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Improving molecular tools for global surveillance of measles virus.

AbstractBACKGROUND:
The genetic characterization of wild-type measles viruses plays an important role in the description of viral transmission pathways and the verification of measles elimination. The 450 nucleotides that encode the carboxyl-terminus of the nucleoprotein (N-450) are routinely sequenced for genotype analysis.
OBJECTIVES:
The objectives of this study were to develop improved primers and controls for RT-PCR reactions used for genotyping of measles samples and to develop a method to provide a convenient, safe, and inexpensive means to distribute measles RNA for RT-PCR assays and practice panels.
STUDY DESIGN:
A newly designed, genetically defined synthetic RNA and RNA isolated from cells infected with currently circulating genotypes were used to compare the sensitivity of primer pairs in RT-PCR and nested PCR. FTA® cards loaded with lysates of measles infected cells were tested for their ability to preserve viral RNA and destroy virus infectivity.
RESULTS:
A new primer pair, MeV216/MeV214, was able to amplify N-450 from viruses representing 10 currently circulating genotypes and a genotype A vaccine strain and demonstrated 100-fold increased sensitivity compared to the previously used primer set. A nested PCR assay further increased the sensitivity of detection from patient samples. A synthetic positive control RNA was developed that produced PCR products that are distinguishable by size from PCR products amplified from clinical samples. FTA® cards completely inactivated measles virus and stabilized RNA for at least six months.
CONCLUSIONS:
These improved molecular tools will advance molecular characterization of circulating measles viruses globally and provide enhanced quality control measures.
AuthorsBettina Bankamp, Lauren A Byrd-Leotis, Elena N Lopareva, Gibson K S Woo, Chunyu Liu, Youngmee Jee, Hinda Ahmed, Wilina W Lim, Nalini Ramamurty, Mick N Mulders, David Featherstone, William J Bellini, Paul A Rota
JournalJournal of clinical virology : the official publication of the Pan American Society for Clinical Virology (J Clin Virol) Vol. 58 Issue 1 Pg. 176-82 (Sep 2013) ISSN: 1873-5967 [Electronic] Netherlands
PMID23806666 (Publication Type: Comparative Study, Evaluation Study, Journal Article, Research Support, Non-U.S. Gov't)
CopyrightPublished by Elsevier B.V.
Chemical References
  • DNA Primers
  • RNA, Viral
Topics
  • DNA Primers (genetics)
  • Epidemiological Monitoring
  • Genotype
  • Global Health
  • Humans
  • Measles (diagnosis, epidemiology, virology)
  • Measles virus (classification, genetics, isolation & purification)
  • Molecular Diagnostic Techniques (methods, standards)
  • Molecular Epidemiology (methods)
  • RNA, Viral (genetics)
  • Reference Standards
  • Reverse Transcriptase Polymerase Chain Reaction (methods, standards)
  • Sensitivity and Specificity

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