The conventional RT-PCR for measles that amplifies a fragment to generate the N-450 sequencing window is used by some laboratories for case confirmation. Several sensitive conventional RT-PCR assays that target the N or H genes for either RNA detection and/or genotyping have been described with lower limits of detection in the range of 1,000-10,000 copies of measles viral RNA per reaction [8]. Real-time RT-PCR assays that target regions of the measles N or H genes can typically detect as few as 10-100 copies of viral RNA per reaction and can provide quantitative results [9,7,10].
Intercountry laboratory workshops sponsored by WHO to support molecular detection and genotyping for measles and rubella may offer different protocols for conventional or real-time RT-PCR. As with EIAs for antibody detection, various validated in-house and commercial kits may demonstrate acceptable performance, but all RT-PCR protocols must meet validation criteria. Regional workshops may utilize different kits, but all workshops emphasize the technical aspects of RT-PCR as well as quality assurance and quality control components. Some examples of the protocols that are widely used are described below.
Many of the intercountry laboratory workshops have provided training for the real-time RT-PCR method utilized at the CDC (Atlanta) that amplifies a 75-nucleotide region of the measles N gene [9]. Kits containing primers, probes and positive control RNA are provided by CDC to GMRLN laboratories. The kits produced at the CDC include two positive controls consisting of synthetic measles RNA of known copy number. The acceptable range of Ct values for both controls are provided in the kit instructions. The primers and probe for detection of the reference gene RNase P are also included. The protocol for the CDC measles real-time RT-PCR is available in pdf format (Annex 6.2).