Challenges Facing Real-Time PCR Characteriztaion of Acute Respiratory Tract Infections
Ian M. Mackay, Katherine E. Arden, Michael D. Nissen and Theo P Sloots
from: Real-Time PCR in Microbiology: From Diagnosis to Characterization (Edited by: Ian M. Mackay). Caister Academic Press, U.K. (2007)
The age of reliance upon in vitro cell culture for routine laboratory diagnosis of respiratory virus infections has well and truly passed. We are much more comfortable with the application of molecular methods to detect and characterise the most common and frequent infectious agents of humans. The increased acceptance of molecular tools is not at the complete expense of other biological or serological methods; they will always have a place in microbiology, however the era of the high-throughput laboratory has driven the use of faster, more sensitive and more specific methods to diagnose of viral pathogens. Unfortunately these methods have some inherent limitations and the use of molecular techniques pose a number of serious problems to overcome, especially apparent in the area of respiratory virus detection and characterization. The scope and diversity of respiratory viruses mean that scientists in this field have to design and evaluate their own assays "in-house" because commercial options are extremely limited. The question of our ability to reliably detect so many viruses and so much subtle nucleotide variation is at the forefront of assay design and implementation problems, and we must address this aggressively. We are also faced with the extreme difficulty of quantifying respiratory viruses from essentially acellular fluids, secreted from within the host onto its surface (albeit a humid, highly convoluted invagination housed within our body) which we sample using a variety of collection methods. Quantification is further complicated by issues of specimen quality, handling and storage. Recently, the appearance of newly identified viruses or NIVs have both challenged and stymied respiratory virus real-time PCR assay designers and will undoubtedly continue to do so into the foreseeable future. We also see that real-time PCR cannot be used in isolation; for maximum success it must be accompanied by nucleotide sequencing, phylogenetic analyses and constant vigilance over the relevant literature. There is also a driving need to delve into the increased co-detection of multiple viral sequences and the ill-defined impact of quasispecies variation on oligonucleotide design. Real-time PCR is a mature technology and an extremely useful one for the study of acute respiratory tract infections yet it is not the perfect tool. In the meantime, we have much work to do in order to make best use of what we have to work with today read more ...