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Article: Reverse Transcriptase PCR Diagnostic Assay for the Coronavirus Associated with Severe Acute Respiratory Syndrome

TitleReverse Transcriptase PCR Diagnostic Assay for the Coronavirus Associated with Severe Acute Respiratory Syndrome
Authors
Issue Date2004
PublisherAmerican Society for Microbiology.
Citation
Journal Of Clinical Microbiology, 2004, v. 42 n. 5, p. 1994-1999 How to Cite?
AbstractRecent outbreaks of severe acute respiratory syndrome (SARS) have spurred intense research efforts around the world to deal with the serious threat to health posed by this novel coronavirus. A rapid, reliable diagnostic assay is needed for monitoring the spread of the disease. Here we report a method for eliminating false-negative results and increasing test sensitivity, based on the hypothesis that the message encoded by the nucleocapsid (N) gene is the most abundant during viral infection. Nasopharyngeal aspirates and stool samples were obtained from suspected SARS patients with major clinical symptoms and a significant history of close contact with infected patients. Total RNAs were extracted in a 96-well format, together with pig kidney epithelial (PK-15) cells as an internal control for extraction efficiency. PCR inhibitors were removed by ethanol precipitation, and a PCR for the pig β-actin gene was used as a positive control for all clinical samples. Samples were analyzed by a reverse transcriptase PCR assay. Northern blot analysis was performed to demonstrate differences in subgenomic transcripts of the virus, and a real-time quantitative PCR was employed to compare the sensitivities of two loci (1b and N). The detection rate of the assay reached 44.4% on day 9 after the onset of the disease. The diagnostic PCR amplifying the N gene gave an average of a 26. 0% (6.3 to 60.0%) stronger intensity signal than that for the lb gene. In conclusion, the nucleocapsid gene represents an additional sensitive molecular marker for the diagnosis of the SARS coronavirus and can be further adapted for use in a high-throughput platform assay.
Persistent Identifierhttp://hdl.handle.net/10722/49242
ISSN
2015 Impact Factor: 3.631
2015 SCImago Journal Rankings: 2.151
PubMed Central ID
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorHui, RKHen_HK
dc.contributor.authorZeng, Fen_HK
dc.contributor.authorChan, CMNen_HK
dc.contributor.authorYuen, KYen_HK
dc.contributor.authorPeiris, JSMen_HK
dc.contributor.authorLeung, FCCen_HK
dc.date.accessioned2008-06-12T06:37:26Z-
dc.date.available2008-06-12T06:37:26Z-
dc.date.issued2004en_HK
dc.identifier.citationJournal Of Clinical Microbiology, 2004, v. 42 n. 5, p. 1994-1999en_HK
dc.identifier.issn0095-1137en_HK
dc.identifier.urihttp://hdl.handle.net/10722/49242-
dc.description.abstractRecent outbreaks of severe acute respiratory syndrome (SARS) have spurred intense research efforts around the world to deal with the serious threat to health posed by this novel coronavirus. A rapid, reliable diagnostic assay is needed for monitoring the spread of the disease. Here we report a method for eliminating false-negative results and increasing test sensitivity, based on the hypothesis that the message encoded by the nucleocapsid (N) gene is the most abundant during viral infection. Nasopharyngeal aspirates and stool samples were obtained from suspected SARS patients with major clinical symptoms and a significant history of close contact with infected patients. Total RNAs were extracted in a 96-well format, together with pig kidney epithelial (PK-15) cells as an internal control for extraction efficiency. PCR inhibitors were removed by ethanol precipitation, and a PCR for the pig β-actin gene was used as a positive control for all clinical samples. Samples were analyzed by a reverse transcriptase PCR assay. Northern blot analysis was performed to demonstrate differences in subgenomic transcripts of the virus, and a real-time quantitative PCR was employed to compare the sensitivities of two loci (1b and N). The detection rate of the assay reached 44.4% on day 9 after the onset of the disease. The diagnostic PCR amplifying the N gene gave an average of a 26. 0% (6.3 to 60.0%) stronger intensity signal than that for the lb gene. In conclusion, the nucleocapsid gene represents an additional sensitive molecular marker for the diagnosis of the SARS coronavirus and can be further adapted for use in a high-throughput platform assay.en_HK
dc.format.extent386 bytes-
dc.format.mimetypetext/html-
dc.languageengen_HK
dc.publisherAmerican Society for Microbiology.en_HK
dc.relation.ispartofJournal of Clinical Microbiologyen_HK
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.rightsJournal of Clinical Microbiology. Copyright © American Society for Microbiology.en_HK
dc.rightsCopyright © American Society for Microbiology, Journal of Clinical Microbiology, 2004, v. 42 n. 5, p. 1994-1999en_HK
dc.subject.meshReverse Transcriptase Polymerase Chain Reaction - methods - statistics & numericalen_HK
dc.subject.meshSARS Virus - genetics - isolation & purificationen_HK
dc.subject.meshSevere Acute Respiratory Syndrome - diagnosis - virologyen_HK
dc.subject.meshDNA Probes - geneticsen_HK
dc.subject.meshDNA, Viral - geneticsen_HK
dc.titleReverse Transcriptase PCR Diagnostic Assay for the Coronavirus Associated with Severe Acute Respiratory Syndromeen_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=0095-1137&volume=42&issue=5&spage=1994&epage=1999&date=2004&atitle=Reverse+transcriptase+PCR+diagnostic+assay+for+the+coronavirus+associated+with+severe+acute+respiratory+syndromeen_HK
dc.identifier.emailHui, RKH: rkhhui@hkucc.hku.hken_HK
dc.identifier.emailYuen, KY: kyyuen@hkucc.hku.hken_HK
dc.identifier.emailPeiris, JSM: malik@hkucc.hku.hken_HK
dc.identifier.emailLeung, FCC: fcleung@hkucc.hku.hken_HK
dc.identifier.authorityHui, RKH=rp00711en_HK
dc.identifier.authorityYuen, KY=rp00366en_HK
dc.identifier.authorityPeiris, JSM=rp00410en_HK
dc.identifier.authorityLeung, FCC=rp00731en_HK
dc.description.naturepublished_or_final_versionen_HK
dc.identifier.doi10.1128/JCM.42.5.1994-1999.2004en_HK
dc.identifier.pmid15131160-
dc.identifier.pmcidPMC404607en_HK
dc.identifier.scopuseid_2-s2.0-2442484802en_HK
dc.identifier.hkuros87945-
dc.identifier.hkuros93608-
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-2442484802&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume42en_HK
dc.identifier.issue5en_HK
dc.identifier.spage1994en_HK
dc.identifier.epage1999en_HK
dc.identifier.isiWOS:000221424100020-
dc.publisher.placeUnited Statesen_HK
dc.identifier.scopusauthoridHui, RKH=7103304764en_HK
dc.identifier.scopusauthoridZeng, F=7202911544en_HK
dc.identifier.scopusauthoridChan, CMN=7404814053en_HK
dc.identifier.scopusauthoridYuen, KY=36078079100en_HK
dc.identifier.scopusauthoridPeiris, JSM=7005486823en_HK
dc.identifier.scopusauthoridLeung, FCC=7103078633en_HK

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