Numerous clinical virology laboratories continue to employ diagnostic algorithms, which incorporate antigen or culture-based methods. The latter have several drawbacks in that they are technically demanding and laborious. Furthermore, not all viruses replicate in cell culture and the panel of susceptible cell lines is limited. The undeniable advantage of this approach is that the recovered viral strains are valuable for further pathogen characterization and antiviral susceptibility testing. Antigen-based methods can be used for both detection and typing of pathogens in clinical samples or in cell culture. Until now, several assays have been developed, although high variability of viruses may pose a problem and lead to false-negative results. Indisputably, the most sensitive and specific methods for detection of respiratory viruses are molecular techniques. Molecular methods may be roughly divided into sensitive and specific assays such as real-time PCR or loop-mediated isothermal amplification methods, and techniques with broad Quinidine specificity that are capable of detecting a wide variety of pathogens. The latter group includes sequence-independent methods, Sequence-independent, single-primer amplification, differential display). The main disadvantage of such an approach is that the sensitivity of the assays is limited. Further, these methods are relatively laborious and high-throughput analysis is unworkable. Some previously developed methods are designed to detect a broad range of targets combine, at least partially, the advantages of these two approaches. In the present study, human ciliated airway epithelial cell cultures were used in combination with a newly developed set of nested PCR assays with broad specificity, which are able to detect all known strains of respiratory viruses while disregarding co-infections, genetic variability, and other material contained in the clinical sample. Assay design was based on all 20(S)-Notoginsenoside-R2 available sequence data for human respiratory viruses; therefore, one may assume that they may enable the detection of previously uncharacterized viral strains. The combination of this highly sensitive culture system with a set of optimized molecular assays should allow improved detection in clinical samples. This hypothesis was verified using a set of clinical samples from patients suffering from respiratory disease. To design the sensitive and specific assays, all available sequences from public databases were analyzed. Primer design was based on sequence analysis facilitating the identification of conserved regions within viral genomes. Primer blasting showed no significant similarity to human genomic sequences and primer pairs with the best thermodynamic parameters were chosen for subsequent studies. HAE cultures constitute a semi-natural environment for the replication of viral pathogens, and pre-amplification of viruses contained in otherwise negative clinical samples resulted in the detection of viral agent in 100% of cases. This observation is compliant with the fact that the HAE model is superior to any other culture system in terms of amplifying respiratory viruses. HAE cultures allow replication of majority of the respiratory viral species, including those that cannot be cultured in cell lines. Again, one may question whether using HAE cultures will bias the results, as it is known that human metapneumovirus, for example, does not replicate in such a model. This assumption is undoubtedly correct, although as mentioned above, this culture model is superior for the majority of respiratory viruses and is unrivalled in terms of virus discovery.