One-Step RT-LATE-PCR for mRNA and Viral RNA Detection and Quantification

2010 ◽  
pp. 153-185 ◽  
Author(s):  
Cristina Hartshorn ◽  
Lawrence J. Wangh
Keyword(s):  
Viral Rna ◽  
Rna Detection ◽  
One Step ◽  
Detection And Quantification

scholarly journals Sensitive Detection and Quantification of SARS-CoV-2 by Multiplex Droplet Digital RT-PCR

2020 ◽  
Author(s):  
Remco de Kock ◽  
Mieke Baselmans ◽  
Volkher Scharnhorst ◽  
Birgit Deiman
Keyword(s):  
Simultaneous Detection ◽  
Housekeeping Gene ◽  
Multiplex Assay ◽  
Viral Rna ◽  
Sensitive Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Assay Sensitivity ◽  
One Step ◽  
Detection And Quantification

Abstract Purpose. We aimed to develop a one-step droplet digital RT-PCR (RT-ddPCR) multiplex assay that allows for sensitive detection of SARS-CoV-2 RNA with respect to human derived RNA and could be used for triage and monitoring of Covid-19 patients. Methods. A one step RT-ddPCR multiplex assay was developed for simultaneous detection of SARS-CoV-2 E, RdRp and N viral RNA, and human Rpp30 DNA and GUSB mRNA, for internal nucleic acid (NA) extraction and RT-PCR control. Dilution series of viral RNA transcripts were prepared in water and total NA extract of Covid-19 negative patients. As reference assay, an E-GUSB duplex RT-PCR was used. Results. Assay sensitivity of the RT-PCR assay drastically decreased when SARS-CoV-2 copies were detected in a background of total NA extract compared to water, while the sensitivity of the RT-ddPCR was not affected by the total NA background. GUSB mRNA detection was used to set validity criteria to assure viral RNA and RT-PCR assay quality. In a background of at least 100 GUSB mRNA copies, 5 copies of viral RNA are reliably detectable and 10 copies viral RNA copies are reliably quantifiable. Conclusion. The present study describes a robust and sensitive one-step RT-ddPCR multiplex assay for reliable detection and quantification of SARS-CoV-2 RNA. By determining the fractional abundance of viral RNA with respect to a human housekeeping gene, viral loads from different samples can be compared, what could be used to investigate the infectiveness and to monitor Covid-19 patients.

scholarly journals Do I Have HIV or Not? Lack of RNA Detection and the Case for Sensitive DNA Testing

2020 ◽  
Vol 7 (11) ◽  
Author(s):  
Sandra A Springer ◽  
Silvina Masciotra ◽  
Jeffrey A Johnson ◽  
Sheldon Campbell
Keyword(s):  
Antiretroviral Therapy ◽  
Hiv Infection ◽  
Viral Rna ◽  
Infection Status ◽  
Test Results ◽  
Dna Testing ◽  
Rna Detection ◽  
Hiv Test

Abstract We present a case of a 20-year-old male who had ambiguous HIV test results after entering new provider care and whose status was later complicated by undetectable viral RNA off antiretroviral therapy (ART). Verifying HIV infection status may occasionally require sensitive DNA testing that might need to be considered in diagnostic guidelines to resolve diagnosis and ensure appropriate ART management.

scholarly journals Direct Viral RNA Detection of SARS-CoV-2 and DENV in Inactivated Samples by Real-Time RT-qPCR: Implications for Diagnosis in Resource Limited Settings with Flavivirus Co-Circulation

Pathogens ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1558
Author(s):  
Zhan Qiu Mao ◽  
Mizuki Fukuta ◽  
Jean Claude Balingit ◽  
Thi Thanh Ngan Nguyen ◽  
Co Thach Nguyen ◽  
...  
Keyword(s):  
Real Time ◽  
Early Stage ◽  
Rna Extraction ◽  
Viral Rna ◽  
Clinical Samples ◽  
Heat Inactivation ◽  
Resource Limited Settings ◽  
Rna Detection ◽  
Resource Limited ◽  
Starting Point

The RT-qPCR method remains the gold standard and first-line diagnostic method for the detection of SARS-CoV-2 and flaviviruses, especially in the early stage of viral infection. Rapid and accurate viral detection is a starting point in the containment of the COVID-19 pandemic and flavivirus outbreaks. However, the shortage of diagnostic reagents and supplies, especially in resource-limited countries that experience co-circulation of SARS-CoV-2 and flaviviruses, are limitations that may result in lesser availability of RT-qPCR-based diagnostic tests. In this study, the utility of RNA-free extraction methods was assessed for the direct detection of SARS-CoV-2 and DENV-2 in heat-inactivated or chemical-inactivated samples. The findings demonstrate that direct real-time RT-qPCR is a feasible option in comparison to conventional real-time RT-qPCR based on viral genome extraction-based methods. The utility of heat-inactivation and direct real-time RT-qPCR for SARS-CoV-2, DENV-2 viral RNA detection was demonstrated by using clinical samples of SARS-CoV-2 and DENV-2 and spiked cell culture samples of SARS-CoV-2 and DENV-2. This study provides a simple alternative workflow for flavivirus and SARS-CoV-2 detection that includes heat inactivation and viral RNA extraction-free protocols, with aims to reduce the risk of exposure during processing of SARS-CoV-2 biological specimens and to overcome the supply-chain bottleneck, particularly in resource limited settings with flavivirus co-circulation.

scholarly journals One-step real-time PCR assay for detection and quantification of RNA HCV to monitor patients under treatment in Brazil

2018 ◽  
Vol 22 (5) ◽  
pp. 418-423
Author(s):  
Elisabete Andrade ◽  
Daniele Rocha ◽  
Marcela Fontana-Maurell ◽  
Elaine Costa ◽  
Marisa Ribeiro ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Assay ◽  
One Step ◽  
Detection And Quantification

scholarly journals SARS-CoV-2 detection using isothermal amplification and a rapid, inexpensive protocol for sample inactivation and purification

2020 ◽  
Vol 117 (39) ◽  
pp. 24450-24458 ◽  
Author(s):  
Brian A. Rabe ◽  
Constance Cepko
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Reverse Transcription ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Viral Rna ◽  
Rna Detection ◽  
Sample Stability ◽  
Rapid Inactivation ◽  
Diagnostic Capabilities ◽  
Purification Protocol

The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has had an enormous impact on society worldwide, threatening the lives and livelihoods of many. The effects will continue to grow and worsen if economies begin to open without the proper precautions, including expanded diagnostic capabilities. To address this need for increased testing, we have developed a sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay compatible with current reagents, which utilizes a colorimetric readout in as little as 30 min. A rapid inactivation protocol capable of inactivating virions, as well as endogenous nucleases, was optimized to increase sensitivity and sample stability. This protocol, combined with the RT-LAMP assay, has a sensitivity of at least 50 viral RNA copies per microliter in a sample. To further increase the sensitivity, a purification protocol compatible with this inactivation method was developed. The inactivation and purification protocol, combined with the RT-LAMP assay, brings the sensitivity to at least 1 viral RNA copy per microliter in a sample. This simple inactivation and purification pipeline is inexpensive and compatible with other downstream RNA detection platforms and uses readily available reagents. It should increase the availability of SARS-CoV-2 testing as well as expand the settings in which this testing can be performed.

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