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Reverse cross blot hybridization assay for rapid detection of PCR-amplified DNA from candida species, Cryptococcus neoformans, and Saccharomyces cerevisiae in clinical samples.

ABSTRACT: A PCR-based assay was developed to detect and identify medically important yeasts in clinical samples. Using a previously described set of primers (G. Morace et al., J. Clin. Microbiol. 35:667-672, 1997), we amplified a fragment of the ERG11 gene for cytochrome P-450 lanosterol 14alpha-demethylase, a crucial enzyme in the biosynthesis of ergosterol. The PCR product was analyzed in a reverse cross blot hybridization assay with species-specific probes directed to a target region of the ERG11 gene of Candida albicans (pCal), C. guilliermondii (pGui), C. (Torulopsis) glabrata (pGla), C. kefyr (pKef), C. krusei (pKru), C. parapsilosis (pPar), C. tropicalis (pTro), the newly described species C. dubliniensis (pDub), Saccharomyces cerevisiae (pSce), and Cryptococcus neoformans (pCry). The PCR-reverse cross blot hybridization assay correctly identified multiple isolates of each species tested. No cross-hybridization was detected with any other fungal, bacteria, or human DNAs tested. The method was tested against conventional identification on 140 different clinical samples, including blood and cerebrospinal fluid, from patients with suspected fungal infections. The results agreed with those of culture and phenotyping for all but six specimens (two of which grew yeasts not included in the PCR panel of probes and four in which PCR positivity-culture negativity was justified by clinical findings). Species identification time was reduced from a mean of 4 days with conventional identification to 7 h with the molecular method. The PCR-reverse cross blot hybridization assay is a rapid method for the direct detection and identification of yeasts in clinical samples.

SUBMITTER: Posteraro B

PROVIDER: S-EPMC86500 | biostudies-literature | 2000 Apr

REPOSITORIES: biostudies-literature

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Reverse cross blot hybridization assay for rapid detection of PCR-amplified DNA from candida species, Cryptococcus neoformans, and Saccharomyces cerevisiae in clinical samples.

Posteraro B B Sanguinetti M M Masucci L L Romano L L Morace G G Fadda G G

Journal of clinical microbiology 20000401 4

A PCR-based assay was developed to detect and identify medically important yeasts in clinical samples. Using a previously described set of primers (G. Morace et al., J. Clin. Microbiol. 35:667-672, 1997), we amplified a fragment of the ERG11 gene for cytochrome P-450 lanosterol 14alpha-demethylase, a crucial enzyme in the biosynthesis of ergosterol. The PCR product was analyzed in a reverse cross blot hybridization assay with species-specific probes directed to a target region of the ERG11 gene ...[more]

PMID: 10747151

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Project description:DNA damage has been considered to be the universal critical lesion in cells after exposure to ionizing radiation. Measuring radiation-induced DNA damage is important to understand the mechanisms of radiation-induced toxicity and monitor DNA damage repairs. Currently the most widely used methods to measure DNA damage are pulsed-field gel electrophoresis (PFGF) and single-cell gel electrophoresis (also known as the comet assay), both of which are technically challenging and time consuming. Long range quantitative polymerase chain reaction (LR-QPCR) has been used successfully to measure nuclear and mitochondrial DNA damage in mammalian and several model organism cells. The principle of this assay is that DNA lesions will slow down or block the progression of DNA polymerase. Therefore, the amplification efficiency of DNA with fewer lesions will be higher than DNA with more lesions under the same reaction condition. Here, we developed the LR-QPCR assay primers and reaction conditions to quantify DNA damage in Cryptococcus neoformans (C. neoformans) and Saccharomyces cerevisiae (S. cerevisiae) after gamma ray exposure. Under these conditions, long DNA targets of C. neoformans H99 and S. cerevisiae BY4741 (17.6 and 16.4 kb for nuclear DNA and 15.3 and 14.6 kb for mitochondrial DNA) were quantitatively amplified using extracted DNA templates, respectively. Two short mitochondrial DNA targets of these two species (207 bp and 154 bp) were also quantitatively amplified and used to monitor the number of mitochondria. Using the LR-QPCR method, we showed that the frequency of radiation-induced mitochondrial and nuclear DNA lesions had a significant linear correlation with the radiation doses (from 500 Gy to 3000 Gy) in both species. Furthermore, the faster disappearance of DNA damage detected in C. neoformans H99S strain compared to H99 strain may help to explain the different radiation sensitivity of these two strains. In summary, we developed a simple, sensitive method to measure radiation-induced DNA damage, which can greatly facilitate the study of radiation-induced toxicity and can be widely used as a dosimetry in radiation-induced cell damage.

Dataset Information

Reverse cross blot hybridization assay for rapid detection of PCR-amplified DNA from candida species, Cryptococcus neoformans, and Saccharomyces cerevisiae in clinical samples.

Publications

Reverse cross blot hybridization assay for rapid detection of PCR-amplified DNA from candida species, Cryptococcus neoformans, and Saccharomyces cerevisiae in clinical samples.

Similar Datasets

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