Sensitive Detection of SARS Coronavirus RNA by a Novel Asymmetric Multiplex Nested RT-PCR Amplification Coupled With Oligonucleotide Microarray Hybridization
Identifieur interne : 001155 ( Ncbi/Checkpoint ); précédent : 001154; suivant : 001156Sensitive Detection of SARS Coronavirus RNA by a Novel Asymmetric Multiplex Nested RT-PCR Amplification Coupled With Oligonucleotide Microarray Hybridization
Auteurs :Source :
- Microarrays in Clinical Diagnostics ; 2005.
Descripteurs français
- KwdFr :
- MESH :
- analyse : ARN viral.
- génétique : Virus du SRAS.
- instrumentation : RT-PCR, Sensibilité et spécificité, Séquençage par oligonucléotides en batterie.
English descriptors
- KwdEn :
- Oligonucleotide Array Sequence Analysis (instrumentation), Oligonucleotide Array Sequence Analysis (methods), RNA, Viral (analysis), Reverse Transcriptase Polymerase Chain Reaction (instrumentation), Reverse Transcriptase Polymerase Chain Reaction (methods), SARS Virus (genetics), Sensitivity and Specificity.
- MESH :
- chemical , analysis : RNA, Viral.
- genetics : SARS Virus.
- instrumentation : Oligonucleotide Array Sequence Analysis, Reverse Transcriptase Polymerase Chain Reaction.
- methods : Oligonucleotide Array Sequence Analysis, Reverse Transcriptase Polymerase Chain Reaction.
- Sensitivity and Specificity.
Abstract
We have developed a sensitive method for the detection of specific genes simultaneously. First, DNA was amplified by a novel asymmetric multiplex PCR with universal primer(s). Second, the 6-carboxytetramethylrhodamine (TAMRA)-labeled PCR products were hybridized specifically with oligonucleotide microarrays. Finally, matched duplexes were detected by using a laser-induced fluorescence scanner. The usefulness of this method was illustrated by analyzing severe acute respiratory syndrome (SARS) coronavirus RNA. The detection limit was 100 copies/µL. The results of the asymmetric multiplex nested reverse transcription-PCR were in agreement with the results of the microarray hybridization; no hybridization signal was lost as happened with applicons from symmetric amplifications. This reliable method can be used to the identification of other microorganisms, screening of genetic diseases, and other applications.
Url:
DOI: 10.1385/1-59259-923-0:59
PubMed: 16156097
PubMed Central: 7122606
Affiliations:
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PMC:7122606Le document en format XML
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First, DNA was amplified by a novel asymmetric multiplex PCR with universal primer(s). Second, the 6-carboxytetramethylrhodamine (TAMRA)-labeled PCR products were hybridized specifically with oligonucleotide microarrays. Finally, matched duplexes were detected by using a laser-induced fluorescence scanner. The usefulness of this method was illustrated by analyzing severe acute respiratory syndrome (SARS) coronavirus RNA. The detection limit was 10<sup>0</sup> copies/µL. The results of the asymmetric multiplex nested reverse transcription-PCR were in agreement with the results of the microarray hybridization; no hybridization signal was lost as happened with applicons from symmetric amplifications. This reliable method can be used to the identification of other microorganisms, screening of genetic diseases, and other applications.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Chamberlain, J S" uniqKey="Chamberlain J">J. S. 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