scholarly journals Speeding Up COVID-19 Detection Using Shaker-Mill Homogenization and a Direct-to-PCR Workflow

2020 ◽  
Author(s):  
Zachary P Morehouse ◽  
Caleb M Proctor ◽  
Gabriella L Ryan ◽  
Rodney J Nash
Keyword(s):  
Polymerase Chain Reaction ◽  
Rna Extraction ◽  
Viral Rna ◽  
Chain Reaction ◽  
Limited Access ◽  
Viral Transport ◽  
Mechanical Disruption ◽  
Global Pandemic ◽  
Polymerase Chain

Accurate and timely testing has become an essential measure in combatting the COVID-19 global pandemic. Currently, polymerase chain reaction (PCR) based assays are the most relied on methods for SARS-CoV-2 detection. This traditional workflow involves a viral RNA extraction from the viral transport media storing nasopharyngeal swabs collected from patients, followed by PCR based detection. While accurate, this methodology is time consuming and resource heavy, causing for delays in receiving results or limited access to testing. Herein, we demonstrate a validated method for SARS-CoV-2 detection from viral transport media using a two-step, direct-to-PCR workflow revolving around shaker-mill homogenization. This method completely bypasses the extraction steps of the traditional workflow, replacing it with 30 seconds of mechanical disruption sufficient to allow for COVID-19 detection with a 96.43% sensitivity and 100% specificity when compared to traditional extraction to PCR based methods.

scholarly journals Improved RNA Extraction from Woody Plants for the Detection of Viral Pathogens by Reverse Transcription-Polymerase Chain Reaction

Plant Disease ◽  
1997 ◽  
Vol 81 (2) ◽  
pp. 222-226 ◽  
Author(s):  
Donald J. MacKenzie ◽  
Morven A. McLean ◽  
Srima Mukerji ◽  
Margaret Green
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Woody Plants ◽  
Rna Extraction ◽  
Viral Rna ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Apple Stem ◽  
Spin Column ◽  
Polymerase Chain

An efficient procedure for the extraction of high-quality RNA from woody plants without the use of phenol, organic solvents, or alcohol precipitation is described. The method employs commercially available spin-column matrices and mitigates the inhibitory effects of plant polysaccharides and polyphenolic compounds commonly observed on subsequent polymerase chain reaction amplification when conventional extraction methods are applied to woody plant species. The method described has been successfully used in the development of highly sensitive reverse transcription-polymerase chain reaction (RT-PCR) techniques for the detection of a number of viruses in their woody hosts. The viruses detected included apple stem grooving capillovirus (ASGV), apple stem pitting virus, Prunus necrotic ringspot ilarvirus (PNRSV), grapevine fanleaf and Arabis mosaic nepoviruses, and grapevine leafroll-associated closterovirus type 3. The method described was equally effective for the extraction of viral RNA from either budwood, leaves, or flower blossoms as determined by the equivalent RT-PCR detection of ASGV and PNRSV from these tissues. Detection of viral RNA in samples of total plant RNA prepared using this method was found to be as sensitive as was previously described for the immunocapture RT-PCR technique.

scholarly journals SARS-CoV-2 direct real-time polymerase chain reaction testing in laboratories with shortage challenges

2021 ◽  
Vol 16 (2) ◽  
pp. 133-139
Author(s):  
Taghreed Alaifan ◽  
Asmaa Altamimi ◽  
Dalia Obeid ◽  
Turki Alshehri ◽  
Shaihana Almatrrouk ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Rna Extraction ◽  
Threshold Value ◽  
Limited Resources ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Viral Transport ◽  
Extraction Step ◽  
Polymerase Chain

Aim: In our study, we propose the use of direct real-time polymerase chain reaction (RT-PCR), this test does not require extraction or a preheating step, which saves a lot of cost, labor, processing time and provides a solution for supply shortage. Materials & methods: We assayed 185 nasopharyngeal samples stored in viral transport media. The indirect method was done with RNA extraction step, and the direct RT-PCR was done without an extraction step, both assays were evaluated on a commercially validated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) kit targeting E gene. Results: Our assay showed a sensitivity of 79%, a specificity of 100% and the agreement between methods was 72%. Conclusion: Overall, this simple direct RT-PCR approach can be utilized as a qualitative diagnostic tool for emergency SARS-CoV-2 surveillance in countries with limited resources and may help laboratories to continue testing and at greater frequency despite supply shortages, although with delay in cycle threshold value in comparison with indirect RT-PCR.

scholarly journals Viral RNA Shedding and Transmission Potential of Asymptomatic and Pauci-symptomatic COVID-19 Patients

2020 ◽  
Author(s):  
Arghadip Samaddar ◽  
Ravisekhar Gadepalli ◽  
Vijaya Lakshmi Nag ◽  
Sanjeev Misra ◽  
Pankaj Bhardwaj ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Viral Rna ◽  
Exhaled Breath ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Transmission Potential ◽  
Polymerase Chain ◽  
Pcr Test

Abstract We studied the pattern and duration of viral RNA shedding in 32 asymptomatic and 11 pauci-symptomatic coronavirus disease 2019 (COVID-19) cases. Viral RNA shedding in exhaled breath progressively diminished and became negative after six days of a positive reverse transcription polymerase chain reaction (RT-PCR) test. Therefore, the duration of isolation can be minimised to six days.

scholarly journals Use of conserved sequences from hepatitis C virus for the detection of viral RNA in infected sera by polymerase chain reaction

Hepatology ◽  
1991 ◽  
Vol 14 (4) ◽  
pp. 595-600 ◽  
Author(s):  
Genevieve Inchauspe ◽  
Kenji Abe ◽  
Suzanne Zebedee ◽  
Marc Nasoff ◽  
Alfred M. Prince
Keyword(s):  
Polymerase Chain Reaction ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Rna ◽  
Chain Reaction ◽  
Conserved Sequences ◽  
Polymerase Chain

scholarly journals Persistence of Porcine Reproductive and Respiratory Syndrome Virus in Serum and Semen of Adult Boars

1995 ◽  
Vol 7 (4) ◽  
pp. 456-464 ◽  
Author(s):  
Jane Christopher-Hennings ◽  
Eric A. Nelson ◽  
Rebecca J. Hines ◽  
Julie K. Nelson ◽  
Sabrina L. Swenson ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Virus Isolation ◽  
Fluorescent Antibody ◽  
Acute Stage ◽  
Viral Rna ◽  
Respiratory Syndrome Virus ◽  
Indirect Fluorescent Antibody ◽  
Chain Reaction ◽  
Serum Samples ◽  
Polymerase Chain

Four seronegative adult boars were intranasally inoculated with porcine reproductive and respiratory syndrome virus (PRRSV) isolate VR-2332. Serum and semen were collected 2-3 times weekly for over 100 days postinoculation (DPI). Serum samples were assayed for PRRSV by virus isolation (VI) and a polymerase chain reaction (PCR) and screened for antibodies to PRRSV using the indirect fluorescent antibody (IFA) and virus neutralization (VN) tests. Semen was assayed for PRRSV RNA by PCR. Virus or viral RNA was detected in the serum of all boars within 1 DPI by VI and/or PCR. However, VI results indicated that viremia was transient and occurred from 1 to 9 DPI. Viral RNA was detected in serum from 1 to 31 DPI. In the acute stage of the infection, PRRSV RNA was detected in serum by PCR prior to the presence of viral RNA in semen. The PRRSV RNA was detected in semen as early as 3 DPI and persisted for 25 DPI in 2 of the boars and 56 and 92 DPI in the remaining 2 boars. Detection of PRRSV RNA in semen occurred 2-8 and 28-35 days prior to the detection of antibodies by IFA and VN, respectively. PRRSV was isolated from the bulbourethral gland of the boar that shed viral RNA in semen for 92 DPI. These results suggest that PRRSV RNA can be detected by PCR in boar serum and semen, and may persist for variable periods of time. Viremia and the serologic status of the boar are not adequate indicators of when PRRSV or PRRSV RNA is being shed in the semen. Preliminary findings also indicated that neither shipping stress nor reinoculation with homologous PRRSV resulted in viremia or viral RNA shedding in semen.

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