scholarly journals Culture-Based Virus Isolation To Evaluate Potential Infectivity of Clinical Specimens Tested for COVID-19

2020 ◽  
Vol 58 (8) ◽  
Author(s):  
Chung-Guei Huang ◽  
Kuo-Ming Lee ◽  
Mei-Jen Hsiao ◽  
Shu-Li Yang ◽  
Peng-Nien Huang ◽  
...  
Keyword(s):  
Real Time ◽  
Viral Genome ◽  
Copy Number ◽  
Virus Isolation ◽  
Rt Pcr ◽  
Clinical Specimens ◽  
Reverse Transcription Pcr ◽  
Copy Numbers ◽  
Genome Copy Number ◽  
Virus Culture

ABSTRACT Real-time reverse transcription-PCR (RT-PCR) is currently the most sensitive method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes coronavirus disease 2019 (COVID-19). However, the correlation between detectable viral RNA and culturable virus in clinical specimens remains unclear. Here, we performed virus culture for 60 specimens that were confirmed to be positive for SARS-CoV-2 RNA by real-time RT-PCR. The virus could be successfully isolated from 12 throat and nine nasopharyngeal swabs and two sputum specimens. The lowest copy number required for virus isolation was determined to be 5.4, 6.0, and 5.7 log10 genome copies/ml sample for detecting the nsp12, E, and N genes, respectively. We further examined the correlation of genome copy number and virus isolation in different regions of the viral genome, demonstrating that culturable specimens are characterized by high copy numbers with a linear correlation observed between copy numbers of amplicons targeting structural and nonstructural regions. Overall, these results indicate that in addition to the copy number, the integrity of the viral genome should be considered when evaluating the infectivity of clinical SARS-CoV-2 specimens.

scholarly journals Detection and Typing of Human Pathogenic Hantaviruses by Real-Time Reverse Transcription-PCR and Pyrosequencing

2007 ◽  
Vol 53 (11) ◽  
pp. 1899-1905 ◽  
Author(s):  
Marit Kramski ◽  
Helga Meisel ◽  
Boris Klempa ◽  
Detlev H Krüger ◽  
Georg Pauli ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Hantaan Virus ◽  
Hantavirus Infection ◽  
Sequence Information ◽  
Rt Pcr ◽  
Clinical Specimens ◽  
Reverse Transcription Pcr ◽  
Pcr Assays

Abstract Background: Because the clinical course of human infections with hantaviruses can vary from subclinical to fatal, rapid and reliable detection of hantaviruses is essential. To date, the diagnosis of hantavirus infection is based mainly on serologic assays, and the detection of hantaviral RNA by the commonly used reverse transcription (RT)-PCR is difficult because of high sequence diversity of hantaviruses and low viral loads in clinical specimens. Methods: We developed 5 real-time RT-PCR assays, 3 of which are specific for the individual European hantaviruses Dobrava, Puumala, or Tula virus. Two additional assays detect the Asian species Hantaan virus together with Seoul virus and the American species Andes virus together with Sin Nombre virus. Pyrosequencing was established to provide characteristic sequence information of the amplified hantavirus for confirmation of the RT-PCR results or for a more detailed virus typing. Results: The real-time RT-PCR assays were specific for the respective hantavirus species and optimized to run on 2 different platforms, the LightCycler and the ABI 7900/7500. Each assay showed a detection limit of 10 copies of a plasmid containing the RT-PCR target region, and pyrosequencing was possible with 10 to 100 copies per reaction. With this assay, viral genome could be detected in 16 of 552 (2.5%) specimens of suspected hantavirus infections of humans and mice. Conclusions: The new assays detect, differentiate, and quantify hantaviruses in clinical specimens from humans and from their natural hosts and may be useful for in vitro studies of hantaviruses.

scholarly journals Antigen-based testing but not real-time PCR correlates with SARS-CoV-2 virus culture

2020 ◽  
Author(s):  
Andrew Pekosz ◽  
Charles K. Cooper ◽  
Valentin Parvu ◽  
Maggie Li ◽  
Jeffrey C. Andrews ◽  
...  
Keyword(s):  
Real Time ◽  
Infectious Virus ◽  
Virus Isolation ◽  
Low Cost ◽  
Test Results ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Effective Implementation ◽  
Virus Culture ◽  
Polymerase Chain

SUMMARYIndividuals can test positive for SARS-CoV-2 by real-time polymerase chain reaction (RT-PCR) after no longer being infectious.1-8 Positive SARS-CoV-2 antigen-based testing exhibits a temporal pattern that corresponds with active, replicating virus and could therefore be a more accurate predictor of an individual’s potential to transmit SARS-CoV-2.2,3,9 Using the BD Veritor System for Rapid Detection of SARS-CoV-2 later flow antigen detection test, we demonstrate a higher concordance of antigen-positive test results with the presence of cultured, infectious virus when compared to RT-PCR. When compared to infectious virus isolation, the sensitivity of antigen-based testing is similar to RT-PCR. The correlation between SARS-CoV-2 antigen and SARS-CoV-2 culture represents a significant advancement in determining the risk for potential transmissibility beyond that which can be achieved by detection of SARS-CoV-2 genomic RNA. Coupled with a rapid time-to-result, low cost, and scalability, antigen-based testing should facilitate effective implementation of testing and public health interventions that will better contain COVID-19.

scholarly journals Detection of SARS Coronavirus in Patients with Severe Acute Respiratory Syndrome by Conventional and Real-Time Quantitative Reverse Transcription-PCR Assays

2004 ◽  
Vol 50 (1) ◽  
pp. 67-72 ◽  
Author(s):  
Leo L M Poon ◽  
Kwok Hung Chan ◽  
On Kei Wong ◽  
Timothy K W Cheung ◽  
Iris Ng ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
N Gene ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
Copy Numbers ◽  
Stool Samples ◽  
Pcr Assays

Abstract Background: A novel coronavirus (CoV) was recently identified as the agent for severe acute respiratory syndrome (SARS). We compared the abilities of conventional and real-time reverse transcription-PCR (RT-PCR) assays to detect SARS CoV in clinical specimens. Methods: RNA samples isolated from nasopharyngeal aspirate (NPA; n = 170) and stool (n = 44) were reverse-transcribed and tested by our in-house conventional RT-PCR assay. We selected 98 NPA and 37 stool samples collected at different times after the onset of disease and tested them in a real-time quantitative RT-PCR specific for the open reading frame (ORF) 1b region of SARS CoV. Detection rates for the conventional and real-time quantitative RT-PCR assays were compared. To investigate the nature of viral RNA molecules in these clinical samples, we determined copy numbers of ORF 1b and nucleocapsid (N) gene sequences of SARS CoV. Results: The quantitative real-time RT-PCR assay was more sensitive than the conventional RT-PCR assay for detecting SARS CoV in samples collected early in the course of the disease. Real-time assays targeted at the ORF 1b region and the N gene revealed that copy numbers of ORF 1b and N gene sequences in clinical samples were similar. Conclusions: NPA and stool samples can be used for early diagnosis of SARS. The real-time quantitative RT-PCR assay for SARS CoV is potentially useful for early detection of SARS CoV. Our results suggest that genomic RNA is the predominant viral RNA species in clinical samples.

scholarly journals Sensitive Multiplex Real-Time Reverse Transcription-PCR Assay for the Detection of Human and Animal Noroviruses in Clinical and Environmental Samples

2007 ◽  
Vol 73 (17) ◽  
pp. 5464-5470 ◽  
Author(s):  
Sandro Wolf ◽  
Wendy M. Williamson ◽  
Joanne Hewitt ◽  
Malet Rivera-Aban ◽  
Susan Lin ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Wide Spectrum ◽  
Clinical Diagnostics ◽  
Target Sequence ◽  
Rt Pcr ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
Copy Numbers ◽  
Treated Drinking Water

ABSTRACT In this study, we developed a triplex real-time reverse transcription-PCR (RT-PCR)-based method that detects and distinguishs between noroviruses belonging to genogroups I, II, and III and that targets the junction between the regions of open reading frame 1 (ORF1) and ORF2. This is the first assay to include all three genogroups and the first real-time RT-PCR-based method developed for the detection of bovine noroviruses. The assay was shown to be broadly reactive against a wide spectrum of norovirus genotypes, including GI/1 through GI/7, GII/1 through GII/8, GII/10, GII/12, and GII/17, in different matrices (including fecal specimens, treated and raw sewage, source water, and treated drinking water). The assay is highly sensitive, detecting low copy numbers of plasmids that carry the target sequence. A new bovine norovirus, Bo/NLV/Norsewood/2006/NZL, was identified by this assay and was further genetically characterized. The results implicate a broad range of possible applications, including clinical diagnostics, tracing of fecal contaminants, and due to its sensitivity and broad reactivity, environmental studies.

Multicenter evaluation of the ENTEROVIRUS R-gene™ real-time RT-PCR assay for the detection of enteroviruses in clinical specimens

2010 ◽  
Vol 47 (1) ◽  
pp. 54-59 ◽  
Author(s):  
Sylvie Pillet ◽  
Geneviève Billaud ◽  
Shabir Omar ◽  
Bruno Lina ◽  
Bruno Pozzetto ◽  
...  
Keyword(s):  
Real Time ◽  
R Gene ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Clinical Specimens

scholarly journals Comparative evaluation of conventional RT-PCR and real-time RT-PCR (RRT-PCR) for detection of avian metapneumovirus subtype A

2009 ◽  
Vol 39 (5) ◽  
pp. 1445-1451 ◽  
Author(s):  
Helena Lage Ferreira ◽  
Fernando Rosado Spilki ◽  
Márcia Mercês Aparecida Bianchi dos Santos ◽  
Renata Servan de Almeida ◽  
Clarice Weis Arns
Keyword(s):  
Real Time ◽  
Comparative Evaluation ◽  
Virus Isolation ◽  
Diagnostic Methods ◽  
N Gene ◽  
Genomic Rna ◽  
Rt Pcr ◽  
Avian Metapneumovirus ◽  
Lower Detection ◽  
Subtype A

Avian metapneumovirus (AMPV) belongs to Metapneumovirus genus of Paramyxoviridae family. Virus isolation, serology, and detection of genomic RNA are used as diagnostic methods for AMPV. The aim of the present study was to compare the detection of six subgroup A AMPV isolates (AMPV/A) viral RNA by using different conventional and real time RT-PCR methods. Two new RT-PCR tests and two real time RT-PCR tests, both detecting fusion (F) gene and nucleocapsid (N) gene were compared with an established test for the attachment (G) gene. All the RT-PCR tested assays were able to detect the AMPV/A. The lower detection limits were observed using the N-, F- based RRT-PCR and F-based conventional RT-PCR (10(0.3) to 10¹ TCID50 mL-1). The present study suggests that the conventional F-based RT-PCR presented similar detection limit when compared to N- and F-based RRT-PCR and they can be successfully used for AMPV/A detection.

scholarly journals Comparison of virus isolation using the Vero E6 cell line with real-time RT-PCR assay for the detection of human metapneumovirus

2010 ◽  
Vol 10 (1) ◽  
Author(s):  
Yoko Matsuzaki ◽  
Katsumi Mizuta ◽  
Emi Takashita ◽  
Michiko Okamoto ◽  
Tsutomu Itagaki ◽  
...  
Keyword(s):  
Cell Line ◽  
Real Time ◽  
Virus Isolation ◽  
Human Metapneumovirus ◽  
Rt Pcr ◽  
Pcr Assay

scholarly journals Nuclear-cytoplasmic balance: whole genome duplications induce elevated organellar genome copy number

2021 ◽  
Author(s):  
Matheus Fernandes Gyorfy ◽  
Emma R Miller ◽  
Justin L Conover ◽  
Corrinne E Grover ◽  
Jonathan F Wendel ◽  
...  
Keyword(s):  
Copy Number ◽  
Nuclear Genome ◽  
Plant Genome ◽  
Whole Genome ◽  
Genome Copy ◽  
Relative Copy Number ◽  
Organellar Genome ◽  
Plastid Genomes ◽  
Copy Numbers ◽  
Genome Copy Number

The plant genome is partitioned across three distinct subcellular compartments: the nucleus, mitochondria, and plastids. Successful coordination of gene expression among these organellar genomes and the nuclear genome is critical for plant function and fitness. Whole genome duplication events (WGDs) in the nucleus have played a major role in the diversification of land plants and are expected to perturb the relative copy number (stoichiometry) of nuclear, mitochondrial, and plastid genomes. Thus, elucidating the mechanisms whereby plant cells respond to the cytonuclear stoichiometric imbalance that follow WGDs represents an important yet underexplored question in understanding the evolutionary consequences of genome doubling. We used droplet digital PCR (ddPCR) to investigate the relationship between nuclear and organellar genome copy numbers in allopolyploids and their diploid progenitors in both wheat and Arabidopsis. Polyploids exhibit elevated organellar genome copy numbers per cell, largely preserving the cytonuclear stoichiometry observed in diploids despite the change in nuclear genome copy number. To investigate the timescale over which cytonuclear stoichiometry may respond to WGD, we also estimated organellar genome copy number in Arabidopsis synthetic autopolyploids and in a haploid-induced diploid line. We observed corresponding changes in organellar genome copy number in these laboratory-generated lines, indicating that at least some of the cellular response to cytonuclear stoichiometric imbalance is immediate following WGD. We conclude that increases in organellar genome copy numbers represent a common response to polyploidization, suggesting that maintenance of cytonuclear stoichiometry is an important component in establishing polyploid lineages.

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