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 [1–3]. While vaccines remain the most effective public health strategy for prevention [4, 5], 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 [8, 9].
Significant advances in molecular biology and human genomic research has paved the way for a host of new genetic diagnostic tests, including gene sequencing, detection, identification and genotyping of organisms using real time polymerase chain reaction (PCR) or other amplification techniques such as multiplex PCR, reverse line blot hybridization (RLB) , Ligase chain reaction (LCR), Rolling Circle Amplification (RCA) [9, 11], microarray [12–14]. Sequencing often serves as a 'gold standard' for the detection of single nucleotide polymorphisms, drug resistance mutations or virus/bacterial typing [15, 16]. However, as sequencing is cumbersome, expensive and less likely to detect low prevalence mutations (mutations consisting less than 30% of total populations) [17, 18], newer alternative techniques such as real time PCR and RCA are being employed [9, 15]. To establish molecular assays, positive controls are a prerequisite to ascertain specificity and sensitivity [19, 20]. However, in many cases it is difficult and cumbersome to acquire appropriate positive controls. It is thus, important to mention that although the commercial oligonucleotide synthesis can generate long oligonucleotides (≥150 mers) and can serve as suitable controls, there are problems as the oligonucleotide length exceeds 100 nucleobases, the yield of desired products often becomes limited by side reactions and even modest inefficiencies within the stepwise chemical reactions can have large effects on the final yield .
Further, in the event of an influenza pandemic, drug resistant strains and their transmission could be clinically highly significant , meaning that sensitive and specific techniques are required for their early and clinically relevant detection. However, due to the low frequency of naturally occurring resistance mutations in influenza infected patients receiving NA inhibitor treatment, the highly pathogenetic nature of influenza A H5N1 strains, and the technical complexity and time consuming nature of generating of NA resistant strains in vitro, collection of all known resistance mutations as positive controls is challenging. Therefore, our approach utilizing the formation of oligonucleotide dimers between two commercially synthesised long single stranded DNA templates (~95 bases each), to generate ~170 bp double-stranded artificial DNA templates, containing resistance mutations is not only innovative, but a more molecularly feasible and durable. In this way, it offers significant advantages in synthesizing even longer double stranded DNA templates, which can be used as positive control templates in molecular diagnostics. In the present study, we have used these double stranded artificial DNA templates with all known genetic mutations associated with influenza A drug resistance as a positive control in the development of a ligase chain reaction (LCR) for detecting NA inhibitor drug resistance mutations in patient samples.