Chapter 6. Detection of viral RNA by RT-PCR for the confirmation of measles and rubella infection

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Conventional or real-time RT-PCR may be used to confirm the isolation of measles or rubella virus following inoculation and passaging of clinical specimens in cell culture, described in chapter 6. However, many of the laboratories in the GMRLN are equipped and trained to detect measles- and rubella-specific RNA directly from clinical specimens by real-time RT-PCR, which is much more sensitive than conventional RT-PCR. Protocols for performing real-time RT-PCR for the laboratory confirmation of measles or rubella infection have been developed and evaluated over many years and the assays continue to be improved.

When appropriate virologic specimens are available for testing, virus-specific RNA detection by RT-PCR for case confirmation is performed in many laboratories in combination with testing serologic or oral fluid specimens for virus-specific IgM. This approach has greatly improved the ability of the laboratory to provide timely confirmation of measles and rubella cases [1-4]. In addition, detection of viral RNA can provide a more reliable method for case confirmation than testing for IgM where disease prevalence is low, particularly in post-elimination settings [5].

Compared to conventional RT-PCR assays, real-time RT-PCR offers improved analytic sensitivity and the capacity for higher throughput without the need for post-amplification processing. However, the incorporation of adequate assay controls and the maintenance of physical separation of activities that reduce the risk of contamination are crucial. While this chapter provides guidance and recommendations for the diagnostic use of real-time RT-PCR, each laboratory in the GMRLN should work with their national surveillance programme and regional laboratory coordinator before incorporating RT-PCR as a tool for routine case confirmation.

Note: The abbreviation, RT-PCR, may be used herein to refer to both conventional (endpoint) reverse transcription PCR and to real-time (kinetic) RT-PCR, when describing characteristics of RT-PCR in general. Because real-time RT-PCR can be used for quantification of RNA in clinical specimens, real-time RT-PCR is sometimes referred to as quantitative real-time RT-PCR and abbreviated as RT-qPCR. However, in the context of the diagnostic use of RNA detection from measles and rubella samples specifically employing the use of real-time RT-PCR protocols, the term real-time RT-PCR will be used.