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PredicateObject
rdf:type
lifeskim:mentions
pubmed:dateCreated
2004-10-27
pubmed:abstractText
Here we describe a LightCycler-based real-time PCR for quantitative detection of EBV DNA in clinical samples such as unfractionated whole blood, serum, or plasma. This assay is based on amplification of a highly conserved 213-bp region of the EBNA-1 gene, a single-copy gene of EBV required for maintenance of the EBV genome within the infected host cell. For real-time detection of amplicons, two internal hybridization probes are added, labeled with the fluoregenic dyes fluorescein and LCRed640, respectively. Simultaneous hybridization of these probes to the amplification products brings them in close proximity. Subsequent excitation of the fluorescein label by filtered excitation light from a light source in the LightCycler device will lead to fluorescence energy transfer (FRET) from the fluorescein label to the LCRed640 label. The light emitted from the LCRed640 label is then measured and correlates to the amount of product generated. The cycle at which the fluorescence exceeds the background is designated the threshold cycle. By comparing the threshold cycle of a clinical specimen with those of standard curve samples, the amount of EBV DNA in clinical samples can be determined. This real-time PCR approach is extremely rapid owing to efficient heat conduction by using glass capillaries, small reaction volumes, and air as heating medium. The "closed-tube" system eliminates the risk of PCR contamination by product carryover and also the need for post-PCR detection.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:issn
1064-3745
pubmed:author
pubmed:issnType
Print
pubmed:volume
292
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
15-26
pubmed:meshHeading
pubmed:year
2005
pubmed:articleTitle
Quantitative detection of Epstein-Barr virus DNA in clinical specimens by rapid real-time PCR targeting a highly conserved region of EBNA-1.
pubmed:affiliation
Department of Pathology, VU Medical Centre, Amsterdam, The Netherlands.
pubmed:publicationType
Journal Article