Development a hydrolysis probe-based quantitative PCR assay for the specific detection and quantification of Candida auris.

ABSTRACT:

Background and purpose

Candida auris is an emerging multidrug-resistant pathogen. The identification of this species with the conventional phenotypic or biochemical mycological methods may lead to misidentification. Molecular-based species-specific identification methods such as quantitative real-time polymerase chain reaction (qPCR) facilitate a more reliable identification of C. auris than mycological methods. Regarding this, the present study aimed to develop a hydrolysis probe-based qPCR assay for the rapid, accurate identification of C. auris.

Materials and methods

The internal transcribed spacer 2 regions in the nuclear ribosomal DNA of C. auris and other related yeasts were assayed to find a specific PCR target for C. auris. A 123-base-pair target was selected, and primers and a probe were designed for hydrolysis probe-based real-time PCR with TaqMan chemistry. Ten-fold serial dilutions of C. auris ranging from 106 to 100 CFU/mL were prepared to establish a standard curve to quantify the yeast.

Results

The qPCR assay was able to identify and quantify C. auris with a detection limit of 1 C. auris CFU per reaction. Specificity was confirmed by the non-amplification of the sequences belonging to other Candida species, yeasts, molds, bacteria, or human DNAs. The standard curve of the assay showed a highly significant linearity between threshold values and dilution rates (R²=0.99; slope=-3.42).

Conclusion

The applied qPCR assay facilitated the rapid and accurate identification and quantification of emerging opportunistic C. auris. Therefore, considering the promising test validation results, we succeeded to develop a rapid and accurate hydrolysis probe- based qPCR assay for the screening and identification of C. auris.

SUBMITTER: Jafarian H 

PROVIDER: S-EPMC8018816 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature