DNA Mold Testing

I understand that one can use DNA methods to determine whether a mold problem exists in one’s house/building and even identify major mold types present to the species level.  Is that correct?  Are these methods superior to other methods such as collection on culture plates or on spore traps?- Anon

            DNA methods commonly referred to as QPCR (quantitative polymerase chain reaction) or MSQPCR (mold specific quantitative polymerase chain reaction) are increasingly being used to identify and quantify mold types in air, surface, and bulk samples.  These methods are so sensitive that they can detect as few as one spore or fungal cell of a given species in a sample.   

            In beginning the analysis process, the DNA is extracted from the mold spores/cells in the collected samples.  They are then processed using a standardized protocol.  The key to the analytical technique is the use of mold specific primers and probes (there are approximately 130 common fungal species for which these are available) and an amplification solution.  A small quantity of the sample DNA is added to this mixture and the process is repeated for each target mold species. Reaction tubes are then placed in a Sequence Detector instrument (seen in the first image below on the right side, the author is in the foreground with Dr.King-Teh Lin, Mycometrics in the background) where the primers and probes bind to mold DNA they recognize.  On such recognition, the probe fluoresces and the fluorescence energy is detected and measured from each reaction mixture (output traces can be seen on the laptop in the following image.  The number of mold spores or more correctly cells can be determined by comparing the observed fluorescence from the sample to the of a standard mold spore suspension.   

            In the following figure, the process starts with a single copy of the mold gene of interest and then proceeds to multiple cycles of priming and amplification to increase the number of gene copies by a factor of 40 (on the order of a billion fold).

            MSQPCR can identify mold spores/cells whether they are alive (viable) or not (non-viable).  Since allergenicity is independent of mold cell viability QPCR can detect and quantify  problem molds whether they are alive or not. As such, QPCR results are a better indicator of potential exposure risks than culture plate methods. 

            For use in air sampling, QPCR analysis is not subject to the sampling time limitations of culture plate (2-5 minutes) or spore trap methods.  Sampling can be extended to hours or even days to provide more than a transient snapshot of airborne mold levels and since these are integrated, they are less variable.

To be continued

 

March 14, 2007