Quantification of Micro-Organisms: Not Human, Not Simple, Not Quick
Ian M. Mackay, Stephen A. Bustin, José Manuel Andrade, Mikael Kubista 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 majority of real-time PCR applications in microbiology are for qualitative (resulting in a yes or no answer) detection of a virus, bacterium, fungus or parasite. In terms of disease relevance, the importance of quantitative PCR (qPCR) to microbiology has been proven, however it is less clear just how punctilious the clinical microbiology laboratory must be to produce relevant quantitative results. Despite a decade of qPCR experience, commercial development of applications is limited and many of our approaches remain entrenched among the PCR techniques used to monitor human mRNA levels rather than addressing adequately the diverse needs of the microbiology field. Real-time PCR has permeated every aspect of microbiology, but its applications have particular value in the clinical microbiology laboratory where the speed, sensitivity, reproducibility and accuracy of this tool help to produce robust data in a clinically relevant timeframe. Other areas within microbiology have also gained from the use of real-time PCR; gene therapy has found benefit from qPCR applications that monitor the production, replication and administration of viral vectors used to transport therapeutic genes into host cells or tissues. Studies of the host's response to microbial replication suggest a vision of the future wherein patient specimens may be used to provide an indication not only of the type of micro-organism present and its replicative status, but the stage of disease and the type of immune response underway. To make such vision reality, we must first discuss and reach consensus on the best, microbiology-specific qPCR approaches to permit the production of comparable microbial load data. This process must include the development of clear definitions associating microbial load with clinical outcome, the production of more reference materials, the development of more quality assessment schemes and of commercial kits. It may be that we find the perfect estimate of micro-organism numbers is not as important as reproducible and clinically relevant data. The increased identification of newly emergent or previously unknown endemic pathogens demands that we must strive harder than ever to expand our understanding of infectious diseases, and for that we need reliable results from reliable tools read more ...