Abstract：
:The goal of this study was to adapt a long RT-PCR technique to amplify large PCR fragments from the genome of hepatitis C virus (HCV) isolates using clinical samples. This was done by using a reverse transcriptase devoid of RNase H activity and a mixture of two antibody-bound thermostable polymerases to combine the high processivity of Taq and the high fidelity of Pwo with its 3'-->5' exonuclease activity. Other modifications included gentle handling during RNA extraction, the absence of tRNA and random primers, a two-step reverse transcription procedure to optimize cDNA synthesis, and increasing the annealing temperature for primers. With this approach, the HCV-1 genome (nucleotides 35-9282) was amplified consistently as two overlapping fragments of 5344 and 4675 bp from a pooled chimpanzee plasma sample containing approximately 10(6) genome copies of HCV RNA/ml. Using the conditions that we identified, 96% of the complete genomic sequence of a distinct HCV genotype 6 variant (km45) was determined from less than 300 microl of serum. This method should prove useful for molecular, epidemiological and clinical studies of hepatitis C where samples are limited but complete virus sequence is required, for example, identifying mutational hot spots of HCV under specific clinical conditions.
journal_name
J Virol Methodsjournal_title
Journal of virological methodsauthors
Lu L,Nakano T,Smallwood GA,Heffron TG,Robertson BH,Hagedorn CHdoi
10.1016/j.jviromet.2005.01.031subject
Has Abstractpub_date
2005-06-01 00:00:00pages
139-48issue
1-2eissn
0166-0934issn
1879-0984pii
S0166-0934(05)00060-1journal_volume
126pub_type
杂志文章abstract：:In preparation for field trials, a nuclear polyhedrosis virus (NPV) of the pine beauty moth, Panolis flammea, was mass produced in vivo in an alternative (heterologous) host, the cabbage moth, Mamestra brassicae. Simultaneously, Mamestra NPV was also produced in M. brassicae. This homologous NPV/host system was a cons...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/0166-0934(88)90019-5
更新日期：1988-03-01 00:00:00
abstract：BACKGROUND:Influenza causes a significant annual disease burden, with characterization of the infecting virus important in clinical and public health settings. Rapid immunoassays are fast but insensitive, whereas real-time RT-PCR is sensitive but susceptible to genetic mutations and often requires multiple serial assay...
journal_title：Journal of virological methods
pub_type： 杂志文章,多中心研究
doi：10.1016/j.jviromet.2019.113686
更新日期：2019-11-01 00:00:00
abstract：:Hepatitis C virus (HCV) entry into target cells is thought to be a multistep process involving several cellular factors. However, their precise role during virus entry is unclear. Investigation of the mechanisms of HCV entry, such as the order of intervention by the cellular receptors, requires synchronizing infection...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2008.11.015
更新日期：2009-04-01 00:00:00
abstract：:Cell membranes were prepared from herpes simplex virus (HSV) type 1-infected cells and solubilized with a low concentration of sodium deoxycholate. The supernatant after ultracentrifugation was used as antigen in a newly developed solid phase assay, diffusion in gel-enzyme-linked immunosorbent assay (DIG-ELISA). Antig...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/0166-0934(82)90045-3
更新日期：1982-04-01 00:00:00
abstract：:We describe a simple method for the incorporation of biotin into nucleic acid probes. This method has been improved and optimized to produce biotinylated DNA probes for the detection of DNA by dot-blot, Southern and colony hybridization techniques. The sensitivity of this method has been particularly improved to allow...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/0166-0934(89)90072-4
更新日期：1989-10-01 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：:A solid-phase enzyme-linked immunoassay using a fluorogenic substrate (4-methylumbelliferyl-beta-D-galactopyranoside) was developed. Antibodies were covalently linked to glutaraldehyde-activated 96-well aminopolystyrene plates. Antigens from test samples were adsorbed to the solid phase and detected using antibodies c...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/0166-0934(81)90050-1
更新日期：1981-10-01 00:00:00
abstract：:Certification represents the first line of defense against fruit tree viruses. For certification or surveys dealing with large number of samples, ELISA is still considered the technique of choice and requires a continuous supply of good quality antibodies. Prune dwarf virus (PDV) is among the major viruses affecting s...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2004.05.013
更新日期：2004-10-01 00:00:00
abstract：:Early and accurate diagnosis of Crimean-Congo hemorrhagic fever (CCHF) is essential for the treatment and outcome of the disease and prevention of its further transmission. Molecular-based diagnostic assays now serve as the front-line tool in the diagnosis of CCHF. However, the development of real-time RT-PCR assay fo...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2005.11.006
更新日期：2006-05-01 00:00:00
abstract：:Latent fruit tree viruses present economic threat to the industry and nurseries as diseases they cause not only reduce fruit quality and production yield, but can also be spread inadvertently through propagation due to the lack of viral symptoms on an infected mother plant. As a result, these viruses require appropria...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2020.113836
更新日期：2020-04-01 00:00:00
abstract：:A molecular diagnostic assay for the rapid, sensitive and specific detection of Apple stem pitting virus (ASPV) in infected samples, utilizing reverse transcription-recombinase polymerase amplification (RT-RPA) at an isothermal constant temperature of 42 °C and the designed target-specific primers, was developed. The ...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2019.113747
更新日期：2019-12-01 00:00:00
abstract：:A single-tube duplex and multiplex PCR was developed for the simultaneous detection of African cassava mosaic virus (ACMV), East African cassava mosaic Cameroon virus (EACMCV), East African cassava mosaic Malawi virus (EACMMV) and East African cassava mosaic Zanzibar virus (EACMZV), four cassava mosaic begomoviruses (...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2012.10.007
更新日期：2013-04-01 00:00:00
abstract：:A new method based on RNA preparation from shellfish by polyethylene glycol (PEG) precipitation and trichloroacetic acid (TCA) extraction was developed for the detection of hepatitis A virus (HAV) by the polymerase chain reaction (PCR). The extraction provides high yield and the extracted RNA is undegraded. This metho...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/0166-0934(93)90091-5
更新日期：1993-06-01 00:00:00
abstract：:Peste des petits ruminants virus (PPRV) causes a devastating disease of small ruminants present across much of Africa and Asia. Recent surveillance activities and phylogenetic analyses have suggested that the virus is an emerging problem as it is now being detected in areas previously free of the disease. As such, the...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2010.11.022
更新日期：2011-02-01 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：:Human papillomavirus (HPV) infection is the primary cause of cervical cancer. The Quantivirus(®) HPV E6/E7 RNA 3.0 assay (DiaCarta, CA, USA) detects E6/E7 mRNA of 13 high risk subtypes and 6 low risk subtypes. Cervical specimens collected in PreservCyt were processed for HPV detection. Cervical biopsies were taken onl...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2012.11.024
更新日期：2013-02-01 00:00:00
abstract：:Avian influenza virus (AIV) is an enveloped virus with segmented RNA that belongs to the Orthomyxoviridae. Recently, avian influenza virus isolates have not only posed a significant threat to the poultry industry but also serious public health concerns. The full-length viral hemagglutinin (HA), neuraminidase (NA) or b...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2011.05.006
更新日期：2011-08-01 00:00:00
abstract：:The increasing use of the baculovirus expression vector system (BEVS) has generated significant interest into techniques for quantifying baculovirus stocks. One method involves the use of quantitative real-time polymerase chain reaction (PCR). This study investigated simplifying baculovirus sample preparation for quan...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2012.03.001
更新日期：2012-06-01 00:00:00
abstract：:A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of Cucurbit chlorotic yellows virus (CCYV). In this procedure, a set of four primers matching a total of six sequences in the coat protein gene region of CCYV was synthesized for the RT-LAMP assay using total...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2013.08.037
更新日期：2014-01-01 00:00:00
abstract：:A method for duplex nested RT-PCR (nRT-PCR) with internal control (IC) for the detection of Nipah virus RNA is described. Incorporation of IC RNA distinguished false and true negative results. The extrinsic RNA was added directly to the PCR master mix and co-amplified with virus specific RNA in a duplex reaction to de...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2006.11.023
更新日期：2007-04-01 00:00:00
abstract：:The major potato virus X (PVS) RNA translation product migrates in Laemmli's electrophoresis system as a 210 kDa polypeptide ('p210'). If a Tris-phosphate-SDS buffer system is used instead of a Tris-glycine-SDS one, the mobility of p210 is higher than that of the largest TMV RNA translation product, the 183 kDa protei...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/0166-0934(89)90136-5
更新日期：1989-02-01 00:00:00
abstract：:A chimeric porcine circovirus type 1-2 (PCV1-2) infectious DNA clone has low transfection efficiency and exhibits low levels of proliferation. Electroporation and lipofection parameters were optimized for PK-15 and Dulac cells with the purpose of increasing the efficiency for rescuing infectious PCV1-2. Titers of PCV1...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2014.08.005
更新日期：2014-11-01 00:00:00
abstract：:In the context of early infant diagnosis (EID) decentralization in sub-Saharan Africa, dried blood spot (DBS) is now widely used for HIV proviral DNA detection in resource-limited settings. A new version of CAP/CTM (version 2) has been introduced, recently by Roche Diagnosis as a new real-time PCR assay to replace pre...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2015.12.006
更新日期：2016-03-01 00:00:00
abstract：:The effective control of foot-and-mouth disease (FMD) requires sensitive, specific and rapid diagnostic tools. However, the control and eradication of FMD in Africa is complicated by, among other factors, the existence of five of the seven FMD virus (FMDV) serotypes, including the SAT-serotypes 1, 2 and 3 that are gen...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2018.02.006
更新日期：2018-05-01 00:00:00
abstract：:An indirect enzyme-linked immunosorbent assay termed rnPRRS ELISA using baculovirus-expressed and affinity-purified viral nucleocapsid protein (rNC) antigen was developed for detecting antibodies against porcine reproductive and respiratory syndrome virus (PRRSV) in swine sera. Sera (1395) originating from different E...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/s0166-0934(97)02186-1
更新日期：1997-05-01 00:00:00
abstract：:Ultra-deep pyrosequencing (UDPS) of targeted amplicons allows to determine a large number of individual sequence reads from a single PCR product, and this approach is thus extremely valuable for analysis of mixed viral populations. A mixture of genetically distinct DNA templates, however, may lead to the generation of...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2010.07.040
更新日期：2010-10-01 00:00:00
abstract：:This report describes the first time entire viral capsids have been purified using fast protein liquid chromatography (FPLC) techniques. The FPLC is used here to separate the two electrophoretic forms of cowpea mosaic virus. The capsid forms are shown to be separated by the Mono-Q column without damaging the capsids. ...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/0166-0934(87)90011-5
更新日期：1987-07-01 00:00:00
abstract：:A simple procedure is described for reproducible detection of double stranded (ds) RNAs in leafroll infected grapevines. The procedure involves the extraction of tissues by a medium which preferentially yields dsRNA. The RNA is purified by CF11 cellulose chromatography and gel electrophoresis. The dsRNAs varied in siz...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/0166-0934(91)90170-5
更新日期：1991-02-01 00:00:00
abstract：:Simian betaretroviruses (formerly Type D retroviruses; SRV) are a group of closely related retroviruses for which the natural host species are Asian monkeys of the genus Macaca. Five serotypes have been identified by classical neutralization assays and three additional untyped variants have been reported (SRV(Tsukuba)...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2009.07.030
更新日期：2009-12-01 00:00:00
abstract：:Feline calicivirus (FCV) has been used by researchers as a surrogate for Norwalk virus (NV), since they share a similar genomic organization, physicochemical characteristics, and are grouped in the same family, Caliciviridae. Unlike NV, however, FCV can grow in established cell lines and produce a syncytial form of cy...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/s0166-0934(02)00214-8
更新日期：2003-02-01 00:00:00
