Background
Positive controls are an integral component of any sensitive molecular diagnostic tool, but this can be affected, if several mutations are being screened in a scenario of a pandemic or newly emerging disease where it can be difficult to acquire all the necessary positive controls from the host. This work describes the development of a synthetic oligo-cassette for positive controls for accurate and highly sensitive diagnosis of several mutations relevant to influenza virus drug resistance.
Results
Using influenza antiviral drug resistance mutations as an example by employing the utility of synthetic paired long oligonucleotides containing complementary sequences at their 3′ ends and utilizing the formation of oligonucleotide dimers and DNA polymerization, we generated ~170bp dsDNA containing several known specific neuraminidase inhibitor (NAI) resistance mutations. These templates were further cloned and successfully applied as positive controls in downstream assays.
Conclusion
This approach significantly improved the development of diagnosis of resistance mutations in terms of time, accuracy, efficiency and sensitivity, which are paramount to monitoring the emergence and spread of antiviral drug resistant influenza strains. Thus, this may have a significantly broader application in molecular diagnostics along with its application in rapid molecular testing of all relevant mutations in an event of pandemic.
Background
Recently, the occurrence in humans of infection with virulent avian influenza A H5N1 and the emergence of swine origin pandemic influenza A H1N1 2009 strain have sparked fear of an ongoing pandemic with novel genetic characters. While vaccines remain the most effective public health strategy for prevention, antiviral drugs such as neuraminidase inhibitors (NAIs), oseltamivir and zanamivir, could play an important role in the response to the early phases of a pandemic, if available in sufficient quantities. However, like other antiviral agents, the emergence of influenza viruses with reduced susceptibility to the NAI is inevitable during treatment. To date, strains with altered susceptibility to NAI have been recovered from approximately 1% of immunocompetent adult patients and up to 18% of pediatric patients. In addition, oseltamivir-resistant influenza A H5N1 and pandemic H1N1 2009 viruses with the H274Y mutation have been reported from patients during oseltamivir treatment.