RT-PCR is a sensitive method and should be performed following strict quality control and quality assurance procedures. A description of the quality assurance programme administered through WHO for molecular external quality assurance (mEQA) is provided in Chapter 12. Quality assurance, quality control, and assessment of laboratory capacity and performance. All the laboratories that use RT-PCR as a method for case confirmation and/or report genotypes to MeaNS and RubeNS participate in the annual mEQA programme. In addition, the WHO accreditation process for the GMRLN includes an assessment of the facilities for RT-PCR and documentation of QA/QC processes for laboratories using molecular techniques.
To avoid cross-contamination of samples tested by RT-PCR, unidirectional workflow proceeding from the area for RNA extraction/reagent preparation (pre-amplification area) to the PCR amplification room (post-amplification area) must be implemented and maintained. It is important that appropriate facilities and dedicated rooms and equipment for RT-PCR pre-amplification and post-amplification steps are provided to reduce the possibility of cross-contamination with amplification products. There should be designated areas for RNA extraction, preparation of the pre-mix, addition of test RNA, and addition of control RNAs. The initial steps of RNA extraction must be performed in a class II BSC because clinical samples are potentially infectious until the RNA lysis buffer is added. Subsequent steps in RNA extraction and RT-PCR can be performed in a BSC or a dedicated PCR cabinet.
Each work area should have dedicated laboratory coats and equipment and the equipment should not be moved from one work area to another. All personnel must be familiar with the protocols and instruments used. Equipment should be properly maintained, with documentation that verifies that routine preventive maintenance and calibration are regularly performed. Following these guidelines will help minimize the chance of false-positive results. In addition, important practices that should be included in RT-PCR SOPs are listed below:
- Maintain separate areas, dedicated equipment (e.g., pipettes, microcentrifuges) and supplies (e.g., microcentrifuge tubes, pipette tips, gowns and gloves) for each of the following activities:
- RNA extraction in a class II BSC
- Preparation of the RT-PCR pre-mix
- RNA template addition
- Addition of positive controls
- RT-PCR amplification
- Manipulation of the products of amplification - Wear clean, previously unworn, disposable gowns and new, powder-free gloves during pre-mix setup and handling of extracted nucleic acids; gloves should be changed frequently
- Store primer/probes and enzyme master mix at appropriate temperatures (see package inserts)
- Do not use reagents beyond their expiration dates
- Keep reagent tubes and reactions capped as much as possible
- Clean work surfaces and equipment (pipettors, tube racks, etc.) using a commercial product that destroys DNA and RNA or a freshly prepared chlorine bleach solution (10%)
- Use aerosol barrier (filter) pipette tips only
The use of appropriate controls is essential for both conventional RT-PCR and real-time RT-PCR assays for detection of measles or rubella. Synthetic RNA controls for conventional RT-PCR are available for GMRLN laboratories (contact the GLC and CDC for instructions). The synthetic positive control RNAs for conventional RT-PCR are designed with insertions or deletions in the target regions. The PCR product from the control reaction will migrate with a slightly different molecular weight in agarose gel electrophoresis. Use of the synthetic positive controls enables the laboratory to visually detect cross-over of the positive control RNA into the test samples or negative control samples when running the amplification products on the agarose gel. Because of this ability to discriminate the synthetic controls from wild or vaccine viruses, the synthetic positive controls should be used exclusively when available.
Real-time RT-PCR assays are usually designed to detect cellular RNA from a reference gene, or “housekeeping gene”. The most frequently targeted reference genes, human ribonuclease P (RNase P) and glyceraldehyde 3-phosphate dehydrogenase (GADPH), are present in all human cells and are used to evaluate the adequacy of the clinical sample and the quality of the RNA extraction. Detection of a reference gene is performed either in a multiplex format or as a separate reaction. Failure to detect the reference gene indicates that insufficient cellular material was collected, inhibitory substances are present in the sample, or the sample was transported or processed improperly. WHO strongly recommends the use of reference genes for real-time RT-PCR assays that are used for case confirmation.
The incorporation of adequate in-house RNA positive controls provides evidence that the sensitivity of the assay was within accepted parameters. Documentation of the use of appropriate controls is necessary to ensure accuracy. Synthetic viral RNA or viral RNA from infected culture are commonly used as positive controls. Negative controls should include sterile, nuclease-free water for no-template controls (NTC) and mock-extracted RNA samples (extraction control). For real-time RT-PCR, multiple virus-specific and single cellular gene-specific reactions are often included in the test protocol. The SOP should identify quality indicators that will be monitored for each assay to help identify trends that may affect assay performance. For example, many laboratories plot the Ct value of one of the standard positive control reactions.