External Synthetic RNA Controls for Ebola
Synthetic RNA can be used to verify technical performance and interpretation of data quality resulting from various molecular diagnostic tests or methods. External spike-in controls are useful for method standardization and validation used by several laboratories and companies in recent years. In general, synthetic RNA controls are custom made. The controls are developed and optimized for their use in different diagnostic platforms and assays, such as in quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR), or microarray assays, as well as others. The use of synthetic RNA controls enable the development of diagnostic assays or testing methods allowing monitoring the spread of viral infections, such as the Ebola epidemic, the flu, SARS, the cold, infections with enteroviruses, or any other virus.
The longer a viral epidemic continues, the greater the chances are that the virus will spread to a wider community or jump over borders into other countries. The present Ebola epidemic illustrates this. As of October 2014, the Ebola outbreak is reported to have infected more than 5,000 people, and the mortality rate of this disease is reported to be close to 70%. The availability of fast and accurate diagnostic tests increases the chances for the early detection of an infection.
The ease of travel today together with the incubation time of eight to 21 days for the Ebola virus, increases the chance that an infected person can bring the virus across borders, thereby spreading the virus outside of Africa. According to Dr. Tom Frieden, MD, MPH, the director of the Center for Disease Control and Prevention, in Atlanta, it is important to rapidly test people for infection who have been traveling to countries that have been affected with the Ebola virus.
Since early recognition of an Ebola infection is critical to prevent further spread of the virus and to increase the chances of a person's survival, the need for reliable diagnostic methods is great. However, in the case of the Ebola Virus the diagnosis of a person infected with the virus is quite difficult during the early days of the infection. Reasons are initial symptoms, such as fever, which are not specific to the infection with Ebola. People infected with malaria or typhoid fever often show similar symptoms. Early signs of infection include fever, headache, sore throat, joint and muscle aches, diarrhea and weakness.
Reasons for testing if the Ebola virus is present in a person are:
- A person shows symptoms mentioned above and is known to have had contact with blood or body fluids of an infected person.
- A person shows symptoms mentioned above and is known to have had contact with infected animals.
- A person shows symptoms mentioned above and is known to have had contact with infected objects such as infected clothes or tools that belonged to an infected person.
Early diagnosis of an infection with the virus can help to avoid further infections. This will help to identify infected persons and isolate them from the community that surrounds them thereby minimizing the spread of the virus.
What is an Ebola virus?
The Ebola virus is a single-stranded, negative-sense mini-genome RNA virus. Zaire Ebola virus is responsible for the recent outbreak in West-Africa. Ebola viruses belong to the filoviridae family, and together with Paramyxoviridae, Rhabdoviridae, and Borna disease virus, Filoviridae viruses belong to the taxonomic order mononegavirales. Mononegavirales is the term used for "nonsegmented negative-strand RNA viruses" (NNSV). These are enveloped viruses that have mini-genomes consisting of a single RNA molecule of negative or anti-mRNA sense. Nucleic acids isolated from negative strand RNA viruses or virus-infected cells cannot infect or initiate an infection cycle when introduced into the host cell. This criterion was used to distinguish “positive’ from “negative”-strand RNA viruses. The viral genome needs to be first transcribed to produce mRNAs. Therefore, the purified virion RNA is not infectious. The virus needs to bring its own RNA polymerase into the cell in order to produce mRNA. To allow the virus to be infective a viral polymerase must be part of the viral particle or virion. In addition, synthetic RNA fragments derived from the viral genome are also not infectious. Therefore, the use of non-infectious synthetic viral RNA allows for the design of PCR primers or probes as well as peptides and recombinant proteins for molecular diagnostics.
What testing methods are presently available?
In general, the development or use of optimal RNA isolation methods together with real-time PCR are a good start for the development of fast and accurate testing or diagnostic methods. Optimized primers and probes for the PCR reaction are needed. The use of external synthetic RNA controls will allow evaluating, validating and standardizing classical as well as newly designed diagnostic testing methods or assays.
The following table shows a list of laboratory or diagnosis tests that are available or need to be developed.
Laboratory or diagnosis tests for Ebola
A few days after symptoms begin
- Antigen-capture enzyme-linked immunosorbent assay (ELISA) testing
- IgM ELISA
- Polymerase chain reaction (PCR)
- Virus isolation
Later in disease course or after recovery
- IgM and IgG antibodies
- Immunohistochemistry testing
- Virus isolation
Retrospectively in deceased patients
- IgM and IgG antibodies
- Immunohistochemistry testing
- Virus isolation
The External RNA Controls Consortium reported in 2005 in their progress report that reference standards to facilitate platform evaluation and comparability are needed. For example, the report showed that such standards may form part of a toolkit to evaluate the key performance characteristics of different qPCR platforms. Furthermore, a panel of RNA controls was developed for use in gene expression applications by the External RNA Controls Consortium (ERCC).
According to the ERCC, certified reference materials (CRM)
- consist of a reference set of approximately 100 well-characterized clones comprising RNA transcripts from random unique sequences as determined by sequence comparison to mouse, rat, human, drosophila, bacteria, and mosquito sequence databases, as well as inclusion of other nonhuman sequences;
- access to clones of the reference set deposited at a public repository;
- publication of all sequence information and test data;
- protocols for the preparation and use of the controls;
- suitable algorithms and bioinformatics tools for quantitative assessment and evaluation.
The next figures illustrate the workflow of an expression assay process and the use of spike-in controls to allow for standardization and validation of the process.
During the assay process sources for variability can be measured at various steps as indicated by the red arrows.
The use of synthetic RNA controls allows evaluation of technical performance throughout the whole assay process as has been pointed out elegantly by the ERCC.
Links to info for the ERCC:
Criteria for the design of new synthetic RNA controls that may need to be considered are:
- Their usefulness for the validation and standardization of testing procedures or assays.
- Controls used need to be non-infectious. Synthetic RNA fragments or mini-genomes derived from published genomic data available from public databases have been shown to be non-infective. The use of these type of controls for diagnostic assays or tests help to protect laboratory personnel and prevent infections.
- Synthetic RNA controls are very useful for qRT-PCR since exact known amounts of the control can be spike-in to allow for the sensitive detection of target sequences.
With over 30 years of experience in synthetic DNA chemistry and Molecular biology BSI offers custom designed synthetic RNA controls.
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