title: Development of a broad-range quantitative polymerase chain reaction assay to detect and identify fungal DNA in equine endometrial samples.
abstract: OBJECTIVE
  To develop a broad-range 28S ribosomal DNA quantitative PCR (qPCR) assay for detection of fungal DNA in equine endometrial samples.
  
  
  METHODS
  12 fungal samples from a clinical diagnostic laboratory and 29 samples obtained from 17 mares.
  
  
  METHODS
  The qPCR assay was optimized with commercially acquired fungal organisms and validated with samples obtained from the clinical diagnostic laboratory. Subsequently, 29 samples from 17 mares suspected of having fungal endometritis were evaluated via the qPCR assay and via traditional fungal culture and endometrial cytology. Amplicons from the qPCR assay were subjected to genetic sequencing to identify the organisms.
  
  
  RESULTS
  The qPCR assay theoretically had a detection threshold of 2 organisms of Candida albicans. Fungal DNA was amplified from all 12 fungal samples from the commercial diagnostic laboratory. Fungal identification by use of genetic sequencing was successful for 34 of 36 amplicons from the 12 samples assayed. A fungal agent was identified via qPCR assay and genetic sequencing in all 12 samples; in contrast, a fungal agent was identified in only 8 of 12 samples via standard fungal culture and biochemical analysis. The qPCR assay detected fungal DNA in samples from 12 of 17 mares suspected of having fungal endometritis.
  
  
  CONCLUSIONS
  A rapid, sensitive, and repeatable qPCR assay was developed for detection of fungal DNA from equine endometrial samples. The qPCR may prove to be clinically useful as an adjunct to microbial culture and cytologic examination to provide identification of fungal organisms in a timely manner.
authors.affiliation: Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80521, USA.. rferris@colostate.edu
authors.forename: Ryan A
authors.initials: RA
authors.lastname: Ferris
authors.forename: Katy
authors.initials: K
authors.lastname: Dern
authors.forename: Julia K
authors.initials: JK
authors.lastname: Veir
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Hawley
authors.forename: Michael R
authors.initials: MR
authors.lastname: Lappin
authors.forename: Patrick M
authors.initials: PM
authors.lastname: McCue
mesh_terms: D000818:Animals; D001232:Aspergillus fumigatus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D002176:Candida albicans / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D004271:DNA, Fungal / Q000032:analysis*; D004275:DNA, Ribosomal / Q000032:analysis; D004715:Endometriosis / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D005260:Female; D005658:Fungi / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D006734:Horse Diseases / Q000175:diagnosis* / Q000382:microbiology; D006736:Horses; D009181:Mycoses / Q000175:diagnosis / Q000382:microbiology; D012339:RNA, Ribosomal, 28S / Q000032:analysis; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000662:veterinary
chemical_list: D004271:DNA, Fungal; D004275:DNA, Ribosomal; D012339:RNA, Ribosomal, 28S