Partial amplification of the measles virus nucleocapsid gene from stored sera and cerebrospinal fluids for molecular epidemiological studies.
The analysis of stored sera for retrospective molecular epidemiological studies provides a powerful tool to investigate strain variation in measles viruses that had circulated up to 20 years ago. For this purpose, a rapid and simple method for extraction of RNA from stored sera and cerebrospinal fluids (CSF) was developed. When used on sera and CSFs that have been frozen for as long as 20 years, this method proved to be more efficient than established techniques. The extracted RNA was reverse transcribed into cDNA by using random hexamer primers. The PCR amplification of the 3' terminus of the nucleocapsid gene (N) was divided into two overlapping fragments of 375 and 384 bp length, covering the entire region of interest. This region is thought to have the highest variability within the MV genome and has previously been shown to be suitable for strain characterization. The resulting PCR fragments were sequenced manually by using standard methods without the need of further clean-up steps.
Kreis
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National Institute for Virology, Johannesburg, South Africa. Stephk@niv.ac.za
Schoub
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D000595:Amino Acid Sequence; D001769:Blood / Q000821:virology; D002555:Cerebrospinal Fluid / Q000821:virology; D018076:DNA, Complementary / Q000032:analysis; D005784:Gene Amplification; D005814:Genes, Viral; D006801:Humans; D008457:Measles / Q000097:blood / Q000134:cerebrospinal fluid / Q000453:epidemiology; D008459:Measles virus / Q000235:genetics* / Q000302:isolation & purification*; D017720:Molecular Epidemiology; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000235:genetics*; D012367:RNA, Viral / Q000302:isolation & purification; D012189:Retrospective Studies; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013048:Specimen Handling
D018076:DNA, Complementary; D019590:Nucleocapsid Proteins; D012367:RNA, Viral