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Updated: Oct 10, 2025
High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR
Published on: November 28, 2016
Reverse-transcription PCR increases sensitivity of broad-range fungal detection in bronchoalveolar lavage fluid
Heather L Glasgow1, Kurtis Cruz1, Sean C Murphy1,2
1Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, 98195, USA.
Abstract:
Broad-range PCR targeting 28S D1-D2 ribosomal DNA (rDNA) identifies numerous fungi but has limited sensitivity in clinical specimens. Ribosomal RNA (rRNA) vastly outnumbers rDNA, suggesting reverse transcription (RT)-PCR could improve detection. Among contrived samples, RT-PCR decreased 28S PCR cycle threshold values by 10--12 cycles and lowered the limit of detection > 2000-fold. Among 32 bronchoalveolar lavage specimens, RT-PCR detected 12/15 (80%) fungal PCR- or culture-positive specimens, versus 6/12 (50%) by 28S PCR, 9/12 (75%) by any fungal PCR, and 13/15 (87%) by culture. RT-PCR newly identified fungi in 4/17 (24%) PCR- and culture-negative specimens. RT substantially increased 28S PCR sensitivity overall.
Lay Summary:
Fungal infection remains difficult to diagnose in the laboratory. Here, we have shown that detecting ribosomal RNA and DNA, rather than only ribosomal DNA, in a broad range fungal assay results in a significant enhancement in the ability to detect and identify fungal pathogens in clinical samples.
