biosensors for COVID‐19 Mass Testing

2020

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Fast and widespread diagnosis is crucial to fighting against the outbreak of COVID-19. This work surveys the landscape of available and emerging biosensor technologies for COVID-19 testing. Molecular diagnostic assays based on quantitative reverse transcription polymerase chain reaction are used in most clinical laboratories. However, the COVID-19 pandemic has overwhelmed testing capacity and motivated the development of fast point-of-care tests and the adoption of isothermal DNA amplification. Antigenic and serological rapid tests based on lateral-flow immunoassays suffer from low sensitivity. Advanced digital systems enhance performance at the expense of speed and the need for large equipment. Emerging technologies, including CRISPR gene-editing tools, benefit from high sensitivity and specificity of molecular diagnostics and the easy use of lateral-flow assays. DNA sequencing and sample pooling strategies are highlighted to bring out the full capacity of the available biosensor technologies and accelerate mass testing.

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Section: Overview Of the Genetic Testing Protocols And Innovationsmentioning

confidence: 99%

Trends and Innovations in Biosensors for COVID‐19 Mass Testing

2020

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“…Trizol extraction followed by isopropanol precipitation provides a high yield of purified RNA [5], however, it requires extensive labor, is difficult to scale to high-throughput, and involves hazardous materials. Simpler isopropanol precipitation methods, in which patient swab samples are first mixed with commercial or homemade lysis solutions, have been reported to give Ct values comparable to those obtained using commercial RNA purification kits [6][7][8].…”

Section: Introductionmentioning

confidence: 99%

Inexpensive, versatile and open-source methods for SARS-CoV-2 detection

Graham

Dugast-Darzacq

Dailey

et al. 2020

Preprint

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Re-opening of communities in the midst of the ongoing COVID-19 pandemic has ignited a second wave of infections in many places around the world. Mitigating the risk of reopening will require widespread SARS-CoV-2 testing, which would be greatly facilitated by simple, rapid, and inexpensive testing methods. To this end, we evaluated several protocols for RNA extraction and RT-qPCR that are simpler and less expensive than prevailing methods. First, we show that isopropanol precipitation provides an effective means of RNA extraction from nasopharyngeal (NP) swab samples. Second, we evaluate direct addition of NP swab samples to RT-qPCR reactions without an RNA extraction step. We describe a simple, inexpensive swab collection solution suitable for direct addition, which we validate using contrived swab samples. Third, we describe an open-source master mix for RT-qPCR and show that it permits detection of viral RNA in NP swab samples. Lastly, we show that an end-point fluorescence measurement provides an accurate diagnostic readout without requiring a qPCR thermocycler. Adoption of these simple, inexpensive methods has the potential to significantly reduce the time and expense of COVID-19 testing.

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“…A two-step testing procedure using primer sets targeting the E gene for initial screening followed by the RdRp gene to confirm positive samples is recommended by the German Consiliary Laboratory for Coronaviruses [ 2 ]. The unprecedented global demand for commercial RNA extraction kits and ensuing shortage of these reagents [ 3 ] led to the establishment of several diagnostic workflows performed on patient samples with or without an intermediate RNA extraction step [ 4 , 5 , 6 , 7 ]. Viral RNA isolation from clinical samples depends on the rapid inactivation of viral particles, typically by detergent solubilization, and on the denaturation of omnipresent RNases [ 8 ].…”

Section: Introductionmentioning

confidence: 99%

SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP

Klein

Müller

Khalid

et al. 2020

Viruses

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Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits, followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and, alternatively, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) using a primer set targeting the N gene, as well as RT-qPCR using a primer set targeting the E gene, showing that the RNA extraction protocol presented here can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.

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Fast SARS-CoV-2 detection protocol based on RNA precipitation and RT-qPCR in nasopharyngeal swab samples

Cited by 9 publications

References 0 publications

Trends and Innovations in Biosensors for COVID‐19 Mass Testing

Trends and Innovations in Biosensors for COVID‐19 Mass Testing

Inexpensive, versatile and open-source methods for SARS-CoV-2 detection

SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP

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