Development of a real-time RT-PCR assay combined with ethidium monoazide treatment for RNA viruses and its application to detect viral RNA after heat exposure

2011 ◽  
Vol 63 (3) ◽  
pp. 502-507 ◽  
Author(s):  
K. Kim ◽  
H. Katayama ◽  
M. Kitajima ◽  
Y. Tohya ◽  
S. Ohgaki
Keyword(s):  
Rna Viruses ◽  
Heat Exposure ◽  
Plaque Assay ◽  
Gel Filtration ◽  
Viral Rna ◽  
Rt Pcr ◽  
Ethidium Monoazide ◽  
Log Reduction ◽  
Spin Column ◽  
Pcr Method

A method was developed for discriminating damaged viruses or naked viral RNA from intact viruses by ethidium monoazide (EMA) treatment before RT-PCR. The applied EMA treatment consisted of three steps: (1) EMA dose, (2) exposure to light, and (3) additional purification by spin-column gel filtration. Approximately 4-log reduction in viral RNA concentration was observed by adding a dose of 10 μg/mL-EMA with 300 s of light irradiation. Although residual EMA can be an inhibitor of RT-PCR, its effect was reduced by spin-column gel filtration or a QIAamp® Viral RNA Mini Kit. EMA-RT-PCR was applied to the thermally treated PV1. Results of EMA-RT-PCR were similar to the plaque assay when PV1 was thermally inactivated. Although this is a preliminary study investigating applicability of the EMA-RT-PCR method for RNA viruses, the results suggest that the method is potentially applicable for the selective detection of epidemiologically important enteric viruses in water such as enteroviruses and noroviruses.

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.

Storage of bovine viral diarrhoea virus samples on filter paper and detection of viral RNA by a RT-PCR method

2001 ◽  
Vol 92 (1) ◽  
pp. 19-22 ◽  
Author(s):  
Štefan Vilček ◽  
Ladislav Strojny ◽  
Branislav Ďurkovič ◽  
Wigbert Rossmanith ◽  
David Paton
Keyword(s):  
Filter Paper ◽  
Bovine Viral Diarrhoea Virus ◽  
Viral Rna ◽  
Bovine Viral Diarrhoea ◽  
Rt Pcr ◽  
Diarrhoea Virus ◽  
Pcr Method

Recovery and Disinfection of Two Human Norovirus Surrogates, Feline Calicivirus and Murine Norovirus, from Hard Nonporous and Soft Porous Surfaces

2015 ◽  
Vol 78 (10) ◽  
pp. 1842-1850 ◽  
Author(s):  
THOMAS YEARGIN ◽  
ANGELA FRASER ◽  
GUOHUI HUANG ◽  
XIUPING JIANG
Keyword(s):  
Sodium Hypochlorite ◽  
Limit Of Detection ◽  
Plaque Assay ◽  
Viral Rna ◽  
Feline Calicivirus ◽  
Murine Norovirus ◽  
Human Norovirus ◽  
Surface Type ◽  
Log Reduction ◽  
Porous Surfaces

Human norovirus is a leading cause of foodborne disease and can be transmitted through many routes, including environmental exposure to fomites. In this study, both the recovery and inactivation of two human norovirus surrogates, feline calicivirus (FCV) and murine norovirus (MNV), on hard nonporous surfaces (glass) and soft porous surfaces (polyester and cotton) were evaluated by both plaque assay and reverse transcription quantitative PCR method. Two disinfectants, sodium hypochlorite (8.25%) and accelerated hydrogen peroxide (AHP, at 4.25%) were evaluated for disinfection efficacy. Five coupons per surface type were used to evaluate the recovery of FCV and MNV by sonication and stomaching and the disinfection of each surface type by using 5 ml of disinfectant for a contact time of 5 min. FCV at an initial titer of ca. 7 log PFU/ml was recovered from glass, cotton, and polyester at 6.2, 5.4, and 3.8 log PFU/ml, respectively, compared with 5.5, 5.2, and 4.1 log PFU/ml, respectively, for MNV with an initial titer of ca. 6 log PFU/ml. The use of sodium hypochlorite (5,000 ppm) was able to inactivate both FCV and MNV (3.1 to 5.5 log PFU/ml) below the limit of detection on all three surface types. AHP (2,656 ppm) inactivated FCV (3.1 to 5.5 log PFU/ml) below the limit of detection for all three surface types but achieved minimal inactivation of MNV (0.17 to 1.37 log PFU/ml). Reduction of viral RNA by sodium hypochlorite corresponded to 2.72 to 4.06 log reduction for FCV and 2.07 to 3.04 log reduction for MNV on all three surface types. Reduction of viral RNA by AHP corresponded to 1.89 to 3.4 log reduction for FCV and 0.54 to 0.85 log reduction for MNV. Our results clearly indicate that both virus and surface types significantly influence recovery efficiency and disinfection efficacy. Based on the performance of our proposed testing method, an improvement in virus recovery will be needed to effectively validate virus disinfection of soft porous surfaces.

scholarly journals Modulation of Dengue Virus Infection in Human Cells by Alpha, Beta, and Gamma Interferons

2000 ◽  
Vol 74 (11) ◽  
pp. 4957-4966 ◽  
Author(s):  
Michael S. Diamond ◽  
T. Guy Roberts ◽  
Dianna Edgil ◽  
Betty Lu ◽  
Joel Ernst ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Plaque Assay ◽  
Cell Types ◽  
Viral Rna ◽  
Rt Pcr ◽  
Cell Infection ◽  
Variable Effect ◽  
Dependent Cell ◽  
Low Passage

ABSTRACT A role for interferon (IFN) in modulating infection by dengue virus (DV) has been suggested by studies in DV-infected patients and IFN receptor-deficient mice. To address how IFN modulates DV type 2 infection, we have assayed IFN-α, -β, and -γ for the ability to enhance or diminish antibody-independent and antibody-dependent cell infection using a competitive, asymmetric reverse transcriptase-mediated PCR (RT-PCR) assay that quantitates positive and negative strands of viral RNA, a flow cytometric assay that measures viral antigen, and a plaque assay that analyzes virion production. Our data suggest that IFN-α and -β protect cells against DV infection in vitro. Treatment of hepatoma cells with IFN-α or -β decreases viral RNA levels greater than 1,000-fold, the percentage of cells infected 90 to 95%, and the amount of infectious virus secreted 150- to 100,000-fold. These results have been reproduced with several cell types and viral strains, including low-passage isolates. In contrast, IFN-γ has a more variable effect depending on the cell type and pathway of infection. Quantitative RT-PCR experiments indicate that IFN inhibits DV infection by preventing the accumulation of negative-strand viral RNA.

Enteric viruses in drinking water supplies

2002 ◽  
Vol 2 (3) ◽  
pp. 17-22
Author(s):  
A.P. Wyn-Jones ◽  
J. Watkins ◽  
C. Francis ◽  
M. Laverick ◽  
J. Sellwood
Keyword(s):  
Drinking Water ◽  
Heavy Rainfall ◽  
Liquid Culture ◽  
Plaque Assay ◽  
Enteric Viruses ◽  
Enteric Virus ◽  
The Other ◽  
Raw Water ◽  
Rt Pcr ◽  
Water Supplies

Two rural spring drinking water supplies were studied for their enteric virus levels. In one, serving about 30 dwellings, the water was chlorinated before distribution; in the other, which served a dairy and six dwellings the water was not treated. Samples of treated (40 l) and untreated (20 l) water were taken under normal and heavy rainfall conditions over a six weeks period and concentrated by adsorption/elution and organic flocculation. Infectious enterovirus in concentrates was detected in liquid culture and enumerated by plaque assay, both in BGM cells, and concentrates were also analysed by RT-PCR. Viruses were found in both raw water supplies. Rural supplies need to be analysed for viruses as well as bacterial and protozoan pathogens if the full microbial hazard is to be determined.

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

scholarly journals Improved real-time RT-PCR method for high-throughput measurements using second derivative calculation and double correction

BioTechniques ◽  
2005 ◽  
Vol 38 (2) ◽  
pp. 287-293 ◽  
Author(s):  
Van Luu-The ◽  
Nathalie Paquet ◽  
Ezequiel Calvo ◽  
Jean Cumps
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Second Derivative ◽  
Rt Pcr ◽  
Pcr Method
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