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Fungal Molecular Diagnostics


Pneumocystis PCR

Slide 23

February 2011

In the next few slides, I will briefly describe our real-time PCR assays for fungal pathogens and will highlight a couple of examples of how they have been useful in our practice. The first assay that I will discuss is the Pneumocystis jirovecii assay. A full description of this assay has been published in Diagnostic Microbiology and Infectious Diseases and I have provided the reference in this slide if you are interested in reading more of the details. So why did we choose to develop a Pneumocystis PCR assay? One reason was that this organism is not easily cultured in the clinical laboratory. Stains and immunofluorescence assays have, therefore, been utilized, but they are often insensitive, subjective, and require experienced readers. Our goal with the PCR assay was to develop a sensitive and objective method to detect this organism. The target selected is a 162-base pair region of the cyclin-dependent kinase or cdc-2 gene in Pneumocystis jiroveci. Cdc-2 is involved in Pneumocystis cell cycle regulation and, therefore, this gene is highly conserved. Following the workflow described on the previous slide, we use cdc-2 sequence-specific FRET hybridization probes to detect any Pneumocystis jirovecii DNA present in the patient’s specimen. Acceptable sources for this assay are respiratory specimens such as bronchoalveolar lavage (BAL) fluid and sputum.

Pneumocystis PCR

 


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