Abstract | BACKGROUND: 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.
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Authors | Bettina 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 |
Journal | Journal 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 |
PMID | 23806666
(Publication Type: Comparative Study, Evaluation Study, Journal Article, Research Support, Non-U.S. Gov't)
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Copyright | Published by Elsevier B.V. |
Chemical References |
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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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