Efficacy of a novel SARS-CoV-2 detection kit without RNA extraction and purification

AbstractRapid detection of SARS-CoV-2 is critical for the diagnosis of coronavirus disease 2019 (COVID-19) and preventing the spread of the virus. A novel “2019 Novel Coronavirus Detection Kit (nCoV-DK)” halves detection time by eliminating the steps of RNA extraction and purification. We evaluated concordance between the nCoV-DK and direct PCR. The virus was detected in 53/71 fresh samples by the direct method and 55/71 corresponding frozen samples by the nCoV-DK. The overall concordance rate of the virus detection between the two methods was 94.4% (95% CI, 86.2-98.4). Concordance rates were 95.2% (95% CI, 83.8-99.4), 95.5% (95% CI, 77.2-99.9), 85.7% (95% CI, 42.1-99.6) in nasopharyngeal swab, saliva, and sputum samples, respectively. These results indicate that the nCoV-DK effectively detects SARS-CoV-2 in all types of the samples including saliva, while reducing time required for detection, labor, and risk of human error.

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A simple RNA preparation method for SARS-CoV-2 detection by RT-qPCR

Scientific Reports ◽

10.1038/s41598-020-73616-w ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Aniela Wozniak ◽

Ariel Cerda ◽

Catalina Ibarra-Henríquez ◽

Valentina Sebastian ◽

Grace Armijo ◽

...

Keyword(s):

Rna Extraction ◽

Molecular Diagnostic ◽

Nasopharyngeal Swab ◽

Diagnostic Laboratory ◽

Public Health Decision ◽

Health Decision Making ◽

Health Decision ◽

Analytical Step ◽

Commercial Kits ◽

Novel Coronavirus

Abstract The technique RT-qPCR for viral RNA detection is the current worldwide strategy used for early detection of the novel coronavirus SARS-CoV-2. RNA extraction is a key pre-analytical step in RT-qPCR, often achieved using commercial kits. However, the magnitude of the COVID-19 pandemic is causing disruptions to the global supply chains used by many diagnostic laboratories to procure the commercial kits required for RNA extraction. Shortage in these essential reagents is even more acute in developing countries with no means to produce kits locally. We sought to find an alternative procedure to replace commercial kits using common reagents found in molecular biology laboratories. Here we report a method for RNA extraction that takes about 40 min to complete ten samples, and is not more laborious than current commercial RNA extraction kits. We demonstrate that this method can be used to process nasopharyngeal swab samples and yields RT-qPCR results comparable to those obtained with commercial kits. Most importantly, this procedure can be easily implemented in any molecular diagnostic laboratory. Frequent testing is crucial for individual patient management as well as for public health decision making in this pandemic. Implementation of this method could maintain crucial testing going despite commercial kit shortages.

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Assessment of the direct quantitation of SARS-CoV-2 by droplet digital PCR

10.21203/rs.3.rs-55725/v1 ◽

2020 ◽

Author(s):

Michela Deiana ◽

Antonio Mori ◽

Chiara Piubelli ◽

Salvatore Scarso ◽

Mose Favarato ◽

...

Keyword(s):

Direct Method ◽

Rna Extraction ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Direct Approach ◽

Heat Inactivation ◽

Nasopharyngeal Swab ◽

Clinical Value ◽

Care Needs ◽

North East

Abstract Droplet digital PCR (ddPCR) is a sensitive and reproducible technology widely used for quantitation of several different viruses. The aim of this study was to compare the 2019-nCoV CDC ddPCR Triplex Probe Assay (BioRad) performance on the direct quantitation of SARS-CoV-2 on nasopharyngeal swab respect to the procedure applied to the extracted RNA. Moreover, the two widely used swab types were compared (UTM 3mL and ESwab 1mL, COPAN). A total of 50 nasopharyngeal swabs (n=25 UTM 3mL and n=25 ESwab 1mL) from SARS-CoV-2 patients collected during the pandemic from IRCCS Sacro Cuore Don Calabria Hospital (Veneto Region, North-East Italy) were used for our purpose. After heat inactivation, an aliquot of swab medium was used in order to perform the direct quantitation. Then, we compared the direct method with the quantitation performed on the RNA purified from nasopharyngeal swab by automated extraction. We observed that the direct approach achieved generally equal RNA copies compared to the RNA extracted. The results with the direct quantitation were more accurate on ESwab with a sensitivity of 93.33% [95% CI, 68.05 to 99.83] and specificity of 100.00% for both N1 and N2. On the other hand, on UTM we observed a higher rate of discordant results for N1 and N2. The human internal amplification control (RPP30) showed 100% of both sensitivity and specificity independent of swabs and approaches.In conclusion, we described a simple and fast approach for the quantitation of SARS-CoV-2 in nasopharyngeal swab. Our approach resulted in an efficient quantitation, without automated RNA extraction and purification. However, special care needs to be taken on the potential bias due to the conservation of samples as we used thawed material and to the heating treatment. Further studies on a larger cohort of samples are warranted to evaluate the clinical value of this direct approach.

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Direct Saliva versus Conventional Nasopharyngeal Swab qRT-PCR to Diagnose SARS – CoV2: Validity Study

Asian Journal of Research in Infectious Diseases ◽

10.9734/ajrid/2021/v6i230193 ◽

2021 ◽

pp. 37-46

Author(s):

Michael L. Tee ◽

Paulyn Jean R. Ubial ◽

Diana Rose E. Ranoa ◽

Cherica A. Tee ◽

Aedrian A. Abrilla ◽

...

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Rna Extraction ◽

Nasopharyngeal Swab ◽

Diagnostic Validity ◽

Real Time Quantitative Pcr ◽

Qrt Pcr ◽

Paired Samples ◽

Feasible Alternative ◽

Novel Coronavirus

Background: Saliva has been demonstrated as a feasible alternative specimen to nasopharyngeal swab for the detection of SARS-CoV-2 using real-time or quantitative reverse transcription polymerase chain reaction (qRT-PCR) method that bypasses the need for explicit viral ribonucleic acid (RNA) extraction. Aim: To assess the diagnostic validity of direct saliva-to-qRT-PCR in the detection of SARS-CoV-2 compared to conventional nasopharyngeal swab qRT-PCR. Methodology: Self-collected saliva samples were processed by heating at 95oC for 30 minutes followed by addition of buffer and detergent while viral RNA from nasopharyngeal swabs were extracted using the Sansure Biotech sample release reagent. Paired samples were used as templates for qRT-PCR using the Sansure Novel Coronavirus (COVID-19) Nucleic Acid Diagnostic Kit and Sansure Biotech MA6000 Real-Time Quantitative PCR System. Direct saliva-to-qRT-PCR was compared to nasopharyngeal swab qRT-PCR in terms of diagnostic validity and agreement parameters, and both platforms were compared separately in terms of similar parameters with a composite reference standard (CRS) wherein the criteria for a positive result is SARS-CoV-2 detection in at least either nasopharyngeal swab or saliva. Results: Of the 238 nasopharyngeal swab-saliva pairs tested, 20 (8.4%) nasopharyngeal swab and 24 (10.1%) saliva specimens tested positive. We documented a sensitivity of 85.0% (95% CI: 62.1%, 96.8%), specificity of 96.8% (95% CI: 93.5%, 98.7%), accuracy of 95.8% (95% CI: 92.4%, 98.0%) and Cohen Kappa of 0.75 (95% CI: 0.60, 0.90) when direct saliva-to-qRT-PCR was compared to the conventional platform. When the two platforms were individually compared to the CRS, numerically higher but not statistically significant sensitivity and accuracy were noted for direct saliva-to-qRT-PCR than for nasopharyngeal swab qRT-PCR. Conclusion: Direct saliva-to-qRT-PCR is non-inferior to nasopharyngeal swab qRT-PCR for detecting SARS-CoV-2 using the Sansure Novel Coronavirus Nucleic Acid Diagnostic Kit.

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Concentration of the cellular material in the nasopharyngeal swabs increases the clinical sensitivity of SARS-CoV2 RT-PCR

10.1101/2020.10.31.20218958 ◽

2020 ◽

Author(s):

Priya Kannian ◽

Pasuvaraj Mahanathi ◽

Veeraraghavan Ashwini

Keyword(s):

Direct Method ◽

Rna Extraction ◽

Direct Methods ◽

N Gene ◽

Nasopharyngeal Swab ◽

Rt Pcr ◽

Clinical Sensitivity ◽

Concentration Method ◽

Viral Transport Medium ◽

Analytical Variable

SummarySevere acute respiratory syndrome - coronavirus 2 (SARS-CoV2) is detected by a highly sensitive molecular method, reverse transcriptase-polymerase chain reaction (RT-PCR) from nasopharyngeal swab (NPS) samples collected in 2-3ml of viral transport medium (VTM). Unlike body fluids, NPS samples are undermined by high variability in the amount of cells that get suspended into the VTM. Hence, the cell density used for RNA extraction becomes an important analytical variable that contributes to the overall sensitivity of the RT-PCR. In this study, we compared the sensitivity of SARS-CoV2 RT-PCR in 50 NPS samples collected from in-patients of the COVID wards using the concentration and direct methods. The concentration method detected the viral RNA in all 50 samples, while the direct method was positive in only 41 (82%) samples (p=0.003). Additionally, the Ct values were lower in the direct method compared to concentration method among the 41 positive samples (p=0.03 for N gene and p=0.04 for RdRp gene). The mean CV% was also ≥10%. Thus, the concentration of the cells prior to RNA extraction drastically improves the sensitivity of detection of SARS-CoV2 in NPS samples.

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Assessment of the direct quantitation of SARS-CoV-2 by droplet digital PCR

Scientific Reports ◽

10.1038/s41598-020-75958-x ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 2

Author(s):

Michela Deiana ◽

Antonio Mori ◽

Chiara Piubelli ◽

Salvatore Scarso ◽

Mosè Favarato ◽

...

Keyword(s):

Direct Method ◽

Rna Extraction ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Direct Approach ◽

Heat Inactivation ◽

Nasopharyngeal Swab ◽

Clinical Value ◽

Care Needs ◽

North East

Abstract Droplet digital PCR (ddPCR) is a sensitive and reproducible technology widely used for quantitation of several viruses. The aim of this study was to evaluate the 2019-nCoV CDC ddPCR Triplex Probe Assay (BioRad) performance, comparing the direct quantitation of SARS-CoV-2 on nasopharyngeal swab with the procedure applied to the extracted RNA. Moreover, two widely used swab types were compared (UTM 3 mL and ESwab 1 mL, COPAN). A total of 50 nasopharyngeal swabs (n = 25 UTM 3 mL and n = 25 ESwab 1 mL) from SARS-CoV-2 patients, collected during the pandemic at IRCCS Sacro Cuore Don Calabria Hospital (Veneto Region, North-East Italy), were used for our purpose. After heat inactivation, an aliquot of swab medium was used for the direct quantitation. Then, we compared the direct method with the quantitation performed on the RNA purified from nasopharyngeal swab by automated extraction. We observed that the direct approach achieved generally equal RNA copies compared to the extracted RNA. The results with the direct quantitation were more accurate on ESwab with a sensitivity of 93.33% [95% CI, 68.05 to 99.83] and specificity of 100.00% for both N1 and N2. On the other hand, on UTM we observed a higher rate of discordant results for N1 and N2. The human internal amplification control (RPP30) showed 100% of both sensitivity and specificity independent of swabs and approaches. In conclusion, we described a direct quantitation of SARS-CoV-2 in nasopharyngeal swab. Our approach resulted in an efficient quantitation, without automated RNA extraction and purification. However, special care needs to be taken on the potential bias due to the conservation of samples and to the heating treatment, as we used thawed and heat inactivated material. Further studies on a larger cohort of samples are warranted to evaluate the clinical value of this direct approach.

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Sensitive extraction-free SARS-CoV-2 RNA virus detection using a novel RNA preparation method

10.1101/2021.01.29.21250790 ◽

2021 ◽

Author(s):

Bin Guan ◽

Karen M. Frank ◽

José O. Maldonado ◽

Margaret Beach ◽

Eileen Pelayo ◽

...

Keyword(s):

Preparation Method ◽

Direct Detection ◽

Rna Virus ◽

Rna Extraction ◽

Virus Detection ◽

Detection Sensitivity ◽

Nasopharyngeal Swab ◽

Reverse Transcription Pcr ◽

Viral Transport Medium ◽

Droplet Pcr

AbstractCurrent conventional detection of SARS-CoV-2 involves collection of a patient sample with a nasopharyngeal swab, storage of the swab during transport in a viral transport medium, extraction of RNA, and quantitative reverse transcription PCR (RT-qPCR). We developed a simplified and novel preparation method using a Chelex resin that obviates RNA extraction during viral testing. Direct detection RT-qPCR and digital-droplet PCR was compared to the current conventional method with RNA extraction for simulated samples and patient specimens. The heat-treatment in the presence of Chelex markedly improved detection sensitivity as compared to heat alone, and lack of RNA extraction shortens the overall diagnostic workflow. Furthermore, the initial sample heating step inactivates SARS-CoV-2 infectivity, thus improving workflow safety. This fast RNA preparation and detection method is versatile for a variety of samples, safe for testing personnel, and suitable for standard clinical collection and testing on high throughput platforms.

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Saliva as a Diagnostic Tool for Detection of Coronavirus: A Review

Journal of Islamabad Medical & Dental College ◽

10.35787/jimdc.v9i3.570 ◽

2020 ◽

Vol 9 (3) ◽

pp. 225-232

Author(s):

Abhishek Lal ◽

Mahnoor Khawaja M. Saleem ◽

Naseer Ahmed

Keyword(s):

Literature Review ◽

Diagnostic Tool ◽

Rapid Detection ◽

Healthcare Professionals ◽

Google Scholar ◽

Nasopharyngeal Swab ◽

Diagnostic Modality ◽

Non Invasive ◽

Saliva Collection ◽

Novel Coronavirus

We aimed in this literature review to demonstrate the association and potential of detecting novel coronavirus in saliva of patients and how its implication in future can aid in diagnosis as a non-invasive diagnostic modality. The specimen can be easily obtained and tested from suspected individuals. Review of available literature in PubMed, Google Scholar, EBSCO, and Semantic Scholar was carried out using keywords and combination of “Coronavirus”, “saliva” and “diagnosis”. Of 1846 articles found, 110 were screened and included in this literature review. Currently, nasopharyngeal swab is the standard diagnostic tool as it has been reported to be accurate and sensitive towards detection of coronavirus. Testing of saliva specimens is now being considered to aid rapid detection, because saliva collection and its testing are relatively simple, cheap, and safe for both patients as well as healthcare professionals. Further research on this will be beneficial to control and contain the virus.

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A simple RNA preparation method for SARS-CoV-2 detection by RT-qPCR

10.1101/2020.05.07.083048 ◽

2020 ◽

Cited By ~ 1

Author(s):

Aniela Wozniak ◽

Ariel Cerda ◽

Catalina Ibarra-Henriquez ◽

Valentina Sebastian ◽

Grace Armijo ◽

...

Keyword(s):

Rna Extraction ◽

Molecular Diagnostic ◽

Nasopharyngeal Swab ◽

Diagnostic Laboratory ◽

Public Health Decision ◽

Health Decision Making ◽

Health Decision ◽

Analytical Step ◽

Commercial Kits ◽

Novel Coronavirus

AbstractThe technique RT-qPCR for viral RNA detection is the current worldwide strategy used for early detection of the novel coronavirus SARS-CoV-2. RNA extraction is a key pre-analytical step in RT-qPCR, often achieved using commercial kits. However, the magnitude of the COVID-19 pandemic is causing disruptions to the global supply chains used by many diagnostic laboratories to procure the commercial kits required for RNA extraction. Shortage in these essential reagents is even more acute in developing countries with no means to produce kits locally. We sought to find an alternative procedure to replace commercial kits using common reagents found in molecular biology laboratories. Here we report a method for RNA extraction that takes about 40 min to complete ten samples, and is not more laborious than current commercial RNA extraction kits. We demonstrate that this method can be used to process nasopharyngeal swab samples and yields RT-qPCR results comparable to those obtained with commercial kits. Most importantly, this procedure can be easily implemented in any molecular diagnostic laboratory. Frequent testing is crucial for individual patient management as well as for public health decision making in this pandemic. Implementation of this method could maintain crucial testing going despite commercial kit shortages.

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A Direct Method for RT-PCR Detection of SARS-CoV-2 in Clinical Samples

Healthcare ◽

10.3390/healthcare9010037 ◽

2021 ◽

Vol 9 (1) ◽

pp. 37

Author(s):

Sherif A. El-Kafrawy ◽

Mai M. El-Daly ◽

Ahmed M. Hassan ◽

Reham M. Kaki ◽

Adel M. Abuzenadah ◽

...

Keyword(s):

Rna Virus ◽

Direct Method ◽

Rna Extraction ◽

Pcr Detection ◽

Clinical Samples ◽

Acid Extraction ◽

Rt Pcr ◽

Direct Pcr ◽

Rdrp Region ◽

Positive Strand Rna

Introduction: the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of acute respiratory disease (COVID-19). SARS-CoV-2 is a positive-strand RNA virus and its genomic characterization has played a vital role in the design of appropriate diagnostics tests. The current RT-PCR protocol for SARS-CoV-2 detects two regions of the viral genome, requiring RNA extraction and several hours. There is a need for fast, simple, and cost-effective detection strategies. Methods: we optimized a protocol for direct RT-PCR detection of SARS-CoV-2 without the need for nucleic acid extraction. Nasopharyngeal samples were diluted to 1:3 using diethyl pyrocarbonate (DEPC)-treated water. The diluted samples were incubated at 95 °C for 5 min in a thermal cycler, followed by a cooling step at 4 °C for 5 min. Samples then underwent reverse transcription real-time RT-PCR in the E and RdRp genes. Results: our direct detection protocol showed 100% concordance with the standard protocol with an average Ct value difference of 4.38 for the E region and 3.85 for the RdRp region. Conclusion: the direct PCR technique was found to be a reliable and sensitive method that can be used to reduce the time and cost of the assay by removing the need for RNA extraction. It enables the use of the assay in research, diagnostics, and screening for COVID-19 in regions with fewer economic resources, where supplies are more limited allowing for wider use for screening.

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Search mode for rapid detection of virus particles

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100085733 ◽

1991 ◽

Vol 49 ◽

pp. 286-287

Author(s):

O. E. Bradfute

Keyword(s):

Electron Microscopy ◽

Rapid Detection ◽

Plant Virus ◽

Low Dose ◽

Search Mode ◽

Virus Diseases ◽

Virus Particles ◽

Focus Image ◽

Time Required ◽

Rapid Switching

Electron microscopy is frequently used in preliminary diagnosis of plant virus diseases by surveying negatively stained preparations of crude extracts of leaf samples. A major limitation of this method is the time required to survey grids when the concentration of virus particles (VPs) is low. A rapid survey of grids for VPs is reported here; the method employs a low magnification, out-of-focus Search Mode similar to that used for low dose electron microscopy of radiation sensitive specimens. A higher magnification, in-focus Confirm Mode is used to photograph or confirm the detection of VPs. Setting up the Search Mode by obtaining an out-of-focus image of the specimen in diffraction (K. H. Downing and W. Chiu, private communications) and pre-aligning the image in Search Mode with the image in Confirm Mode facilitates rapid switching between Modes.

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