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 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 DIRECT RT-qPCR DETECTION OF SARS-CoV-2 RNA FROM PATIENT NASOPHARYNGEAL SWABS WITHOUT AN RNA EXTRACTION STEP

2020 ◽  
Author(s):  
Emily A. Bruce ◽  
Meei-Li Huang ◽  
Garrett A. Perchetti ◽  
Scott Tighe ◽  
Pheobe Laaguiby ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Diagnostic Testing ◽  
Rna Extraction ◽  
World Health ◽  
Clinical Samples ◽  
Extraction Step ◽  
The World ◽  
Control And Prevention ◽  
Polymerase Chain ◽  
Health Organization

ABSTRACTThe ongoing COVID-19 pandemic has caused an unprecedented need for rapid diagnostic testing. The Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) recommend a standard assay that includes an RNA extraction step from a nasopharyngeal (NP) swab followed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect the purified SARS-CoV-2 RNA. The current global shortage of RNA extraction kits has caused a severe bottleneck to COVID-19 testing. We hypothesized that SARS-CoV-2 RNA could be detected from NP samples via a direct RT-qPCR assay that omits the RNA extraction step altogether, and tested this hypothesis on a series of blinded clinical samples. The direct RT-qPCR approach correctly identified 92% of NP samples (n = 155) demonstrated to be positive for SARS-CoV-2 RNA by traditional clinical diagnostic RT-qPCR that included an RNA extraction. Thus, direct RT-qPCR could be a front-line approach to identify the substantial majority of COVID-19 patients, reserving a repeat test with RNA extraction for those individuals with high suspicion of infection but an initial negative result. This strategy would drastically ease supply chokepoints of COVID-19 testing and should be applicable throughout the world.

scholarly journals Low-cost protocol for rapid detection of ZIKV from patient and mosquito samples using a direct-RT-qPCR assay without RNA extraction step

2021 ◽  
Author(s):  
Severino Silva ◽  
Renata Mendes ◽  
Jurandy Magalhães ◽  
Elisa Azevedo ◽  
Marília Sena ◽  
...  
Keyword(s):  
Rapid Detection ◽  
Low Cost ◽  
Rna Extraction ◽  
Qpcr Assay ◽  
Extraction Step

scholarly journals SARS-CoV-2 Direct Detection Without RNA Isolation With Loop-Mediated Isothermal Amplification (LAMP) and CRISPR-Cas12

2021 ◽  
Vol 8 ◽  
Author(s):  
Alfredo Garcia-Venzor ◽  
Bertha Rueda-Zarazua ◽  
Eduardo Marquez-Garcia ◽  
Vilma Maldonado ◽  
Angelica Moncada-Morales ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Direct Detection ◽  
Direct Method ◽  
Rna Extraction ◽  
Rna Isolation ◽  
High Sensitivity ◽  
High Specificity ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification

As to date, more than 49 million confirmed cases of Coronavirus Disease 19 (COVID-19) have been reported worldwide. Current diagnostic protocols use qRT-PCR for viral RNA detection, which is expensive and requires sophisticated equipment, trained personnel and previous RNA extraction. For this reason, we need a faster, direct and more versatile detection method for better epidemiological management of the COVID-19 outbreak. In this work, we propose a direct method without RNA extraction, based on the Loop-mediated isothermal amplification (LAMP) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein (CRISPR-Cas12) technique that allows the fast detection of SARS-CoV-2 from patient samples with high sensitivity and specificity. We obtained a limit of detection of 16 copies/μL with high specificity and at an affordable cost. The diagnostic test readout can be done with a real-time PCR thermocycler or with the naked eye in a blue-light transilluminator. Our method has been evaluated on a small set of clinical samples with promising results.

scholarly journals A mild-symptom COVID-19 patient showed consistently high pharyngeal viral loads in a period of ten days after hospitalization

2020 ◽  
Author(s):  
Dong Chen ◽  
Shuqing Han ◽  
Yue Sun ◽  
Yisong Shen ◽  
Xiaofei Zhou ◽  
...  
Keyword(s):  
Health Care ◽  
Rna Extraction ◽  
High Viral Load ◽  
Pcr Analysis ◽  
Peak Time ◽  
Upper Respiratory Tract ◽  
Viral Loads ◽  
Care Workers ◽  
Extraction Step ◽  
Temporal Profiles

Abstract Background: The pandemics of coronavirus disease 2019 (COVID-19) threatens both human lives and health care system. COVID-19 patients may differ in their capability in spreading the causative virus, the severe acute respiratory syndrome-corona virus 2 (SARS-CoV-2). Methods: In this study, oropharyngeal swabs specimens from 43 patients admitted to our hospital during the COVID-19 peak time in Wuhan, China were obtained to survey temporal profiles of the viral loads in their upper respiratory tract. An internal and an absolute mRNA control were included in the real-time RT-PCR analysis and RNA extraction step to remove the potential influence of experimental variations on the result interpretation. Results: We found about one third of the hospitalized COVID-19 patients were never tested as SARS-CoV-2 positive during the course of this study. One patient with mild symptoms displayed constant high levels of viral loads after hospitalization, which were orders of magnitude higher than all other positive patients. Conclusions: We propose that if pharyngeal viral loads in a patient could indicate its ability in spreading the virus to others, then identification and strict separation of the high viral load patients should provide an effective mean in restricting viral spreading and protect health care workers from infection.

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 Detection of Viable Cryptosporidium parvum in Soil by Reverse Transcription–Real-Time PCR Targetinghsp70mRNA

2011 ◽  
Vol 77 (18) ◽  
pp. 6476-6485 ◽  
Author(s):  
Zhanbei Liang ◽  
Ann Keeley
Keyword(s):  
Cryptosporidium Parvum ◽  
Reverse Transcription ◽  
Rna Binding ◽  
Direct Detection ◽  
Rna Extraction ◽  
Milk Powder ◽  
Extraction Methods ◽  
Content Type ◽  
Total Rna ◽  
Rna Extraction Methods

ABSTRACTExtraction of high-quality mRNA fromCryptosporidium parvumis a key step in PCR detection of viable oocysts in environmental samples. Current methods for monitoring oocysts are limited to water samples; therefore, the goal of this study was to develop a rapid and sensitive procedure forCryptosporidiumdetection in soil samples. The efficiencies of five RNA extraction methods were compared (mRNA extraction with the Dynabeads mRNA Direct kit after chemical and physical sample treatments, and total RNA extraction methods using the FastRNA Pro Soil-Direct, PowerSoil Total RNA, E.Z.N.A. soil RNA, and Norgen soil RNA purification kits) for the direct detection ofCryptosporidiumwith oocyst-spiked sandy, loamy, and clay soils by using TaqMan reverse transcription-PCR. The study also evaluated the presence of inhibitors by synthesis and incorporation of an internal positive control (IPC) RNA into reverse transcription amplifications, used different facilitators (bovine serum albumin, yeast RNA, salmon DNA, skim milk powder, casein, polyvinylpyrrolidone, sodium hexametaphosphate, andSalmonella entericaserovar Typhi) to mitigate RNA binding on soil components, and applied various treatments (β-mercaptoethanol and bead beating) to inactivate RNase and ensure the complete lysis of oocysts. The results of spiking studies showed thatSalmonellacells most efficiently relieved binding of RNA. With the inclusion ofSalmonelladuring extraction, the most efficient mRNA method was Dynabeads, with a detection limit of 6 × 102oocysts g−1of sandy soil. The most efficient total RNA method was PowerSoil, with detection limits of 1.5 × 102, 1.5 × 103, and 1.5 × 104C. parvumoocysts g−1soil for sandy, loamy, and clay samples, respectively.

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.

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