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Improving molecular tools for global surveillance of measles virus
Bettina Bankamp1, Lauren A Byrd-Leotis, Elena N Lopareva
1Measles, Mumps, Rubella and Herpes Virus Laboratory Branch, Division of Viral Diseases, Centers for Disease Control and Prevention, MS C-22, 1600 Clifton Road, Atlanta, GA 30333, USA. bbankamp@cdc.gov
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.
