Detection of rubella virus gene sequences by enzymatic amplification and direct sequencing of amplified DNA

F. A. Eggerding, J. Peters, Richard K Lee, C. B. Inderlied

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

We developed a rapid and sensitive polymerase chain reaction (PCR) assay for detecting and identifying rubella virus (RV). A segment of the RV gene which encodes the E1 membrane glycoprotein of RV was selected as a target for PCR amplification. Single-stranded viral RNA, extracted from infected cells or released from virions, was used as a template for reverse transcription followed by PCR amplification with two different sets of primer pairs, one nested within the other. The amplified E1 gene sequences were detected in ethidium bromide-stained agarose minigels, and their identities were verified by restriction enzyme digestion and hybridization to a probe directed at a site within the PCR target. Single-stranded DNA generated by asymmetric amplification of the target was directly sequenced by using fluorescent dideoxy-terminators and an automated procedure in order to confirm the target sequence. This PCR assay provides a rapid confirmatory test for the detection of RV by cell culture and appears to have considerable potential for the direct detection of RV in clinical specimens. The strategy used in the development of this PCR assay should be useful for developing other diagnostic PCR assays for viruses.

Original languageEnglish
Pages (from-to)945-952
Number of pages8
JournalJournal of Clinical Microbiology
Volume29
Issue number5
StatePublished - May 24 1991
Externally publishedYes

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Rubella virus
DNA Sequence Analysis
Polymerase Chain Reaction
Genes
Ethidium
Single-Stranded DNA
Membrane Glycoproteins
Viral RNA
Virion
Sepharose
Reverse Transcription
Digestion
Cell Culture Techniques
Viruses
Enzymes

ASJC Scopus subject areas

  • Microbiology (medical)
  • Microbiology

Cite this

Detection of rubella virus gene sequences by enzymatic amplification and direct sequencing of amplified DNA. / Eggerding, F. A.; Peters, J.; Lee, Richard K; Inderlied, C. B.

In: Journal of Clinical Microbiology, Vol. 29, No. 5, 24.05.1991, p. 945-952.

Research output: Contribution to journalArticle

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