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Article: Analytic validation of a competitive polymerase chain reaction assay for measuring Epstein-Barr viral load

TitleAnalytic validation of a competitive polymerase chain reaction assay for measuring Epstein-Barr viral load
Authors
Issue Date2001
PublisherLippincott Williams & Wilkins. The Journal's web site is located at http://www.molecularpathology.com
Citation
Diagnostic Molecular Pathology, 2001, v. 10 n. 4, p. 255-264 How to Cite?
AbstractEpstein-Barr virus (EBV) is associated with several benign and malignant diseases, and blood tests for EBV viral load show promise as markers of disease burden in affected patients. A commercial quantitative PCR method (BioSource International) was recently introduced to facilitate measuring viral load. It relies on coamplification of EBV DNA and a spiked competitor in plasma or serum, followed by semiautomated product detection on enzyme-linked immunosorbent assay (ELISA) plates. In the current study, analytic performance characteristics were assessed, and the authors describe several methodologic improvements to facilitate laboratory implementation. Rapid DNA extraction was accomplished using commercial silica spin columns, heat-labile uracil-N-glycosylase was used to inhibit amplicon contamination, and inexpensive agarose gels were used to screen for polymerase chain reaction products requiring ELISA plate quantitation. Accuracy and precision were verified using EBV DNA standards derived from two cell lines and plasmid containing viral sequences. The assay was sensitive to as few as five template copies per polymerase chain reaction and was linear across four orders of magnitude (correlation coefficient 0.995). When applied to matched plasma and serum samples from 15 patients with nasopharyngeal carcinoma, both sample types yielded similar viral load results. This commercial EBV viral load assay provides sensitive and quantitative detection of EBV DNA using equipment already available in many molecular diagnostic laboratories.
Persistent Identifierhttp://hdl.handle.net/10722/88576
ISSN
2015 Impact Factor: 1.474
2015 SCImago Journal Rankings: 0.887
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorFan, Hen_HK
dc.contributor.authorSchichman, SAen_HK
dc.contributor.authorSwinnen, LJen_HK
dc.contributor.authorNicholls, JMen_HK
dc.contributor.authorEagan, PAen_HK
dc.contributor.authorLuther, Men_HK
dc.contributor.authorGulley, MLen_HK
dc.date.accessioned2010-09-06T09:45:11Z-
dc.date.available2010-09-06T09:45:11Z-
dc.date.issued2001en_HK
dc.identifier.citationDiagnostic Molecular Pathology, 2001, v. 10 n. 4, p. 255-264en_HK
dc.identifier.issn1052-9551en_HK
dc.identifier.urihttp://hdl.handle.net/10722/88576-
dc.description.abstractEpstein-Barr virus (EBV) is associated with several benign and malignant diseases, and blood tests for EBV viral load show promise as markers of disease burden in affected patients. A commercial quantitative PCR method (BioSource International) was recently introduced to facilitate measuring viral load. It relies on coamplification of EBV DNA and a spiked competitor in plasma or serum, followed by semiautomated product detection on enzyme-linked immunosorbent assay (ELISA) plates. In the current study, analytic performance characteristics were assessed, and the authors describe several methodologic improvements to facilitate laboratory implementation. Rapid DNA extraction was accomplished using commercial silica spin columns, heat-labile uracil-N-glycosylase was used to inhibit amplicon contamination, and inexpensive agarose gels were used to screen for polymerase chain reaction products requiring ELISA plate quantitation. Accuracy and precision were verified using EBV DNA standards derived from two cell lines and plasmid containing viral sequences. The assay was sensitive to as few as five template copies per polymerase chain reaction and was linear across four orders of magnitude (correlation coefficient 0.995). When applied to matched plasma and serum samples from 15 patients with nasopharyngeal carcinoma, both sample types yielded similar viral load results. This commercial EBV viral load assay provides sensitive and quantitative detection of EBV DNA using equipment already available in many molecular diagnostic laboratories.en_HK
dc.languageengen_HK
dc.publisherLippincott Williams & Wilkins. The Journal's web site is located at http://www.molecularpathology.comen_HK
dc.relation.ispartofDiagnostic Molecular Pathologyen_HK
dc.rightsDiagnostic Molecular Pathology. Copyright © Lippincott Williams & Wilkins.en_HK
dc.subject.meshDNA, Viral - analysisen_HK
dc.subject.meshEnzyme-Linked Immunosorbent Assayen_HK
dc.subject.meshHerpesvirus 4, Human - genetics - isolation & purificationen_HK
dc.subject.meshHumansen_HK
dc.subject.meshPolymerase Chain Reactionen_HK
dc.subject.meshReagent Kits, Diagnosticen_HK
dc.subject.meshReproducibility of Resultsen_HK
dc.subject.meshSensitivity and Specificityen_HK
dc.subject.meshTumor Cells, Cultureden_HK
dc.subject.meshViral Load - methodsen_HK
dc.titleAnalytic validation of a competitive polymerase chain reaction assay for measuring Epstein-Barr viral loaden_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=1052-9551&volume=10&issue=4&spage=255&epage=264&date=2001&atitle=Analytic+validation+of+a+competitive+polymerase+chain+reaction+assay+for+measuring+Epstein-Barr+viral+loaden_HK
dc.identifier.emailNicholls, JM:nicholls@pathology.hku.hken_HK
dc.identifier.authorityNicholls, JM=rp00364en_HK
dc.description.naturelink_to_subscribed_fulltext-
dc.identifier.doi10.1097/00019606-200112000-00008en_HK
dc.identifier.pmid11763317en_HK
dc.identifier.scopuseid_2-s2.0-0035204510en_HK
dc.identifier.hkuros66024en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0035204510&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume10en_HK
dc.identifier.issue4en_HK
dc.identifier.spage255en_HK
dc.identifier.epage264en_HK
dc.identifier.isiWOS:000172388900008-
dc.publisher.placeUnited Statesen_HK

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