Quantification of pathogens by molecular methodsSubmit Manuscript on this topic
The advent of molecular biology tools had a tremendous impact on the quantification of the real number of microorganisms present in different matrices including food and environmental samples.
Traditional methods for quantification of microorganisms rely on counting serially diluted plates assays (bacteria), direct counting by optical microscopy chambers (bacteria and protozoa), transmission electron microscopy (viruses), plaque assays (viruses and bacteria), endpoint dilution and hemagglutination assays (viruses). More recently, flow cytometric assays and particularly quantitative polymerase chain reaction (qPCR) have been extensively used in this context, providing more robust, faster and many times more sensitive quantifications than conventional methodologies.
The qPCR (synonymous designation real time PCR could be also used) is currently the preferred tool of choice to quantify the number of different microorganisms (including not only bacteria, but mainly viruses as well as fungi). However, qPCR in basic setup cannot distinguish between viable and dead cells, which represents the issue in diagnostics of bacteria and partially in fungi. Therefore, the quantity of cells must be confirmed by culture in these cases. But, assuming that not all bacteria on the Earth can be cultured and with regard to the non-cultivable bacteria, qPCR-based methods represent the only tool to detect and quantify such bacteria.
There are no general rules for determination of microbial quantity by qPCR. The most common approach uses quantification standard derived from plasmid harbouring DNA target insert, pure DNA extracted from the microorganism of interest or PCR products. Subsequently, quantity of DNA in such standards is frequently determined by spectrophotometric or fluorometric approaches. However, if the DNA is not purified properly, it often leads to overestimation of DNA quantity. Confirmation of standard DNA quantity by the correlation with culture is not universally usable because each method determines the quantity in different way.
Recent developments in PCR technologies provided large scale of new techniques exploitable in quantification of microorganisms. Digital PCR (dPCR) currently attracts the interest of scientific community as it enables determination of DNA quantity in the sample by qPCR without necessity of using any kind of quantification standard. The method is based on limiting dilution and Poisson statistical analysis.
Vibrational spectroscopic based techniques, namely infrared and Raman spectroscopies, are also valuable techniques for an accurate microbial quantification, enabling the discrimination of bacteria in different physiological states. Their use in Microbiology has been widely expanded in the last 15 years as they are able to provide information in a non-destructive and reliable way and at a single-cell level with minimal preparation of samples. These methods are particularly useful because their coupling with multivariate analysis enables the monitoring biological processes in real time and without the necessity of exogenous chemical reagents, which saves costs, efforts and time in an environmentally friendly way.
The aim of this work is to provide a platform, in which researchers can discuss about the limits of the qPCR and dPCR in the field of microorganisms quantification taking into account the MIQE guidelines (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) available for both qPCR and dPCR. The potentiality of infrared and Raman spectroscopies in bacterial quantification will be also addressed.