The development and use of molecular testing specifically for case confirmation (virus-specific RNA detection) involves a wider range of RT-PCR methodologies than those used for genotyping. Different protocols produce amplicons of various lengths and may target different viral genes. Both conventional RT-PCR and real-time RT-PCR may be used to detect measles- or rubella- specific RNA in clinical specimens. Regardless of the assay selected, the specific target amplified by the primers (for either measles or rubella virus) must be demonstrated to be sensitive for detection of all genotypes.
Conventional RT-PCR is limited for diagnostic use due to its relative lack of sensitivity compared to real-time RT-PCR. In addition, the requirement for post-amplification analysis by agarose gel electrophoresis for conventional RT-PCR increases the turn-around time and is not amenable to high specimen throughput. While nested RT-PCR reactions usually offer a substantial increase in sensitivity over single round conventional RT-PCR, the greater potential for cross-contamination from positive specimens included in the test is a major concern. In general, nested PCR reactions are performed only in Global Specialized or Regional Reference Laboratories for molecular surveillance purposes to obtain a genotype from specimens that have a low copy number of viral RNA. Nested RT-PCR assays should not be used for case confirmation.
The reagents and equipment required for real-time RT-PCR are more expensive than the materials required for conventional RT-PCR. Real-time RT-PCR assays require a dye-labelled oligonucleotide probe. Synthesis of the probes is more complex than synthesis of non-labelled primers and it is critical to find a reliable source for probe synthesis. Because of the increased sensitivity of real-time RT-PCR assays compared to conventional assays, precautions to minimize the possibility of contamination most be followed rigorously. This requires strict adherence to directional workflow and the use of appropriate controls.
Most real-time RT-PCR assays are designed as one-step assays which combine the reverse transcription and PCR amplification steps in a single reaction. This reduces the repeated pipetting steps that increase the likelihood of cross-contamination. An advantage to real-time RT-PCR is that the assays can be configured as multiplex assays for detection of multiple targets in the same reaction [7]. For example, multiplexed real-time RT-PCR assays have been developed to simultaneously detect measles- and rubella-specific RNA and to target reference (cellular) genes.
Whether utilizing conventional RT-PCR or real-time RT-PCR, it is important to use a validated assay and to include positive and negative controls in all RT-PCR assays. The negative controls, or no-template controls (NTCs) are essential to detect extraneous nucleic acid contamination. Laboratories should develop SOPs using validated RT-PCR protocols with established performance characteristics:
- Defined lower limit of detection: the analytic sensitivity has been measured using samples with a known RNA copy number or virus concentration (e.g., pfu/ml, copies/reaction)
- Diagnostic sensitivity: the demonstrated ability to detect measles or rubella RNA in clinical samples from infected individuals
- Specificity: the absence of a signal when tested against other pathogens causing febrile rash illness and other respiratory pathogens. Specificity can be evaluated in silico by submitting the primer sequences to a BLAST search on GenBank
- Repeatability and reproducibility: results show limited intraassay and interassay variation
- Reaction kits include positive control RNAs of known sequence, preferably with genetic markers that clearly identify control reactions
- Optimized and defined reaction conditions
- Demonstrated ability to detect all known circulating genotypes of measles and rubella
- Flexible platform/chemistry
The laboratory must ensure that validation and appropriate QC/QA plans are in place. The quality indicators to be monitored for each assay are indicated in the laboratory SOP.