Abstract:
:A spot nested RT-PCR-RFLP method to detect and identify all members of the Tobamovirus genus is described. It involves a one-step RT-PCR, in which the combination of degenerate deoxyinosine (dI)-substituted primers amplified part of the polymerase region of tobamoviruses, followed by a nested PCR amplification that increased specificity and sensitivity of detection. Virus species differentiation was achieved by subsequent restriction enzyme analysis. The sensitivity of the method was increased further when along with one primer containing many dIs, another homologous primer in which dIs were substituted by dGs was used. The homologous primer was shorter than the dI-containing primer and with lower degeneracy, resulting in higher overall amplification efficiency due to the increased stability of the primer-target duplex. With this strategy, highly degenerate primers containing many dIs can be used effectively to improve detection sensitivity, alleviating problems of primer-target duplex destabilisation that can occur due to many dI substitutions. This method can be useful on the diagnosis, epidemiological investigation, and characterisation of known and unidentified Tobamovirus species.
journal_name
J Virol Methodsjournal_title
Journal of virological methodsauthors
Dovas CI,Efthimiou K,Katis NIdoi
10.1016/j.jviromet.2004.01.004subject
Has Abstractpub_date
2004-05-01 00:00:00pages
137-44issue
2eissn
0166-0934issn
1879-0984pii
S0166093404000060journal_volume
117pub_type
杂志文章abstract::Four IgG(1kappa) monoclonal antibodies (mAbs) against Influenza A/Chicken/Vietnam/8/2004 (H5N1) virus are described. Three of these showed neutralizing activities against H5N1 strains from clades 1, 2 and 3 using a retroviral pseudotype or live virus microneutralization assay. In the pseudotype assay, the IC(90) neutr...
journal_title:Journal of virological methods
pub_type: 杂志文章
doi:10.1016/j.jviromet.2008.12.016
更新日期:2009-05-01 00:00:00
abstract::The development of a quantitative-competitive reverse transcription-PCR (RT-PCR) assay to quantify dengue virus (DEN) genome (vRNA) and its replicative intermediate RNA (vRI) is described. A highly conserved region located on the DEN capsid-premembrane genes was used to produce a competitor RNA molecule which contains...
journal_title:Journal of virological methods
pub_type: 杂志文章
doi:10.1016/j.jviromet.2008.05.007
更新日期:2008-09-01 00:00:00
abstract::Adaptation through fixation of spontaneous mutations in the viral genome is considered to be one of the important factors that enable recurrent West Nile virus (WNV) outbreaks in the U.S. Genetic variations can alter viral phenotype and virulence, and degrade the performance of diagnostic and screening assays, vaccine...
journal_title:Journal of virological methods
pub_type: 杂志文章
doi:10.1016/j.jviromet.2008.09.015
更新日期:2008-12-01 00:00:00
abstract::Determining whether animals have been infected with foot-and-mouth disease virus or vaccinated is important because infected animals frequently become carriers of the virus, shed it intermittently and thus may be the source of new outbreaks of the disease. We had shown previously that the sera of convalescent animals ...
journal_title:Journal of virological methods
pub_type: 杂志文章
doi:10.1016/s0166-0934(96)02165-9
更新日期:1997-04-01 00:00:00
abstract::A colorimetric reverse transcriptase assay (cRT assay) was developed for quantitative detection of HIV-1. In this format, reverse transcriptase incorporates biotin-labeled dUTP onto oligo-dT primers hybridized to poly A templates. The templates are covalently bound to the surface of microtiter wells. The amount of inc...
journal_title:Journal of virological methods
pub_type: 杂志文章
doi:10.1016/0166-0934(95)00073-5
更新日期:1995-11-01 00:00:00
abstract::The clinical sensitivity of the current anti-HIV assays is based for an important part on their reactivity with seroconversion panels. The most sensitive assay closes the seroconversion window as much as possible, thereby reducing the risk of transmitting false negative donations obtained from individuals infected rec...
journal_title:Journal of virological methods
pub_type: 杂志文章
doi:10.1016/s0166-0934(98)00126-8
更新日期:1998-12-01 00:00:00
abstract::Monitoring the serum hepatitis B viral DNA levels with sensitive realtime PCR assays is strongly recommended for the management of patients with chronic HBV infection. This study compares the performance of two realtime HBV quantitative PCR assays with samples from chronic HBeAg(+) and HBeAg(-) patients. ...
journal_title:Journal of virological methods
pub_type: 杂志文章
doi:10.1016/j.jviromet.2013.06.036
更新日期:2013-11-01 00:00:00
abstract::Real-time polymerase chain reaction (real-time PCR) is an accurate, rapid and reliable method that can be used for the detection and also for the quantitation of specific DNA molecules. It can be non-specific, with intercalating dyes (SYBR Green I dye) able to bind to any dsDNA, or specific with a probe (TaqMan), wher...
journal_title:Journal of virological methods
pub_type: 杂志文章
doi:10.1016/j.jviromet.2012.05.010
更新日期:2012-09-01 00:00:00
abstract::Since there are indications of an increasing amount of non-B subtypes in Western Europe it was decided to assess the performance of the ViroSeq HIV-1 Genotyping System on a set of samples from the AIDS Reference Laboratory at the University Hospitals Leuven, a hospital with an increasing number of patients infected wi...
journal_title:Journal of virological methods
pub_type: 杂志文章
doi:10.1016/j.jviromet.2004.02.005
更新日期:2004-07-01 00:00:00
abstract::Rapid characterization of herpesvirus isolates exemplified by equine herpesviruses is described. Total DNAs were isolated from virus infected small scale cell cultures. The DNA fragments obtained after restriction enzyme digestion were separated on agarose gels, transferred and immobilized on filter membranes. A radio...
journal_title:Journal of virological methods
pub_type: 杂志文章
doi:10.1016/0166-0934(86)90030-3
更新日期:1986-11-01 00:00:00
abstract::Peste des petits ruminants (PPR) is a viral disease of small ruminants that is caused by the PPR virus (PPRV) and is a significant burden on subsistence farmers across the developing world. Loop-mediated isothermal amplification (LAMP) provides cost-effective, rapid, specific and sensitive detection of nucleic acid an...
journal_title:Journal of virological methods
pub_type: 杂志文章
doi:10.1016/j.jviromet.2019.113730
更新日期:2019-12-01 00:00:00
abstract::The Bacille Calmette Guerin (BCG), long valued for its role as a live vaccine for the prevention of tuberculosis, is being used as a recombinant delivery vehicle for foreign antigens, principally, for inducing long-lived specific humoral and cellular immunity. Hepatitis B and its sequelae are major public health probl...
journal_title:Journal of virological methods
pub_type: 杂志文章
doi:10.1016/j.jviromet.2004.11.026
更新日期:2005-04-01 00:00:00
abstract::Human enteroviruses are among the most common viruses infecting humans. These viruses are known to be able to infect a wide range of tissues and are believed to establish persistent infections. Enteroviruses are positive-sense single-stranded RNA viruses whose replication involves the synthesis of negative strand inte...
journal_title:Journal of virological methods
pub_type: 杂志文章
doi:10.1016/j.jviromet.2008.07.010
更新日期:2008-11-01 00:00:00
abstract::5-min boiling of 3-fold diluted serum leads to the quantitative release of immune-complexed HIV antigen (Ag) that can be readily measured. The method is capable of detecting as little as 4 to 5 pg/ml Ag p24 depending on the Ag test used. Of a panel of 50 anti-HIV-positive sera, 30 (60%) were Ag-positive after heat den...
journal_title:Journal of virological methods
pub_type: 杂志文章
doi:10.1016/0166-0934(93)90080-b
更新日期:1993-07-01 00:00:00
abstract::The development of a rapid and sensitive assay for detection of replication-competent adenoviruses (RCAs) is described. This RCA assay consists of an incubation step of 4 days of adenoviral vectors on A549 cells in a microcarrier cell culture system followed by detection of amplified RCAs by E1-specific quantitative P...
journal_title:Journal of virological methods
pub_type: 杂志文章
doi:10.1016/j.jviromet.2007.05.010
更新日期:2007-11-01 00:00:00
abstract::A method is described for detecting enteroviruses in both freshwater and marine sediments. Viruses were recovered from sediments by elution into 6% beef extract at pH 9.0 and concentration with polyethylene glycol 6000. The recovery efficiency ranged from 6 to 55% for marine sediments and 16 to 77% for freshwater sedi...
journal_title:Journal of virological methods
pub_type: 杂志文章
doi:10.1016/0166-0934(85)90101-6
更新日期:1985-02-01 00:00:00
abstract::We have developed a sensitive method to detect and localize HPV-6 viral DNA, mRNA and protein in biopsy specimens of genital and respiratory tract lesions by using in situ hybridization and immunoperoxidase assays on sections of plastic-embedded tissue. This modified in situ hybridization technique, using ultrathin se...
journal_title:Journal of virological methods
pub_type: 杂志文章
doi:10.1016/0166-0934(89)90098-0
更新日期:1989-07-01 00:00:00
abstract::Porcine reproductive and respiratory syndrome virus (PRRSV) became a significant pathogen of swine upon its emergence in the late 1980s and since then has exemplified a rapidly evolving, constantly re-emerging pathogen. In addition to the challenges faced in development of vaccines and diagnostics, research on the bas...
journal_title:Journal of virological methods
pub_type: 杂志文章
doi:10.1016/j.jviromet.2015.02.007
更新日期:2015-06-15 00:00:00
abstract::Hepatitis A virus (HAV) infection is the leading worldwide cause of acute viral hepatitis. An important aspect of viral control is rapid diagnosis. Epidemiological studies have linked hepatitis A outbreaks to the consumption of drinking water or soft fruits exposed to faecal contamination. Real-time reverse transcript...
journal_title:Journal of virological methods
pub_type: 杂志文章
doi:10.1016/j.jviromet.2010.02.017
更新日期:2010-06-01 00:00:00
abstract::Chronic HCV infection is one of the leading causes of liver-related death and in many countries it is a primary reason for having a liver transplant. HCV genotype identification has long been used in the clinical practice, since different genotypes have different response rates and required different doses and duratio...
journal_title:Journal of virological methods
pub_type: 杂志文章
doi:10.1016/j.jviromet.2017.10.005
更新日期:2018-01-01 00:00:00
abstract::Commercially marketed kits are now available for rapid viral assay of clinical specimens. This study was conducted to determine the suitability of these kits for use in environmental testing. Eight rotavirus kits and one enteric adenovirus kit were screened for sensitivity using simian rotavirus SA11, human rotavirus ...
journal_title:Journal of virological methods
pub_type: 杂志文章
doi:10.1016/0166-0934(93)90098-c
更新日期:1993-12-15 00:00:00
abstract::A microtiter cell-culture assay is described for measuring neutralizing antibody activity directed against the human immunodeficiency virus (HIV). The assay relies upon inhibition of HIV-mediated cell killing of infected MT-4 lymphoid cells. The assay exhibits comparable sensitivity to two other methods used for such ...
journal_title:Journal of virological methods
pub_type: 杂志文章
doi:10.1016/0166-0934(88)90123-1
更新日期:1988-07-01 00:00:00
abstract::Simultaneous detection of two antigens by immunostaining usually requires primary antibodies from two different species or a hapten modification of one of the antibodies if they are from the same species. A novel double staining method is described for immunodetection of two independent antigens using two mouse monocl...
journal_title:Journal of virological methods
pub_type: 杂志文章
doi:10.1016/s0166-0934(98)00048-2
更新日期:1998-07-01 00:00:00
abstract::Cyprinid herpesvirus-3 (CyHV-3) is an etiological agent of a notifiable disease that causes high mortality rates affecting both the common and koi carp Cyprinus carpio L. There is no current treatment strategy to save CyHV-3 infected fish. RNA mediated interference (RNAi) is an emerging strategy used for understanding...
journal_title:Journal of virological methods
pub_type: 杂志文章
doi:10.1016/j.jviromet.2014.05.022
更新日期:2014-09-01 00:00:00
abstract::The surface details of resin embedded viruses can be greatly enhanced by the use of uranyl acetate block staining. The virions are embedded in agar immediately after fixation and block stained with 3% uranyl acetate in methanol during dehydration. This technique allows those particles outside their host cells to show ...
journal_title:Journal of virological methods
pub_type: 杂志文章
doi:10.1016/0166-0934(84)90040-5
更新日期:1984-02-01 00:00:00
abstract::Rapid and accurate detection of measles virus is important for case diagnosis and public health management. This study compared the performance of two monoplex RT-PCR reactions targeting the H and N genes to a duplex RT-PCR targeting both genes simultaneously. The duplex simplified processing without compromising assa...
journal_title:Journal of virological methods
pub_type: 杂志文章
doi:10.1016/j.jviromet.2016.11.003
更新日期:2017-01-01 00:00:00
abstract::Two methods for denaturing double stranded (ds)RNA of infectious bursal disease virus for the purpose of reverse transcribing it were compared: Heat denaturation at 65 degrees C in the presence of DMSO and in the absence of DMSO. As part of the analysis, the nature of cDNA in the two preparations was examined by polym...
journal_title:Journal of virological methods
pub_type: 杂志文章
doi:10.1016/0166-0934(94)90081-7
更新日期:1994-04-01 00:00:00
abstract::A simple, sensitive and specific method using the polymerase chain reaction (PCR) for amplification of human immunodeficiency virus type 1 (HIV-1) is described. The method involves minimal manipulations. Peripheral blood mononuclear cells (PBMC) were prepared by a rapid Ficoll-Paque gradient method. Lymphocytes were l...
journal_title:Journal of virological methods
pub_type: 杂志文章
doi:10.1016/0166-0934(91)90058-8
更新日期:1991-05-01 00:00:00
abstract::Enzyme immunoassays based on the use of monoclonal antibodies (MAbs) were developed for the detection of the barley stripe mosaic virus (BSMV) and the beet necrotic yellow vein virus (BNYVV). Assays employing conjugates of MAbs to horseradish peroxidase (HRP) were compared to systems with biotinylated MAbs and strepta...
journal_title:Journal of virological methods
pub_type: 杂志文章
doi:10.1016/0166-0934(95)01962-6
更新日期:1996-02-01 00:00:00
abstract::Congenital human cytomegalovirus infections are the major infectious cause of birth defects in the United States. How this virus crosses the placenta and causes fetal disease is poorly understood. Guinea pig cytomegalovirus (GPCMV) is a related virus that provides an important model for studying cytomegaloviral congen...
journal_title:Journal of virological methods
pub_type: 杂志文章
doi:10.1016/j.jviromet.2008.01.031
更新日期:2008-05-01 00:00:00