SARS-CoV-2 Isolation From Ocular Secretions of a Patient With COVID-19 in Italy With Prolonged Viral RNA Detection

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.

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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 ◽

10.3390/pathogens10121558 ◽

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.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2011221117 ◽

2020 ◽

Vol 117 (39) ◽

pp. 24450-24458 ◽

Cited By ~ 10

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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DEVELOPMENT AND ASSESSMENT OF A REAGENT KIT FOR RNA DETECTION OF CRIMEAN-CONGO HEMORRHAGIC FEVER VIRUS WITH USING REVERSE TRANSCRIPTION LOOP-MEDIATED ISOTHERMAL AMPLIFICATION METHOD

Russian Clinical Laboratory Diagnostics ◽

10.18821/0869-2084-2019-64-9-571-577 ◽

2019 ◽

Vol 64 (9) ◽

pp. 571-577

Author(s):

V. A. Ternovoi ◽

Yu. V. Kononova ◽

A. V. Zaykovskaya ◽

E. V. Chub ◽

A. S. Volynkina ◽

...

Keyword(s):

Reverse Transcription ◽

High Speed ◽

Hemorrhagic Fever ◽

Fever Virus ◽

Isothermal Amplification ◽

Viral Rna ◽

Rna Detection ◽

Crimean Congo Hemorrhagic Fever ◽

Loop Mediated Isothermal Amplification ◽

Hemorrhagic Fever Virus

This study presents the results of laboratory trials of the reagent kit for the rapid detection of RNA of the Crimean-Congo hemorrhagic fever virus (CCHFV) using loop-mediated isothermal amplification with reverse transcription (RT-LAMP). The developed RT-LAMP reagent kit was used to detect the CCHFV and showed a sensitivity of 103 GE/ml of viral RNA, which is sufficient for detection of the CCHFV in the early stage of human infections. The kit showed high specificity and no cross-reactivity with viral panel from the State collection of viruses of the FBRI SRC VB «Vector» (arboviruses and hemorrhagic fever viruses). Laboratory trials of the RT-LAMP kit are showed a high analytical and diagnostic sensitivity and specificity for RNA detection of the CCHFV and high speed of the analysis (60-70 min with sample preparation) compared to real-time PCR. Approbation of the kit field version has showed the possibility of setting the RT-LAMP reaction and viral RNA detection without the using of analytical equipments.

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Viral RNA detection by RIG-I-like receptors

Current Opinion in Immunology ◽

10.1016/j.coi.2014.12.012 ◽

2015 ◽

Vol 32 ◽

pp. 48-53 ◽

Cited By ~ 228

Author(s):

Mitsutoshi Yoneyama ◽

Koji Onomoto ◽

Michihiko Jogi ◽

Teppei Akaboshi ◽

Takashi Fujita

Keyword(s):

Viral Rna ◽

Rna Detection

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Inexpensive and colorimetric RNA detection by E. coli cell-free protein synthesis platform at room temperature

10.1101/2021.11.29.21267025 ◽

2021 ◽

Author(s):

Michela Notarangelo ◽

Alessandro Quattrone ◽

Massimo Pizzato ◽

Sheref S. Mansy ◽

O. Duhan Toparlak

Keyword(s):

Room Temperature ◽

Colorimetric Detection ◽

In Vitro Transcription ◽

Viral Rna ◽

Rna Sequences ◽

Rna Detection ◽

E Coli ◽

Free Gene ◽

Cell Free Protein Synthesis

We report colorimetric detection of SARS-CoV-2 viral RNA by an in vitro transcription/translation assay with crude E. coli extracts at room temperature, with the aid of body heat. Clinically-relevant concentrations of viral RNA (ca. 600 copies/test) were detected from synthetic RNA samples. The activation of cell-free gene expression was achieved by toehold-switch-mediated riboregulatory elements that are specific to viral RNA sequences. The colorimetric output was generated by the α-complementation of β-galactosidase ω-fragment (LacZ-ω) with cell-free expressed LacZ-α, using an X-gal analogue as a substrate. The estimated cost of single reaction is less than 1 euro/test, which may facilitate diagnostic kit accessibility in developing countries.

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Saliva sampling is an excellent option to increase the number of SARS CoV2 diagnostic tests in settings with supply shortages

10.1101/2020.06.24.170324 ◽

2020 ◽

Author(s):

Joaquín Moreno-Contreras ◽

Marco A. Espinoza ◽

Carlos Sandoval-Jaime ◽

Marco A. Cantú-Cuevas ◽

Héctor Barón-Olivares ◽

...

Keyword(s):

Diagnostic Tests ◽

Viral Genome ◽

Laboratory Testing ◽

Diagnostic Procedure ◽

Viral Rna ◽

Healthcare Personnel ◽

High Demand ◽

Rna Detection ◽

Golden Standard ◽

Classical Protocol

AbstractAs part of any plan to lift or ease the confinement restrictions that are in place in many different countries, there is an urgent need to increase the capacity of laboratory testing for SARS CoV-2. Detection of the viral genome through RT-qPCR is the golden standard for this test, however, the high demand of the materials and reagents needed to sample individuals, purify the viral RNA, and perform the RT-qPCR test has resulted in a worldwide shortage of several of these supplies. Here, we show that directly lysed saliva samples can serve as a suitable source for viral RNA detection that is cheaper and can be as efficient as the classical protocol that involves column purification of the viral RNA. In addition, it surpasses the need for swab sampling, decreases the risk of the healthcare personnel involved in this process, and accelerates the diagnostic procedure.

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