Discussion Real-Time PCR technologies combine the sensitivity of conventional PCR with the generation of a quantifiable fluorescent signal and have been increasingly used to assess viability of microorganisms [11, 29–31]. Quantitative real-time PCR allows for the detection of PCR products produced at each step of the reaction, since an increase in reporter fluorescent signal is directly proportional to the number of amplicons OICR-9429 generated. As we have done in this work, PCR products can be quantitated by generating a standard curve, in which the absolute concentration
of the plasmid standard is known. In this study we measured the effect of anti-fungal agents against mature biofilms with a real-time RT-PCR assay based on the quantification of EFB1 transcript copy numbers in biofilm cells. The EFB1 gene is constitutively expressed under most growth conditions and is frequently used as a normalization gene in real-time RT-PCR quantification of other Candida genes [32–37]. By designing sense primers that span an intron splice site in the EFB1 sequence, we expected that only intact mRNA molecules would serve as a template in the RT-PCR assay and that
these molecules would be degraded following the death of the organisms in the biofilm. Our results with this molecular assay are consistent with our expectations and show that it is highly quantitative in a wider range of seeding fungal cell densities and that it more accurately measures small-moderate mature biofilm changes in response to stressors, compared to the traditional XTT assay. We have also shown that this assay is particularly well
suited for fungal check details biofilm viability estimates in complex Montelukast Sodium biological systems containing immune effectors or mucosal cell cultures. This may be partly due to the fact that mammalian cells also metabolize XTT, which further limits substrate availability . Compared to the XTT assay, the real-time assay is more technically demanding, more prone to experimental errors due to the multiple additional steps required in sample preparation, more costly, and significantly more time consuming. Thus it should be reserved for susceptibility testing of mature biofilms growing in complex biological model systems containing immune effectors or mucosal cells or used as a confirmatory assay when small changes in mature biofilms are detected with the XTT assay. Conclusions In conclusion, our results indicate that the XTT assay has to be applied with caution to biological systems containing large numbers of organisms alone or in combination with mammalian cells. We also conclude that molecular assessment of biofilms based on quantitation of EFB1 transcripts is a sensitive, reproducible and quantitative method to measure the damaging effect of anti-fungal agents against mature biofilms. The new quantitative assay will aid in further investigations of the mechanisms of Candida biofilm resistance to immune effector cells, which are PU-H71 presently unknown.