Real-time RT-qPCR 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 for measles and rubella may offer different protocols for RT-qPCR. As with EIAs for antibody detection, various validated in-house and commercial kits may demonstrate acceptable performance, but all RT-qPCR protocols must meet validation criteria. Regional workshops may utilize different kits, but all workshops emphasize the technical aspects of RT-qPCR as well as quality assurance and quality control components.
Many of the intercountry laboratory workshops have provided training for the RT-qPCR 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 RT-qPCR is available in pdf format (Annex B11.2).
RT-qPCR is often used in parallel with IgM serological testing, particularly to confirm cases when IgM results are inconclusive or negative, such as in the early phase of illness or in settings with low measles prevalence. This dual approach increases diagnostic accuracy, supports case classification, and informs public health response.
Recent advances include the availability of multiplex RT-qPCR assays that allow simultaneous detection of measles, rubella, and a human cellular control gene. These platforms increase laboratory throughput and provide internal validation of sample quality and extraction efficiency within each reaction.