共查询到20条相似文献,搜索用时 31 毫秒
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Evaluation of real-time reverse transcription polymerase chain reaction assays for the detection of swine vesicular disease virus 总被引:7,自引:0,他引:7
Reid SM Ferris NP Hutchings GH King DP Alexandersen S 《Journal of virological methods》2004,116(2):169-176
Differential detection of swine vesicular disease virus (SVDV) from the other vesicular disease viruses of foot-and-mouth disease (FMD), vesicular stomatitis (VS) and vesivirus is important as the vesicular lesions produced by these viruses are indistinguishable in pigs. Two independent sets of primers and probe, designed from nucleotide sequences within the 5' untranslated region (UTR) of the SVDV genome, were evaluated in a real-time (5' nuclease probe-based or fluorogenic) PCR format. Although both primers/probe sets failed to detect one isolate, the assays successfully amplified RNA extracted from epithelial suspensions (ES) and cell culture grown virus preparations from clinical samples representing all currently designated phylogenetic groups of SVDV. Furthermore, no cross-reactivity was demonstrated when these primer/probe sets were tested with RNA prepared from all seven serotypes of FMD virus (FMDV) and from selected isolates of VS virus (VSV), vesivirus and teschoviruses. These assays provide sensitive and rapid alternatives to supplement the routine procedures of ELISA and virus isolation for SVDV diagnosis. The two independent sets of primers/probe can be used routinely while only one of the primers/probe sets would typically be used in SVDV diagnosis during an outbreak. 相似文献
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Development and evaluation of serotype- and group-specific fluorogenic reverse transcriptase PCR (TaqMan) assays for dengue virus. 总被引:16,自引:0,他引:16 下载免费PDF全文
Johnny D. Callahan Shuenn-Jue L. Wu Amanda Dion-Schultz Beverly E. Mangold Leonard F. Peruski Douglas M. Watts Kevin R. Porter Gerald R. Murphy Wuryadi Suharyono Chwan-Chuen King Curtis G. Hayes Joseph J. Temenak 《Journal of clinical microbiology》2001,39(11):4119-4124
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S. M. Reid N. P. Ferris G. H. Hutchings K. De Clercq B. J. Newman N. J. Knowles A. R. Samuel 《Archives of virology》2001,146(12):2421-2434
Summary. The results of type-specific RT-PCR diagnostic assays on foot-and-mouth disease (FMD) viruses in clinical samples were mapped
onto serotype-specific dendrograms representing the degree of nucleotide sequence variation between the FMD virus isolates.
This novel approach assisted the selection of suitable PCR primer sets for the diagnosis of FMD virus isolates belonging to
different topotypes within each serotype. These interpretations were qualified by using a universal (FMD virus group) specific
primer to confirm that FMD virus RNA had been extracted from the samples under investigation. The analyses showed that the
design of primer sets for the detection of FMD virus serotypes O, A, Asia 1, SAT 1 and SAT 3 were generally satisfactory,
as most virus isolates within the major virus sub-groupings were successfully detected. However, the FMD virus serotype C
and SAT 2 specific primers were less efficient as certain virus sub-groups were not detected. This identified the need for
additional or alternative primers to improve RT-PCR procedures for more comprehensive detection of divergent virus strains
within these serotypes. There were some examples where not all virus isolates from the same outbreak reacted with particular
type-specific primers which suggested that either further minor refinements may be necessary in the primer design or that
there were shortcomings in the RT-PCR methodology.
Received February 5, 2001 Accepted August 8, 2001 相似文献
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Comparison of a multiplex reverse transcription-PCR-enzyme hybridization assay with conventional viral culture and immunofluorescence techniques for the detection of seven viral respiratory pathogens 总被引:7,自引:0,他引:7 下载免费PDF全文
Liolios L Jenney A Spelman D Kotsimbos T Catton M Wesselingh S 《Journal of clinical microbiology》2001,39(8):2779-2783
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Sane J Kurkela S Levanov L Nikkari S Vaheri A Vapalahti O 《Journal of virological methods》2012,179(1):185-188
Sindbis virus (SINV) is an arthropod-borne alphavirus found widely in Eurasia, Africa and Oceania. Clinical SINV infection, characterized by rash and arthritis, is reported primarily in Northern Europe. The laboratory diagnosis of SINV infection is based currently on serology. A one-step TaqMan® real-time RT-PCR assay was developed for the detection of SINV and evaluated its clinical performance with acute-phase serum samples. The specificity and sensitivity of the real-time PCR assay were assessed using cell cultured Finnish SINV strains. The applicability of the assay for diagnostic use was evaluated using 58 serum samples from patients infected with SINV. The real-time RT-PCR assay was specific and sensitive for the detection of SINV in cell culture supernatants with a 95% detection limit of 9 genome copies/reaction determined by probit analysis. However, in the assay only 7/58 (12%) of serum samples were positive of which two were also positive by conventional nested PCR assay and none by virus isolation. This novel assay is specific and sensitive for detection of SINV and can be used for example for screening SINV in wildlife. However, molecular diagnostic techniques using serum samples seem to be of limited value for the diagnosis of human SINV infection due to the short and low viraemia of infection with SINV. 相似文献
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Julie D Fox Song Han Agneta Samuelson Yadan Zhang M Lynne Neale Diana Westmoreland 《Journal of clinical virology》2002,24(1-2):117-130
BACKGROUND: Molecular methods based on RNA amplification are needed for sensitive detection of enteroviruses in clinical samples. Many 'in house' methods based on reverse-transcribed PCR (RT-PCR) could be difficult to use in the routine diagnostic laboratory since they tend to be time-consuming, use reagents from many different suppliers and include non-routine procedures. OBJECTIVES: The aim of this study was to develop and evaluate methods based on nucleic acid sequence based amplification (NASBA) for detection of enterovirus sequences. STUDY DESIGN: 'In house' prepared and commercially available reagents were utilised to develop enterovirus-specific NASBA assays. Optimised methods were evaluated using clinical samples (cerebrospinal fluid, respiratory and stool samples), titred virus controls and in vitro produced synthetic RNA. Results for NASBA were compared with RT-PCR and virus culture. RESULTS: Kit-based reagents gave an equivalent sensitivity to the more laborious 'in house' molecular assays (NASBA and RT-PCR) on clinical material and controls. All molecular methods picked up enterovirus positive clinical samples that were not identified by culture. End point detection sensitivity for the NASBA assay based on the NucliSens Basic Kit was 相似文献
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Reid SM King DP Shaw AE Knowles NJ Hutchings GH Cooper EJ Smith AW Ferris NP 《Journal of virological methods》2007,140(1-2):166-173
Marine caliciviruses form a distinct lineage within the genus Vesivirus (family Caliciviridae). This group includes vesicular exanthema of swine virus (VESV) and San Miguel sea lion virus (SMSV) and other related viruses which have been proposed to be marine in origin isolated from a variety of terrestrial and marine animals. Rapid and reliable detection of marine caliciviruses is important as these viruses appear to be widespread and can cause vesicular disease in a wide variety of susceptible hosts including pigs and experimentally infected cattle where clinical signs cannot be easily distinguished from foot-and-mouth disease (FMD), swine vesicular disease (SVD) and vesicular stomatitis (VS). A real-time RT-PCR assay targeting conserved nucleotide sequences in the RNA-dependent RNA polymerase (3D) region of the genome successfully detected cell culture-grown virus preparations of more than thirty marine calicivirus serotypes. Only the atypical SMSV serotypes 8 and 12 failed to be detected, which provided further indication of genetic divergence between these and the other calicivirus serotypes said to be marine in origin. The real-time RT-PCR assay also specifically amplified RNA from samples collected following experimental inoculation of pigs with VESV. No cross-reactivity was demonstrated when the assay was tested with RNA prepared from representative viruses of FMD, SVD and VS. The real-time RT-PCR assay described is a sensitive and specific tool for detection and differential diagnosis of these viruses from other vesicular-disease causing viruses. 相似文献