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Diagnostic Allele-Specific PCR for the Identification of Candida auris Clades.

ABSTRACT: Candida auris is an opportunistic pathogenic yeast that emerged worldwide during the past decade. This fungal pathogen poses a significant public health threat due to common multidrug resistance (MDR), alarming hospital outbreaks, and frequent misidentification. Genomic analyses have identified five distinct clades that are linked to five geographic areas of origin and characterized by differences in several phenotypic traits such as virulence and drug resistance. Typing of C. auris strains and the identification of clades can be a powerful tool in molecular epidemiology and might be of clinical importance by estimating outbreak and MDR potential. As C. auris has caused global outbreaks, including in low-income countries, typing C. auris strains quickly and inexpensively is highly valuable. We report five allele-specific polymerase chain reaction (AS-PCR) assays for the identification of C. auris and each of the five described clades of C. auris based on conserved mutations in the internal transcribed spacer (ITS) rDNA region and a clade-specific gene cluster. This PCR method provides a fast, cheap, sequencing-free diagnostic tool for the identification of C. auris, C. auris clades, and potentially, the discovery of new clades.

SUBMITTER: Carolus H

PROVIDER: S-EPMC8471779 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: The JAK2 V617F mutation is the most frequent somatic change in myeloproliferative neoplasms, making it an important tumour-specific marker for diagnostic purposes and for the detection of minimal residual disease. Sensitive quantitative assays are required for both applications, particularly for the monitoring of minimal residual disease, which requires not only high sensitivity but also very high specificity. METHODS: We developed a highly sensitive probe-free quantitative mutant-allele detection method, Quantitative Threefold Allele-Specific PCR (QuanTAS-PCR), that is performed in a closed-tube system, thus eliminating the manipulation of PCR products. QuantTAS-PCR uses a threefold approach to ensure allele-specific amplification of the mutant sequence: (i) a mutant allele-specific primer, (ii) a 3'dideoxy blocker to suppress false-positive amplification from the wild-type template and (iii) a PCR specificity enhancer, also to suppress false-positive amplification from the wild-type template. Mutant alleles were quantified relative to exon 9 of JAK2. RESULTS: We showed that the addition of the 3'dideoxy blocker suppressed but did not eliminate false-positive amplification from the wild-type template. However, the addition of the PCR specificity enhancer near eliminated false-positive amplification from the wild-type allele. Further discrimination between true and false positives was enabled by using the quantification cycle (Cq) value of a single mutant template as a cut-off point, thus enabling robust distinction between true and false positives. As 10,000 JAK2 templates were used per replicate, the assay had a sensitivity of 1/10(-4) per replicate. Greater sensitivity could be reached by increasing the number of replicates analysed. Variation in replicates when low mutant-allele templates were present necessitated the use of a statistics-based approach to estimate the load of mutant JAK2 copies. QuanTAS-PCR showed comparable quantitative results when validated against a commercial assay. CONCLUSIONS: QuanTAS-PCR is a simple, cost-efficient, closed-tube method for JAK2 V617F mutation quantification that can detect very low levels of the mutant allele, thus enabling analysis of minimal residual disease. The approach can be extended to the detection of other recurrent single nucleotide somatic changes in cancer.

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Diagnostic Allele-Specific PCR for the Identification of Candida auris Clades.

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