scholarly journals A Rapid SARS-CoV-2 RT-PCR Assay for Low Resource Settings

Diagnostics ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 739
Author(s):  
Arunkumar Arumugam ◽  
Matthew L. Faron ◽  
Peter Yu ◽  
Cole Markham ◽  
Michelle Wu ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Qpcr Assay ◽  
Water Bath ◽  
Raspberry Pi ◽  
Rt Pcr ◽  
Clinical Specimens ◽  
Low Resource Settings ◽  
Clinical Sensitivity ◽  
Low Resource ◽  
Extraction Step

Quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay is the gold standard recommended to test for acute SARS-CoV-2 infection. However, it generally requires expensive equipment such as RNA isolation instruments and real-time PCR thermal cyclers. As a pandemic, COVID-19 has spread indiscriminately, and many low resource settings and developing countries do not have the means for fast and accurate COVID-19 detection to control the outbreak. Additionally, long assay times, in part caused by slow sample preparation steps, have created a large backlog when testing patient samples suspected of COVID-19. With many PCR-based molecular assays including an extraction step, this can take a significant amount of time and labor, especially if the extraction is performed manually. Using COVID-19 clinical specimens, we have collected evidence that the RT-qPCR assay can feasibly be performed directly on patient sample material in virus transport medium (VTM) without an RNA extraction step, while still producing sensitive test results. If RNA extraction steps can be omitted without significantly affecting clinical sensitivity, the turn-around time of COVID-19 tests, and the backlog we currently experience can be reduced drastically. Furthermore, our data suggest that rapid RT-PCR can be implemented for sensitive and specific molecular diagnosis of COVID-19 in locations where sophisticated laboratory instruments are not available. Our USD 300 set up achieved rapid RT-PCR using thin-walled PCR tubes and a water bath setup using sous vide immersion heaters, a Raspberry Pi computer, and a single servo motor that can process up to 96 samples at a time. Using COVID-19 positive clinical specimens, we demonstrated that RT-PCR assays can be performed in as little as 12 min using untreated samples, heat-inactivated samples, or extracted RNA templates with our low-cost water bath setup. These findings can help rapid COVID-19 testing to become more accessible and attainable across the globe.

scholarly journals Evaluating the efficacy of RT-qPCR SARS-CoV-2 direct approaches in comparison to RNA extraction

2020 ◽  
Author(s):  
Ofir Israeli ◽  
Adi Beth-Din ◽  
Nir Paran ◽  
Dana Stein ◽  
Shirley Lazar ◽  
...  
Keyword(s):  
Gold Standard ◽  
Rna Extraction ◽  
Extraction Procedure ◽  
Qpcr Assay ◽  
Viral Rna ◽  
Genetic Identification ◽  
Clinical Specimens ◽  
Extraction Step

ABSTRACTSARS-CoV-2 genetic identification is based on viral RNA extraction prior to RT-qPCR assay, however recent studies support the elimination of the extraction step. Herein, we assessed the RNA extraction necessity, by comparing RT-qPCR efficacy in several direct approaches vs. the gold standard RNA extraction, in detection of SARS-CoV-2 from laboratory samples as well as clinical Oro-nasopharyngeal SARS-CoV-2 swabs. Our findings show advantage for the extraction procedure, however a direct no-buffer approach might be an alternative, since it identified up to 70% of positive clinical specimens.

scholarly journals The Potential Use of Unprocessed Sample for RT-qPCR Detection of COVID-19 without an RNA Extraction Step

2020 ◽  
Author(s):  
Arunkumar Arumugam ◽  
Season Wong
Keyword(s):  
Direct Detection ◽  
Rna Extraction ◽  
Qpcr Assay ◽  
Virus Transport ◽  
Clinical Sensitivity ◽  
Extraction Step ◽  
Control And Prevention ◽  
Assay Time ◽  
Testing Patient ◽  
Virus Transport Medium

ABSTRACTQuantitative reverse transcription polymerase chain reaction (RT-qPCR) assay is the gold standard recommended to test for acute SARS-CoV-2 infection.1–4 It has been used by the Centers for Disease Control and Prevention (CDC) and several other companies in their Emergency Use Authorization (EUA) assays. With many PCR-based molecular assays, an extraction step is routinely used as part of the protocol. This step can take up a significant amount of time and labor, especially if the extraction is performed manually. Long assay time, partly caused by slow sample preparation steps, has created a large backlog when testing patient samples suspected of COVID-19. Using flu and RSV clinical specimens, we have collected evidence that the RT-qPCR assay can be performed directly on patient sample material from a nasal swab immersed in virus transport medium (VTM) without an RNA extraction step. We have also used this approach to test for the direct detection of SARS-CoV-2 reference materials spiked in VTM. Our data, while preliminary, suggest that using a few microliters of these untreated samples still can lead to sensitive test results. If RNA extraction steps can be omitted without significantly affecting clinical sensitivity, the turn-around time of COVID-19 tests and the backlog we currently experience can be reduced drastically. Next, we will confirm our findings using patient samples.

scholarly journals Wide application of minimally processed saliva on multiple RT-PCR kits for SARS-CoV-2 detection in Indonesia

2021 ◽  
Author(s):  
Caroline Mahendra ◽  
Maria M. M. Kaisar ◽  
Suraj R. Vasandani ◽  
Sem Samuel Surja ◽  
Enty Tjoa ◽  
...  
Keyword(s):  
Target Genes ◽  
Local Population ◽  
Rna Extraction ◽  
Human Saliva ◽  
Attractive Alternative ◽  
Rt Pcr ◽  
Sampling Device ◽  
Agreement Rate ◽  
Extraction Step ◽  
Oropharyngeal Swab

Saliva as a sample matrix has been an attractive alternative for the detection of SARS-CoV-2. However, due to potential variability in collection and processing steps, it is recommended to evaluate a proposed workflow amongst the local population. Here, we aim to validate collection and treatment of human saliva as a direct specimen for RT-qPCR based detection of SARS-CoV-2 in Indonesia. We demonstrated that SARS-CoV-2 target genes were detected in saliva specimen and remained stable for five days refrigerated or room temperature storage. The method of processing saliva specimen described in this report is free from RNA-extraction step, thereby reduces cost, time, and manpower required for processing samples. The developed method was validated for use on three COVID-19 RT-PCR kits commercially available. Our developed method achieved 85% agreement rate when compared to paired nasopharyngeal and oropharyngeal swab specimens (NPOP). With the assistance of a specimen sampling device, QuickSpit(TM), collection was found to be more convenient for individuals and improved agreement rate to 90%.

scholarly journals An Improved Multiplex IC-RT-PCR Assay Distinguishes Nine Strains of Potato virus Y

Plant Disease ◽  
2013 ◽  
Vol 97 (10) ◽  
pp. 1370-1374 ◽  
Author(s):  
Mohamad Chikh-Ali ◽  
Stewart M. Gray ◽  
Alexander V. Karasev
Keyword(s):  
Potato Virus ◽  
Potato Virus Y ◽  
Rna Extraction ◽  
Leaf Tissue ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Potato Leaf ◽  
Extraction Step ◽  
First Time ◽  
Virus Testing

A multiplex reverse-transcription polymerase chain reaction (RT-PCR) assay was previously developed to identify a group of Potato virus Y (PVY) isolates with unusual recombinant structures (e.g., PVYNTN-NW and SYR-III) and to differentiate them from other PVY strains. In the present study, the efficiency of this multiplex RT-PCR assay was validated and extended considerably to include five additional strains and strain groups not tested before. To make the multiplex RT-PCR assay more applicable and suitable for routine virus testing and typing, it was modified by replacing the conventional RNA extraction step with the immunocapture (IC) procedure. The results obtained using well-characterized reference isolates revealed, for the first time, that this multiplex RT-PCR assay is an accurate and robust method to identify and differentiate the nine PVY strains reported to date, including PVYO (both PVYO and PVYO-O5), PVYN, PVYNA-N, PVYNTN, PVYZ, PVYE, PVY-NE11, PVYN-Wi, and PVYN:O, which is not possible by any of the previously reported RT-PCR procedures. This would make the IC-RT-PCR procedure presented here a method of choice to identify PVY strains and assess the strain composition of PVY in a given area. The IC-RT-PCR protocol was successfully applied to typing PVY isolates in potato leaf tissue collected in the field.

Development of a TaqMan® RT-PCR assay without RNA extraction step for the detection and quantification of African Chikungunya viruses

2005 ◽  
Vol 124 (1-2) ◽  
pp. 65-71 ◽  
Author(s):  
Boris Pastorino ◽  
Maël Bessaud ◽  
Marc Grandadam ◽  
Severine Murri ◽  
Hugues J. Tolou ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Extraction Step ◽  
Detection And Quantification

scholarly journals Clinical Characteristics of the First 100 Patients of COVID-19 in Tobruk, Libya: A Brief Report from Low-Resource Settings

2021 ◽  
pp. 1-10
Author(s):  
Faisal Ismail ◽  
Atiya Farag ◽  
Soghra Haq ◽  
Mohammad Amjad Kamal
Keyword(s):  
Rt Pcr ◽  
Antibody Testing ◽  
Low Resource Settings ◽  
Low Resource ◽  
Presenting Symptoms ◽  
Negative Results ◽  
Chest X Ray ◽  
Screening Clinic ◽  
Loss Of Appetite ◽  
Rule Out

ABSTRACT Objective: This study aims to report the clinical features of a cohort of patients with suspected COVID-19 from Tobruk, Libya and reflect upon the diagnosis challenge in low-resource settings. Methods: A descriptive report of the first 100 patients with suspected COVID-19 who have visited the SARS-Cov-2 (Severe Acute Respiratory Syndrome-related Coronavirus-2) (COVID-19) screening clinic at the National Centre for Disease Control (NCDC) in Tobruk, Libya. Results: The most common presenting symptoms were fever (90%), cough (89%), dyspnoea (85%), sore throat (79%), fatigue (78%), headache (64%), loss of smell (52%), loss of taste (53%), loss of appetite (43), nausea and vomiting (26%), diarrhoea (22%), and rhinorrhea (16%). 51% of the patients had lymphocytopenia while 13% had thrombocytopenia. Bilateral infiltrates was the most common radiologic finding on chest X-ray (76%), and COVID-19 IgM and /or IgG antibodies were detected in 80% of the patients, while only 37% of the patients were tested positive by the RT-PCR. Conclusions: The disease continued its spread across the region. Fever, cough and dyspnoea were the main symptoms. 21% of the patients did not have any CXR abnormalities. Initial negative results for either antibody testing or RT-PCR-testing for COVID-19 do not rule out the infection.

scholarly journals Rapid molecular detection of Zika virus in urine using the recombinase polymerase amplification assay

2016 ◽  
Author(s):  
Ahmed Abd El Wahed ◽  
Sabri S. Sanabani ◽  
Oumar Faye ◽  
Rodrigo Pessôa ◽  
João Veras Patriota ◽  
...  
Keyword(s):  
Real Time ◽  
Zika Virus ◽  
Recombinase Polymerase Amplification ◽  
Control Measures ◽  
Mosquito Vector ◽  
Rt Pcr ◽  
Low Resource Settings ◽  
Low Resource ◽  
Amplification Assay ◽  
Recombinase Polymerase Amplification Assay

AbstractBackgroundCurrently the detection of Zika virus (ZIKV) in patient samples is done by real-time RT-PCR. Samples collected from rural area are sent to highly equipped laboratories for screening. A rapid point-of-care test is needed to detect the virus, especially at low resource settings.Methodology/Principal FindingsIn this report, we describe the development of a reverse transcription isothermal recombinase polymerase amplification (RT-RPA) assay for the identification of ZIKV. RT-RPA assay was portable, sensitive (21 RNA molecules), and rapid (3-15 minutes). No cross-reactivity was detected to other flaviviruses, alphaviruses and arboviruses. Compared to real-time RT-PCR, the diagnostic sensitivity was 92%, while the specificity was 100%.Conclusions/SignificanceThe developed assay is a promising platform for rapid point of need detection of ZIKV in low resource settings and elsewhere (e.g. during mass gathering).Author SummaryCurrently, Dengue (DENV), Zika (ZIKV) and Chikungunya (CHIKV) viruses represent a global threat. The clinical picture of the acute febrile diseases caused by DENV, ZIKV and CHIKV is very similar, in addition, the same mosquito vector is involved in the transmission cycle. The differentiation between them is of great importance as supportive treatment differs and the identification of any of the three viruses prompts implementation of control measures to avoid spreading of an outbreak. We have developed an assay for the detection of ZIKV genome. The assay based on isothermal “recombinase polymerase amplification” assay, which was performed at one temperature (42°C). The result was obtained in maximum of 15 minutes. Moreover, the assay is easy to be implemented at low resource settings.

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

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.

scholarly journals A Rapid COVID-19 RT-PCR Detection Assay for Low Resource Settings

2020 ◽  
Author(s):  
Arunkumar Arumugam ◽  
Matthew L. Faron ◽  
Peter Yu ◽  
Cole Markham ◽  
Season Wong
Keyword(s):  
Gold Standard ◽  
Rna Isolation ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Low Resource Settings ◽  
Low Resource ◽  
Detection Assay ◽  
The World ◽  
Control And Prevention ◽  
Polymerase Chain

ABSTRACTQuantitative reverse transcription polymerase chain reaction (RT-qPCR) assay is the gold standard recommended to test for acute SARS-CoV-2 infection. It has been used by the Centers for Disease Control and Prevention (CDC) and several other companies in their Emergency Use Authorization (EUA) assays. RT-qPCR requires expensive equipment such as RNA isolation instruments and real-time PCR thermal cyclers, which are not available in many low resource settings and developing countries. As a pandemic, COVID-19 has quickly spread to the rest of the world. Many underdeveloped and developing counties do not have the means for fast and accurate COVID-19 detection to control this outbreak. Using COVID-19 positive clinical specimens, we demonstrated that RT-PCR assays can be performed in as little as 12 minutes using untreated samples, heat-inactivated samples, or extracted RNA templates. Rapid RT-PCR was achieved using thin-walled PCR tubes and a setup including sous vide immersion heaters/circulators. Our data suggest that rapid RT-PCR can be implemented for sensitive and specific molecular diagnosis of COVID-19 in situations where sophisticated laboratory instruments are not available.

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.

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