Development of a broad-range quantitative polymerase chain reaction assay to detect and identify fungal DNA in equine endometrial samples.

The research presents a successful development of a broad-range quantitative PCR (qPCR) assay for detecting fungal DNA in horse endometrial samples. This new method was found to be faster, more sensitive, and consistent in identifying fungal infections in horse uteruses compared to traditional culture and biochemical analysis techniques.

Methods

• The research began by preparing and optimizing a 28S ribosomal DNA quantitative PCR (qPCR) assay. This method was used to detect the presence of fungal DNA in equine endometrial samples.
• 12 fungal samples from a clinical diagnostic laboratory and 29 samples from 17 mares suspected of having fungal endometritis were collected for testing.
• The qPCR assay was first validated using commercially acquired fungal organisms, and later on, clinically obtained samples.
• Traditional fungal culture and endometrial cytology methods were used to compare the results of the qPCR assay.
• The fragments of DNA produced from the qPCR (amplicons) were sequenced to identify the type of fungal organisms present.

Results

• The qPCR assay could theoretically detect as few as two organisms of Candida albicans, a common fungus.
• The assay was able to amplify fungal DNA from all 12 samples obtained from the clinical laboratory.
• In almost all cases (34 of 36 amplicons), the genetic sequencing successfully identified the fungal organism involved.
• The results highlighted an advantage of the qPCR assay over standard fungal culture and biochemical analysis. In all 12 samples, the qPCR assay and genetic sequencing identified a fungal agent while only 8 of these 12 samples were positively identified by the standard methods.
• The qPCR assay also detected fungal DNA in samples from 12 out of 17 mares that were suspected of having fungal endometritis.

Conclusions

• The study successfully developed a rapid, sensitive, and repeatable qPCR assay for the detection of fungal DNA from equine endometrial samples.
• Given its advantages, the qPCR method could be clinically useful as complement to microbial culture and cytologic examination to identify fungal organisms in a timely manner.