scholarly journals Rapid processing of SARS-CoV-2 containing specimens for direct RT-PCR

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0246867
Author(s):  
Piotr Chomczynski ◽  
Peter W. Chomczynski ◽  
Amy Kennedy ◽  
Michal Rymaszewski ◽  
William W. Wilfinger ◽  
...  
Keyword(s):  
Room Temperature ◽  
Diagnostic Testing ◽  
Viral Rna ◽  
Rt Pcr ◽  
Purification Step ◽  
Biological Specimen ◽  
Rna Detection ◽  
Rna Purification ◽  
Rapid Processing ◽  
Effect Of Inhibitors

Widespread diagnostic testing is needed to reduce transmission of COVID-19 and manage the pandemic. Effective mass screening requires robust and sensitive tests that reliably detect the SARS-CoV-2 virus, including asymptomatic and pre-symptomatic infections with a low viral count. Currently, the most accurate tests are based on detection of viral RNA by RT-PCR. We developed a method to process COVID-19 specimens that simplifies and increases the sensitivity of viral RNA detection by direct RT-qPCR, performed without RNA purification. In the method, termed Alkaline-Glycol Processing (AG Processing), a SARS-CoV-2-containing biological specimen, such as saliva or a swab-collected suspension, is processed at pH 12.2 to 12.8 for 5 min at room temperature. An aliquot of the AG-processed specimen is used for detection of SARS-CoV-2 RNA by direct RT-qPCR. AG processing effectively lyses viruses and reduces the effect of inhibitors of RT-PCR that are present in biological specimens. The sensitivity of detecting viral RNA using AG processing is on par with methods that include a viral RNA purification step. One copy of SARS-CoV-2 virus per reaction, equivalent to 300 copies per ml of saliva, is detectable in the AG-processed saliva. The LOD is 300 viral copies per ml of initial saliva specimen. AG processing works with saliva specimens or swab specimens collected into Universal Transport Medium, is compatible with heat treatment of saliva, and was confirmed to work with a range of CDC-approved RT-qPCR products and kits. Detection of SARS-CoV-2 RNA using AG processing with direct RT-qPCR provides a reliable and scalable diagnostic test for COVID-19 that can be integrated into a range of workflows, including automated settings.

scholarly journals Rapid processing of SARS-CoV-2 containing specimens for direct RT-PCR

2020 ◽  
Author(s):  
Piotr Chomczynski
Keyword(s):  
Room Temperature ◽  
Diagnostic Testing ◽  
Viral Rna ◽  
Rt Pcr ◽  
Purification Step ◽  
Biological Specimen ◽  
Rna Detection ◽  
Rna Purification ◽  
Rapid Processing ◽  
Effect Of Inhibitors

Widespread diagnostic testing is needed to reduce transmission of COVID-19 and manage the pandemic. Effective mass screening requires robust and sensitive tests that reliably detect the SARS-CoV-2 virus, including asymptomatic and pre-symptomatic infections with a low viral count. Currently, the most accurate tests are based on detection of viral RNA by RT-PCR. We developed a method to process COVID-19 specimens that simplifies and increases the sensitivity of viral RNA detection by direct RT-qPCR, performed without RNA purification. In the method, termed Alkaline-Glycol Processing (AG processing), a SARS-CoV-2-containing biological specimen, such as saliva or a swab-collected suspension, is processed at pH 12.2 to 12.8 for 5 min at room temperature. An aliquot of the AG-processed specimen is used for detection of SARS-CoV-2 RNA by direct RT-qPCR. AG processing effectively lyses viruses and reduces the effect of inhibitors of RT-PCR that are present in biological specimens. The sensitivity of detecting viral RNA using AG processing is on par with methods that include a viral RNA purification step. One copy of SARS-CoV-2 virus per reaction, equivalent to 300 copies per ml of saliva, is detectable in the AG-processed saliva. The LOD calculated following U.S. FDA guidelines is 600 viral copies per ml of initial saliva specimen. AG processing works with saliva specimens or swab specimens collected into Universal Transport Medium (UTM), is compatible with heat treatment, and was confirmed to work with a range of CDC-approved RT-qPCR products and kits. Detection of SARS-CoV-2 RNA using AG processing with direct RT-qPCR provides a reliable and scalable diagnostic test for COVID-19 that can be integrated into a range of workflows, including automated settings.

scholarly journals Inexpensive and colorimetric RNA detection by E. coli cell-free protein synthesis platform at room temperature

2021 ◽  
Author(s):  
Michela Notarangelo ◽  
Alessandro Quattrone ◽  
Massimo Pizzato ◽  
Sheref S. Mansy ◽  
O. Duhan Toparlak
Keyword(s):  
Room Temperature ◽  
Colorimetric Detection ◽  
In Vitro Transcription ◽  
Viral Rna ◽  
Rna Sequences ◽  
Rna Detection ◽  
E Coli ◽  
Free Gene ◽  
Cell Free Protein Synthesis

We report colorimetric detection of SARS-CoV-2 viral RNA by an in vitro transcription/translation assay with crude E. coli extracts at room temperature, with the aid of body heat. Clinically-relevant concentrations of viral RNA (ca. 600 copies/test) were detected from synthetic RNA samples. The activation of cell-free gene expression was achieved by toehold-switch-mediated riboregulatory elements that are specific to viral RNA sequences. The colorimetric output was generated by the α-complementation of β-galactosidase ω-fragment (LacZ-ω) with cell-free expressed LacZ-α, using an X-gal analogue as a substrate. The estimated cost of single reaction is less than 1 euro/test, which may facilitate diagnostic kit accessibility in developing countries.

scholarly journals Short communication: Stability and integrity of classical swine fever virus RNA stored at room temperature

2017 ◽  
Vol 15 (3) ◽  
pp. e05SC03 ◽  
Author(s):  
Damarys Relova ◽  
Lester J. Pérez ◽  
Liliam Ríos ◽  
Liani Coronado ◽  
Yoandry Hinojosa ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Room Temperature ◽  
Storage Temperature ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Viral Rna ◽  
Nucleotide Sequence Analysis ◽  
Original Material ◽  
Rt Pcr ◽  
The Stability

Worldwide cooperation between laboratories working with classical swine fever virus (CSFV) requires exchange of virus isolates. For this purpose, shipment of CSFV RNA is a safe alternative to the exchange of infectious material. New techniques using desiccation have been developed to store RNA at room temperature and are reported as effective means of preserving RNA integrity. In this study, we evaluated the stability and integrity of dried CSFV RNA stored at room temperature. First, we determined the stability of CSFV RNA covering CSFV genome regions used typically for the detection of viral RNA in diagnostic samples by reverse transcription-polymerase chain reaction (RT-PCR). To this end, different concentrations of in vitro-transcribed RNAs of the 5’-untranslated region and of the NS5B gene were stored as dried RNA at 4, 20, and 37oC for two months. Aliquots were analyzed every week by CSFV-specific quantitative real-time RT-PCR. Neither the RNA concentration nor the storage temperature did affect CSFV RNA yields at any of the time evaluated until the end of the experiment. Furthermore, it was possible to recover infectious CSFV after transfection of SK-6 cells with dried viral RNA stored at room temperature for one week. The full-length E2 of CSFV was amplified from all the recovered viruses, and nucleotide sequence analysis revealed 100% identity with the corresponding sequence obtained from RNA of the original material. These results show that CSFV RNA stored as dried RNA at room temperature is stable, maintaining its integrity for downstream analyses and applications.

scholarly journals Viral RNA load quantification as helpful tool to arbitrate SARS–CoV-2 detection results in respiratory samples

2020 ◽  
Author(s):  
Flora Marzia Liotti ◽  
Giulia Menchinelli ◽  
Simona Marchetti ◽  
Grazia Angela Morandotti ◽  
Maurizio Sanguinetti ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
False Negative ◽  
Negative Association ◽  
Viral Rna ◽  
N Gene ◽  
Rt Pcr ◽  
Rna Detection ◽  
Direct Assay ◽  
Future Clinical Practice ◽  
Oropharyngeal Swab

Abstract Purpose: The increasing COVID-19 widespread has created the necessity to assess the diagnostic accuracy of newly introduced (RT-PCR based) assays for SARS–CoV-2 RNA detection in respiratory tract samples.Methods: We compared the results of the Allplex™ 2019-nCoV assay with those of the Simplexa™ COVID-19 Direct assay, both performed on 125 nasal/oropharyngeal swab samples of patients with COVID-19 suspicion.Results: Fifty-four samples tested positive (CT below 40) and 71 negative (CT above 40) with the Allplex™ 2019-nCoV assay, whereas 47 of 54 samples were also positive with the Simplexa™ COVID-19 Direct assay. Eight results were discordant, resulting in 93.6% agreement between the assays. We used the Quanty COVID-19 assay—developed to detect and quantify SARS–CoV-2 in respiratory tract samples—to arbitrate these results. One Allplex™ 2019-nCoV negative (but Simplexa™ COVID-19 positive) and seven Simplexa™ COVID-19 negative samples were truly false negative. Interestingly, a Spearman’s negative association was found between the viral RNA loads quantified by the Quanty COVID-19 assay and the CT values of RT PCRs performed with either the Allplex™ 2019–nCoV assay or the Simplexa™ COVID-19 Direct assay. However, the strength of this association was higher for the Allplex™ 2019–nCoV assay (N gene, ρ = −0.92; RdRP gene, ρ = −0.91) than for the Simplexa™ COVID-19 Direct assay (ORF1ab gene, ρ = −0.65; S gene, ρ = −0.80).Conclusion: The Allplex™ 2019–nCoV and Simplexa™ COVID-19 Direct assays yielded comparable results. However, the role these assays might play in future clinical practice warrants larger comparison studies.

scholarly journals SARS-CoV-2 RNA detection in nasal mid-turbinate swabs

2020 ◽  
Author(s):  
Laura Dioni ◽  
Benedetta Albetti ◽  
Federica Rota ◽  
Valentina Bollati
Keyword(s):  
Rna Extraction ◽  
Positive Control ◽  
Viral Rna ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Rna Detection ◽  
Nasal Swabs ◽  
Internal Positive Control ◽  
Polymerase Chain ◽  
Genomic Regions

Abstract In this protocol, we describe a method to investigate the presence of SARS-CoV-2 RNA in nasal swabs, using a commercially available high-throughput Real-Time Polymerase Chain Reaction (RT-PCR) assay (TaqPath™ Covid-19 kit, ThermoFisher Scientific) in a 384-Well Plate. After Viral RNA extraction with QIAamp Viral RNA Mini kit, reverse transcription and cDNA amplification, all samples are assessed for the presence of three specific SARS-CoV-2 viral genomic regions and an internal positive control (IPC), in one Multiplex RT-PCR reaction.

scholarly journals A protocol for the detection of genetic markers in saliva by polymerase chain reaction without a nucleic acid purification step: examples of SARS-CoV-2 and GAPDH markers.

2021 ◽  
Author(s):  
Surya Kannan ◽  
Johan Ericsson ◽  
Nazariy Souchelnytskyi ◽  
Serhiy Souchelnytskyi
Keyword(s):  
Nucleic Acids ◽  
Genetic Markers ◽  
Room Temperature ◽  
Direct Detection ◽  
Sodium Dodecyl ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Purification Step ◽  
Efficient Detection ◽  
Nucleic Acid Purification

Abstract Background: The objective of this study was to develop a protocol for direct use of saliva in tests for genetic markers, without purification of nucleic acids. Currently, diagnostic tests use purified nucleic acids from clinical samples. This purification step adds time, cost, and affects the quality of testing. Multiple attempts to remove the purification step were reported. Results: We report a protocol for the direct detection of genetic markers in saliva. The protocol is based on collection of saliva in a solution containing a detergent and ethanol, and is compatible with isothermal amplification (LAMP), real-time RT-PCR and RT-PCR. SARS-CoV-2 and GAPDH markers were used as reference markers. We observed that mild detergents allow efficient detection of markers (e.g. GAPDH and SARS-CoV-2), while strong detergent, e.g. sodium dodecyl sulfate, inhibited the PCR reaction. Under these conditions, saliva samples can be stored for 24 h at +40C or -180C with preservation of the markers. Storage at room temperature led to deterioration of marker detection. Snap heating of saliva samples at the time of collection, followed by a storage at the room temperature, provided partial protection.Conclusions: The protocol presented in this report describes collection and storage of saliva for direct detection of genetic markers and is compatible with PCR and LAMP tests.

scholarly journals Does sampling saliva increase detection of SARS-CoV-2 by RT-PCR? Comparing saliva with oro-nasopharyngeal swabs

2020 ◽  
Author(s):  
Ozlem Akgun Dogan ◽  
Betsi Kose ◽  
Nihat Bugra Agaoglu ◽  
Jale Yildiz ◽  
Gizem Alkurt ◽  
...  
Keyword(s):  
Gold Standard ◽  
Viral Rna ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Standard Samples ◽  
Rna Detection ◽  
Detection Rates ◽  
Gold Standard Method ◽  
Non Invasive ◽  
Isolation Period

The gold standard method in the diagnosis of SARS-CoV-2 infection is the detection of viral RNA in nasopharyngeal sample by RT-PCR. Recently, saliva samples has been suggested as an alternative due to being fast, reliable and non-invasive, rather than nasopharyngeal samples. We compared RT-PCR results in nasopharyngeal, oro-nasopharyngeal and saliva samples of COVID-19 patients. 98 of 200 patients were positive in RT-PCR analysis performed before the hospitalization. In day 0, at least one sample was positive in 67% of 98 patients. Positivity rate was 83% for both oro-nasopharyngeal and nasopharyngeal samples, while it was 63% for saliva samples (p<0.001). On day 5, RT-PCR was performed in 59 patients, 34% had at least one positive result. The positivity rate was 55% for saliva and nasopharyngeal samples, while it was 60% for oro-nasopharyngeal samples. Our study shows that the sampling saliva does not increase the sensitivity of RT-PCR tests at early stages of infection. However, on 5th day, viral RNA detection rates in saliva were similar to nasopharyngeal and oro-nasopharyngeal samples. In conclusion, we suggest that, in patients receiving treatment, virus presence in saliva, in addition to the standard samples, is important to determine the isolation period and to control the transmission.

Effect of Disinfectants on SARS-CoV-2 RNA Detection in Swabs from Various Surfaces

2021 ◽  
Vol 37 (4) ◽  
pp. 78-84
Author(s):  
E.A. Lazareva ◽  
S.P. Yatsentyuk
Keyword(s):  
Hydrogen Peroxide ◽  
Uv Irradiation ◽  
Sodium Salt ◽  
Viral Rna ◽  
Rt Pcr ◽  
Rna Detection ◽  
The World ◽  
Porous Surfaces ◽  
Rna Fragments ◽  
Dichloroisocyanuric Acid

The COVID-19 pandemic has spread rapidly around the world, and some countries have introduced controls on imported products, including testing for viral nucleic acids. In this work, the influence of disinfectants for treatment of various SARS-CoV-2-contaminated surfaces on the detection of viral RNA fragments in swabs from these surfaces was analyzed using quantitative RT-PCR. The effect of disinfectants based on quaternary ammonium salt, hydrogen peroxide, 1-propanol, sodium salt of dichloroisocyanuric acid and ultraviolet radiation was investigated. Our results show that without the exposure to disinfectants, viral RNA can be detected on the surface of all examined materials for at least three days. UV irradiation or irrigation with a disinfectant containing 0.2% active chlorine had the greatest effect on the decontamination of non-porous surfaces as measured by RT-PCR of swabs from these surfaces. Irrigation of porous surfaces (cardboard) with disinfectants had practically no effect on the detection of SARS-CoV-2 RNA by RT-PCR. Key words: SARS-CoV-2, viral RNA, RT-PCR, disinfectants, UV irradiation, surface swabs Funding - This work was supported by the Federal Service of Veterinary and Phytosanitary Surveillance (Rosselkhoznadzor).

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