scholarly journals A simple magnetic nanoparticles-based viral RNA extraction method for efficient detection of SARS-CoV-2

2020 ◽  
Author(s):  
Zhen Zhao ◽  
Haodong Cui ◽  
Wenxing Song ◽  
Xiaoling Ru ◽  
Wenhua Zhou ◽  
...  
Keyword(s):  
Magnetic Nanoparticles ◽  
Clinical Diagnosis ◽  
Molecular Diagnosis ◽  
Extraction Method ◽  
Rna Extraction ◽  
Viral Rna ◽  
Rt Pcr ◽  
Efficient Detection ◽  
Novel Coronavirus ◽  
Rna Complexes

1AbstractThe ongoing outbreak of the novel coronavirus disease 2019 (COVID-19) originating from Wuhan, China, draws worldwide concerns due to its long incubation period and strong infectivity. Although RT-PCR-based molecular diagnosis techniques are being widely applied for clinical diagnosis currently, timely and accurate diagnosis are still limited due to labour intensive and time-consuming operations of these techniques. To address the issue, herein we report the synthesis of poly (amino ester) with carboxyl groups (PC)-coated magnetic nanoparticles (pcMNPs), and the development of pcMNPs-based viral RNA extraction method for the sensitive detection of COVID-19 causing virus, the SARS-CoV-2. This method combines the lysis and binding steps into one step, and the pcMNPs-RNA complexes can be directly introduced into subsequent RT-PCR reactions. The simplified process can purify viral RNA from multiple samples within 20 min using a simple manual method or an automated high-throughput approach. By identifying two different regions (ORFlab and N gene) of viral RNA, a 10-copy sensitivity and a strong linear correlation between 10 and 105 copies of SARS-CoV-2 pseudovirus particles are achieved. Benefitting from the simplicity and excellent performances, this new extraction method can dramatically reduce the turn-around time and operational requirements in current molecular diagnosis of COVID-19, in particular for the early clinical diagnosis.

scholarly journals Detection of Schmallenberg Virus RNA in Bull Semen in Poland

2016 ◽  
Vol 19 (3) ◽  
pp. 655-657 ◽  
Author(s):  
J. Kęsik-Maliszewska ◽  
M. Larska
Keyword(s):  
Extraction Method ◽  
Rna Extraction ◽  
Viral Rna ◽  
Schmallenberg Virus ◽  
Rt Pcr ◽  
Bovine Semen ◽  
Bull Semen ◽  
Virus Rna ◽  
Virus Excretion ◽  
Venereal Transmission

Abstract The detection of Schmallenberg virus (SBV) in the breeding bull semen raised the question of the possibility of venereal transmission of SBV which could result in cost-intensive restrictions in the trade of bovine semen. In order to evaluate the presence of SBV RNA in bovine semen, 131 bull semen samples from four locations in Poland collected between 2013 and 2015 were analysed by RT-PCR for viral RNA. SBV RNA was detected in 5.3% of the samples. The study has revealed that application of an appropriate RNA extraction method is crucial to detect virus excretion via semen.

Microelectrothermofluidic packaging for single chip integrated viral RNA extraction and RT-PCR microdevices

2010 ◽  
Author(s):  
Tae Goo Kang ◽  
Hong Miao Ji ◽  
Siow Pin Melvin Tan ◽  
Guang Kai Ignatius Tay ◽  
Ming Yi Daniel Ang ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Viral Rna ◽  
Single Chip ◽  
Rt Pcr

A survey of the prevalence of infectious bronchitis virus type 4/91 in Iran

2004 ◽  
Vol 52 (2) ◽  
pp. 163-166 ◽  
Author(s):  
M. R. Seyfi Abad Shapouri ◽  
M. Mayahi ◽  
K. Assasi ◽  
S. Charkhkar
Keyword(s):  
Virus Type ◽  
Infectious Bronchitis Virus ◽  
Nested Pcr ◽  
Vaccination Programme ◽  
Rna Extraction ◽  
Viral Rna ◽  
Rt Pcr ◽  
Infectious Bronchitis ◽  
High Prevalence

To evaluate the prevalence of infectious bronchitis virus (IBV) type 4/91 in Iran, tracheal swabs from 77 broiler flocks in 16 provinces were collected at the slaughterhouse. Swabs were subjected to RNA extraction and tested by RT-PCR, followed by a type-specific nested PCR. The viral RNA was detected in 33 samples (42.8%) from different provinces. The results indicate a relatively high prevalence of IBV type 4/91 in Iran and necessitate revising the vaccination programme against this disease.

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 Biosafety level-2 laboratory diagnosis of Zaire Ebola virus disease imported from Liberia to Nigeria

2016 ◽  
Vol 5 (1) ◽  
Author(s):  
Olumuyiwa B. Salu ◽  
Ayorinde B. James ◽  
Bamidele O. Oke ◽  
Mercy R. Orenolu ◽  
Roosevelt A. Anyanwu ◽  
...  
Keyword(s):  
Ebola Virus ◽  
Virus Disease ◽  
Rna Extraction ◽  
Molecular Techniques ◽  
Viral Rna ◽  
Sub Saharan Africa ◽  
Ebola Virus Disease ◽  
Rt Pcr ◽  
Route Of Transmission ◽  
Biosafety Level

Introduction: Global travel is an efficient route of transmission for highly infectious pathogens and increases the chances of such pathogens moving from high disease-endemic areas to new regions. We describe the rapid and safe identification of the first imported case of Ebola virus disease in a traveler to Lagos, Nigeria, using conventional reverse transcription polymerase chain reaction (RT-PCR) in a biosafety level (BSL)-2 facility.Case presentation: On 20 July 2014, a traveler arrived from Liberia at Lagos International Airport and was admitted to a private hospital in Lagos, with clinical suspicion of Ebola virus disease.Methodology and Outcome: Blood and urine specimens were collected, transported to the Virology Unit Laboratory at the College of Medicine, University of Lagos, and processed under stringent biosafety conditions for viral RNA extraction. RT-PCR was set-up to query the Ebola, Lassa and Dengue fever viruses. Amplicons for pan-filoviruses were detected as 300 bp bands on a 1.5% agarose gel image; there were no detectable bands for Lassa and Dengue viral RNA. Nucleotide BLAST and phylogenetic analysis of sequence data of the RNA-dependent RNA polymerase (L) gene confirmed the sequence to be Zaire ebolavirus (EBOV/Hsap/ NGA/2014/LIB-NIG 01072014; Genbank: KM251803.1).Conclusion: Our BSL-2 facility in Lagos, Nigeria, was able to safely detect Ebola virus disease using molecular techniques, supporting the reliability of molecular detection of highly infectious viral pathogens under stringent safety guidelines in BSL-2 laboratories. This is a significant lesson for the many under-facilitated laboratories in resource-limited settings, as is predominantly found in sub-Saharan Africa.

scholarly journals Molecular diagnosis of SARS-CoV-2 in seminal fluid

2021 ◽  
Author(s):  
D. Paoli ◽  
F. Pallotti ◽  
G. Nigro ◽  
L. Mazzuti ◽  
M. N. Hirsch ◽  
...  
Keyword(s):  
Molecular Diagnosis ◽  
Seminal Plasma ◽  
Reproductive Medicine ◽  
Seminal Fluid ◽  
Rna Extraction ◽  
Nasopharyngeal Swab ◽  
Control Panel ◽  
Rt Pcr ◽  
Sperm Bank ◽  
University Of Rome

Abstract Purpose Due to relevant repercussions on reproductive medicine, we aimed to evaluate feasibility of RT-PCR as a detection method of SARS-CoV-2 RNA in seminal fluid. Methods A qualitative determination of the RT-PCR assays in semen was performed through different approaches: (1) efficiency of RNA extraction from sperm and seminal plasma was determined using PRM1 and PRM2 mRNA and a heterologous system as control; (2) samples obtained by diluting viral preparation from a SARS-CoV-2 panel (virus cultured in Vero E6 cell lines) were tested; (3) viral presence in different fractions of seminal fluid (whole sample, seminal plasma and post-centrifugation pellet) was evaluated. Semen samples from mild and recovered COVID-19 subjects were collected by patients referring to the Infectious Disease Department of the Policlinico Umberto I Hospital - “Sapienza” University of Rome. Control subjects were recruited at the Laboratory of Seminology-Sperm Bank “Loredana Gandini'' of the same hospital. Results The control panel using viral preparations diluted in saline and seminal fluid showed the capability to detect viral RNA presence with Ct values depending on the initial viral concentration. All tested semen samples were negative for SARS-CoV-2, regardless of the nasopharyngeal swab result or seminal fluid fraction. Conclusion These preliminary data show that RT-PCR for SARS-CoV-2 RNA testing appears to be a feasible method for the molecular diagnosis of SARS-CoV-2 in seminal fluid, supported by results of the control panel. The ability to detect SARS-CoV-2 in semen is extremely important for reproductive medicine, especially in assisted reproductive technology and sperm cryopreservation.

scholarly journals Surveillance and correlation of SARS-CoV-2 viral RNA, antigen, virus isolation, and self-reported symptoms in a longitudinal study with daily sampling

2021 ◽  
Author(s):  
Gaston Bonenfant ◽  
Jessica Deyoe ◽  
Terianne Wong ◽  
Carlos G. Grijalva ◽  
H. Keipp Talbot ◽  
...  
Keyword(s):  
Positive Culture ◽  
Viral Rna ◽  
Rt Pcr ◽  
Predictive Values ◽  
Qrt Pcr ◽  
Household Transmission ◽  
Daily Sampling ◽  
Diagnostic Panel ◽  
Pcr Assays ◽  
Novel Coronavirus

The novel coronavirus pandemic incited unprecedented demand for assays that detect viral nucleic acids, viral proteins, and corresponding antibodies. The 320 molecular diagnostics in receipt of FDA emergency use authorization mainly focus on viral detection; however, no currently approved test can be used to infer infectiousness, i.e., the presence of replicable virus. As the number of tests conducted increased, persistent SARS-CoV-2 RNA positivity by RT-PCR in some individuals led to concerns over quarantine guidelines. To this end, we attempted to design an assay that reduces the frequency of positive test results from individuals who do not shed culturable virus. We describe multiplex quantitative RT-PCR (qRT-PCR) assays that detect genomic RNA (gRNA) and subgenomic RNA (sgRNA) species of SARS-CoV-2, including spike (S), nucleocapsid (N), membrane (M), envelope (E), and ORF8. The absolute copy number of each RNA target was determined in longitudinal specimens from a household transmission study. Calculated viral RNA levels over the 14-day follow up period were compared with antigen testing and self-reported symptoms to characterize the clinical and molecular dynamics of infection and infer predictive values of these qRT-PCR assays relative to culture isolation. When detection of sgS RNA was added to the CDC 2019-Novel Coronavirus Real-Time RT-PCR Diagnostic Panel, we found a qRT-PCR positive result was 98% predictive of a positive culture (negative predictive value was 94%). Our findings suggest sgRNA presence correlates with active infection, may help identify individuals shedding culturable virus, and that similar multiplex assays can be adapted to current and future variants.

scholarly journals Evaluation of the efficacy of LAMP-based SARS-CoV-2 detection with simple RNA extraction from nasopharyngeal swabs: A prospective observational study

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0260732
Author(s):  
Masaki Karino ◽  
Mizuki Harada ◽  
Chihiro Yamada ◽  
Kyoko Fukuoka ◽  
Megumi Sugo ◽  
...  
Keyword(s):  
Extraction Method ◽  
Prospective Observational Study ◽  
Medical Center ◽  
Rna Extraction ◽  
Extraction Methods ◽  
Viral Rna ◽  
Nasopharyngeal Swab ◽  
Measurement Principle ◽  
Comparison Of The Results ◽  
Reliable Testing

The Loopamp SARS-CoV-2 Detection Kit is used for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Loop-mediated isothermal amplification (LAMP) is based on a measurement principle that can be used with a relatively simple device. Detection using this kit requires viral RNA extraction from samples with the QIAGEN QIAamp Viral Mini Kit (QIAGEN extraction) or the Loopamp Viral RNA Extraction Kit (Eiken extraction), which are recommended by the manufacturer. However, the efficacy of LAMP-based SARS-CoV-2 detection using these extraction methods has not been compared. In this study, we aimed to compare the results of genome extraction and detection from nasopharyngeal swab samples using the QIAGEN and Eiken extraction kits. The present study involved patients who presented to the Rinku General Medical Center with suspected COVID-19 (25 positive and 26 negative cases). A comparison of the results obtained using each extraction method with those obtained via PCR showed that the positive, negative, and overall concordance rates between QIAGEN extraction and PCR were 96.0% (24/25 samples), 100% (26/26), and 98.0% (50/51; κ = 0.96, 95% CI = 0.69–1.00), respectively. Results with Eiken extraction were also favorable, with positive, negative, and overall concordance rates of 88.0% (22/25), 100% (26/26), and 94.1% (48/51; κ = 0.88, 95% CI = 0.61–1.00), respectively. Favorable results were obtained using both QIAGEN and Eiken extraction kits. Since Eiken extraction can be completed in a few minutes, it enables prompt and reliable testing for SARS-CoV-2 detection.

scholarly journals Isolation and Detection of Enterovirus RNA from Large-Volume Water Samples by Using the NucliSens miniMAG System and Real-Time Nucleic Acid Sequence-Based Amplification

2005 ◽  
Vol 71 (7) ◽  
pp. 3734-3740 ◽  
Author(s):  
Saskia A. Rutjes ◽  
Ronald Italiaander ◽  
Harold H. J. L. van den Berg ◽  
Willemijn J. Lodder ◽  
Ana Maria de Roda Husman
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Large Volume ◽  
Water Samples ◽  
Extraction Method ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Rt Pcr ◽  
Virus Rna ◽  
Nasba Assay

ABSTRACT Concentration of water samples is a prerequisite for the detection of the low virus levels that are present in water and may present a public health hazard. The aim of this study was to develop a rapid, standardized molecular method for the detection of enteroviruses in large-volume surface water samples, using a concentration method suitable for the detection of infectious viruses as well as virus RNA. Concentration of water was achieved by a conventional filter adsorption-elution method and ultrafiltration, resulting in a 10,000-fold concentration of the sample. Isolation of virus RNA by a silica-based RNA extraction method was compared with the nonmagnetic and magnetic NucliSens RNA isolation methods. By using the silica-based RNA extraction method in two out of five samples, enterovirus RNA was detected, whereas four out of five samples were positive following RNA isolation with magnetic silica beads. Moreover, estimated RNA levels increased at least 100 to 500 times. Furthermore, we compared enterovirus detection by an in-house reverse transcription (RT)-PCR with a novel commercially available real-time nucleic acid sequence-based amplification (NASBA) assay. We found that the rapid real-time NASBA assay was slightly less sensitive than our in-house RT-PCR. The advantages, however, of a commercial real-time NASBA assay, like the presence of an internal control RNA, standardization, and enormous decrease in turnaround time, makes it an attractive alternative to RT-PCR.

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