Development of a rapid, internally controlled, two target, real-time RT-PCR for detection of measles virus.

Vaccination has greatly reduced global measles incidence, however measles remains endemic in many regions worldwide. Measles surveillance relies on high performance molecular detection of the virus. We have developed and validated a multiplex rRT-PCR assay for the detection of measles virus. The assay includes three independent probes with unique reporter dyes for the simultaneous detection of the measles hemagglutinin gene, nucleoprotein gene and endogenous RNaseP control. Using dilution series of synthetic RNAs the limits of detection were determined to be approximately 20 copies of measles RNA. The assay is extremely reproducible with very low intra-assay and inter-assay coefficients of varation for both the N and the H targets. After testing 68 confirmed measles positive and 86 measles negative archival clinical samples our data shows the multiplex assay has a sensitivity and specificity of 100 %, and a 100 % concordance with the expected results. No cross reactivity was identified with clinical specimens positive for six other viruses. According to the WHO, currently only the B3, D4, D8, H1 measles genotypes of the 24 recognized genotypes continue to circulate and this new multiplex assay successfully detected all four of those genotypes as well as six other genotypes.