Early identification of Candida isolates to the species level is necessary for effective antifungal therapy, and can also facilitate control of hospital infections. Phenotype-based methods for identifying Candida species are often difficult and time-consuming. Molecular biological techniques provide a useful alternative approach. In the present study, the ITS1-5.8S-ITS2 regions of fungal rRNA genes were amplified with universal primers in 20 standard strains. Digestion of the PCR products with one restriction enzyme, MspI, allowed discrimination of medically important Candida species, including C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, and C. guilliermondii. Using this method, we successfully identified 137 clinical isolates of Candida. Among them, C. albicans was identified as the most common species, followed by C. parapsilosis, C. tropicalis, C. glabrata, C. krusei, and C. guilliermondii. This method is a simple, rapid, and cost-effective method for differentiation between species that is applicable in clinical laboratories.

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A one-enzyme pcr-rflp assay for identification of six medically important candida species

An assessment of the efficiency of fungal DNA extraction methods for maximizing the detection of medically important fungi using PCR.

A simple PCR-RFLP method for identification and differentiation of 11 Malassezia species.

Invasive fungal infection has become a major cause of morbidity and mortality in immunocompromised patients. Rapid identification of pathogenic fungi to species level is critical for disease treatment. A real-time LightCycler assay aiming at rapid detection and species identification of pathogenic fungi from clinical isolates was developed. Template DNAs of different species were amplified and detected in real time by employing SYBR Green fluorescent dye. The target sequences for species-level detection were located between the 18S and 28S rDNA. Seven fungal species encountered frequently in the clinical setting, Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis, Candida guilliermondii and Cryptococcus neoformans, could be discriminated by species-specific primers and confirmed by melting-curve analyses. The range of linearity was from 1 ng to 1 pg (μl−1 water) and the sensitivity was 1 pg fungal DNA μl−1. Identification by this real-time PCR method matched biochemical identification for all 58 clinical strains. Therefore, the method is simple, rapid and sensitive enough for detection and identification of several fungal species.

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Species identification of medically important fungi by use of real-time LightCycler PCR.

The development of real-time PCR thermal cycles in the late 1990s has opened up the possibility of accurate quantification of microorganisms in clinical, environmental, and food samples. However, a lack of suitable sample preparation methods that allow rapid quantification of the nucleic acids, remove PCR inhibitors, and prevent false-positive results due to DNA originating from dead cells has limited the use of quantitative PCR. We have used for the first time a new variant of density gradient centrifugation, called flotation, as a user-friendly sample preparation method prior to PCR. This paper describes the use of this sample preparation method, without DNA purification, for direct detection and quantification of Yersinia enterocolitica in PCR-inhibitory meat juice from pork. Flotation combined with qPCR could overcome PCR interference in juice from pork, as was shown by amplification efficiencies of 1.006 ± 0.021 and 1.007 ± 0.025, which are comparable to the amplification efficiency obtained for purified DNA samples (1.005 ± 0.059). Applying flotation to meat juice samples containing natural background flora and spiked with different levels of Y. enterocolitica showed that direct quantification of Y. enterocolitica was possible down to a level of at least 4.2 × 103 CFU per ml of meat juice, even in the presence of 106 CFU of background flora per ml. Finally, the results showed that samples containing large amounts of Y. enterocolitica DNA did not result in a positive PCR signal. This indicates that the risk of false-positive results due to detection of DNA originating from dead cells can be greatly reduced by using flotation prior to PCR.

/pdf/rapid-quantification-of-yersinia-enterocolitica-in-pork-3tuwq9tc6i.pdf

Rapid quantification of yersinia enterocolitica in pork samples by a novel sample preparation method, flotation, prior to real-time PCR

Methods of rapidly extracting chromosomal DNA from human pathogenic yeasts were used in mitochondrial DNA (mtDNA) studies. This paper is concerned with rapid and reliable methods of extracting mtDNA for sequence analysis for species or strain identification, and epidemiological study of medically important fungi and fungal infections. To determine the optimal method of mtDNA extraction without isolating mitochondria, we examined three commonly used methods: 1). boiling, 2). glass bead disruption, and 3). a commercially available kit. We assessed the amount and quality of DNAs using a spectrophotometer and specific polymerase chain reaction (PCR). The DNA yield depended on the extraction method used and the yeast species. An adequate amount of mtDNA was obtained with both glass beads and a commercially available kit to amplify the mitochondrial gene using PCR without isolating the mitochondria. These techniques are convenient for extracting DNA from a variety of small-scale samples.

Hossain Merhendi

Papers

Comparison of different methods for extraction of mitochondrial dna from human pathogenic yeasts