Improving molecular tools for global surveillance of measles virus

J Clin Virol. 2013 Sep;58(1):176-82. doi: 10.1016/j.jcv.2013.05.018. Epub 2013 Jun 24.

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.

Keywords: CDC; Centers for Disease Control and Prevention; Ct; FTA(®) cards; Genotyping; LabNet; MeV; Measles; Molecular diagnostics; RT; WHO; WHO Measles and Rubella Laboratory Network; World Health Organization; cpe; cytopathic effect; measles virus; nt; nucleotides; room temperature; threshold cycle.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Primers / genetics
  • Epidemiological Monitoring
  • Genotype
  • Global Health
  • Humans
  • Measles / diagnosis*
  • Measles / epidemiology*
  • Measles / virology
  • Measles virus / classification
  • Measles virus / genetics*
  • Measles virus / isolation & purification*
  • Molecular Diagnostic Techniques / methods*
  • Molecular Diagnostic Techniques / standards
  • Molecular Epidemiology / methods
  • RNA, Viral / genetics
  • Reference Standards
  • Reverse Transcriptase Polymerase Chain Reaction / methods
  • Reverse Transcriptase Polymerase Chain Reaction / standards
  • Sensitivity and Specificity

Substances

  • DNA Primers
  • RNA, Viral