Phenol-chloroform-based RNA purification for detection of SARS-CoV-2 by RT-qPCR: comparison with automated systems

Dimke, Henrik; Larsen, Sanne Løkkegaard; Skov, Marianne Nielsine; Larsen, Hanne; Hartmeyer, Gitte Nyvang; Møeller, Jesper Bonnet

doi:10.1101/2020.05.26.20099440

Cited by 6 publications

(6 citation statements)

References 14 publications

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“…During this study, Isohelix swabs were selected over Copan swabs due to easier handling and higher sensitivity for sample collection, and this approach has been successfully used by other studies (12). Furthermore, our results demonstrated that automated RNA extraction was as efficient (13, 14) as manual kits for extracting synthetic SARS-CoV-2, which has been previously noted using phenol-chloroform (15).…”

Section: Discussionsupporting

confidence: 72%

End-to-End Protocol for the Detection of SARS-CoV-2 from Built Environments

Parker

Singh

Tighe

et al. 2020

Preprint

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019, is a respiratory virus primarily transmitted from person to person through inhalation of droplets or aerosols, laden with viral particles. However, as some studies have shown, virions can remain infectious for up to 72 hours on surfaces, which can lead to transmission through contact. For this reason, a comprehensive study was conducted to determine the efficiency of protocols to recover SARS-CoV-2 from surfaces in built environments. This end-to-end (E2E) study showed that the effective combination of monitoring SARS-CoV-2 on surfaces include using an Isohelix swab as a collection tool, DNA/RNA Shield as a preservative, an automated system for RNA extraction, and reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) as the detection assay. Using this E2E approach, this study showed that, in some cases, SARS-CoV-2 viral standards were still recovered from surfaces as detected by RT-qPCR for as long as eight days even after bleach treatment. Additionally, debris associated with specific built environment surfaces appeared to negatively impact the recovery of RNA, with Amerstat inhibition as high as 90% when challenged with an inactivated viral control. Overall, it was determined that this E2E protocol required a minimum of 1,000 viral particles per 25 cm2 to successfully detect virus from test surfaces. When this method was employed to evaluate 368 samples collected from various built environmental surfaces, all samples tested negative, indicating that the surfaces were either void of virus or below the detection limit of the assay.

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Section: Discussionsupporting

confidence: 72%

End-to-End Protocol for the Detection of SARS-CoV-2 from Built Environments

Parker

Singh

Tighe

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…During this study, Isohelix swabs were selected over Copan swabs due to easier handling and higher sensitivity for sample collection, and this approach has been successfully used by other studies (12). Furthermore, our results demonstrated that automated RNA extraction was as efficient (13,14) as manual kits for extracting SARS-CoV-2, which has been previously noted using phenol-chloroform (15).…”

Section: Discussionsupporting

confidence: 55%

End-to-End Protocol for the Detection of SARS-CoV-2 from Built Environments

et al. 2020

View full text Add to dashboard Cite

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019, is a respiratory virus primarily transmitted person to person through inhalation of droplets or aerosols, laden with viral particles. However, as recent studies have shown, virions can remain infectious for up to 72 h on surfaces, which can lead to transmission through contact. Thus, a comprehensive study was conducted to determine the efficiency of protocols to recover SARS-CoV-2 from surfaces in built environments. This end-to-end (E2E) study showed that the effective combination for monitoring SARS-CoV-2 on surfaces includes using an Isohelix swab collection tool, DNA/RNA Shield as a preservative, an automated system for RNA extraction, and reverse transcriptase quantitative PCR (RT-qPCR) as the detection assay. Using this E2E approach, this study showed that, in some cases, noninfectious viral fragments of SARS-CoV-2 persisted on surfaces for as long as 8 days even after bleach treatment. Additionally, debris associated with specific built environment surfaces appeared to inhibit and negatively impact the recovery of RNA; Amerstat demonstrated the highest inhibition (>90%) when challenged with an inactivated viral control. Overall, it was determined that this E2E protocol required a minimum of 1,000 viral particles per 25 cm2 to successfully detect virus from test surfaces. Despite our findings of viral fragment longevity on surfaces, when this method was employed to evaluate 368 samples collected from various built environmental surfaces, all samples tested negative, indicating that the surfaces were either void of virus or below the detection limit of the assay. IMPORTANCE The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (the virus responsible for coronavirus disease 2019 [COVID-19]) pandemic has led to a global slowdown with far-reaching financial and social impacts. The SARS-CoV-2 respiratory virus is primarily transmitted from person to person through inhalation of infected droplets or aerosols. However, some studies have shown that virions can remain infectious on surfaces for days and can lead to human infection from contact with infected surfaces. Thus, a comprehensive study was conducted to determine the efficiency of protocols to recover SARS-CoV-2 from surfaces in built environments. This end-to-end study showed that the effective combination for monitoring SARS-CoV-2 on surfaces required a minimum of 1,000 viral particles per 25 cm2 to successfully detect virus from surfaces. This comprehensive study can provide valuable information regarding surface monitoring of various materials as well as the capacity to retain viral RNA and allow for effective disinfection.

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“…It requires widely available chemicals at a low cost. The AGPC methods has been found to match the performance of commercial kits and automated systems in SARS-CoV-2 rRT-PCR detection(7,8). In these studies, swabs were first collected in VTM or cell culture media, which were then used in AGPC RNA isolation.…”

Section: Introductionmentioning

confidence: 93%

A Robust, Safe and Scalable Magnetic Nanoparticle Workflow for RNA Extraction of Pathogens from Clinical and Environmental Samples

Ramos‐Mandujano

Salunke

Mfarrej

et al. 2020

Preprint

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Diagnosis and surveillance of emerging pathogens such as SARS-CoV-2 depend on nucleic acid isolation from clinical and environmental samples. Under normal circumstances, samples would be processed using commercial proprietary reagents in Biosafety 2 (BSL-2) or higher facilities. A pandemic at the scale of COVID-19 has caused a global shortage of proprietary reagents and BSL-2 laboratories to safely perform testing. Therefore, alternative solutions are urgently needed to address these challenges. We developed an open-source method called Magnetic- nanoparticle-Aided Viral RNA Isolation of Contagious Samples (MAVRICS) that is built upon reagents that are either readily available or can be synthesized in any molecular biology laboratory with basic equipment. Unlike conventional methods, MAVRICS works directly in samples inactivated in acid guanidinium thiocyanate-phenol-chloroform (e.g., TRIzol), thus allowing infectious samples to be handled safely without biocontainment facilities. Using 36 COVID-19 patient samples, 2 wastewater samples and 1 human pathogens control sample, we showed that MAVRICS rivals commercial kits in validated diagnostic tests of SARS-CoV-2, influenza viruses, and respiratory syncytial virus. MAVRICS is scalable and thus could become an enabling technology for widespread community testing and wastewater monitoring in the current and future pandemics.

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Phenol-chloroform-based RNA purification for detection of SARS-CoV-2 by RT-qPCR: comparison with automated systems

Cited by 6 publications

References 14 publications

End-to-End Protocol for the Detection of SARS-CoV-2 from Built Environments

End-to-End Protocol for the Detection of SARS-CoV-2 from Built Environments

End-to-End Protocol for the Detection of SARS-CoV-2 from Built Environments

A Robust, Safe and Scalable Magnetic Nanoparticle Workflow for RNA Extraction of Pathogens from Clinical and Environmental Samples

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