Comparison of PCR- and HinfI Restriction Endonuclease-Based Methods for Typing of Candida krusei Isolates (original) (raw)
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Species-specific identification of Candida krusei by hybridization with the CkF1,2 DNA probe
Journal of clinical microbiology, 1996
The species specificity of the Candida krusei DNA fingerprinting probe CkF1,2 has been investigated. A total of 149 pathogenic and nonpathogenic fungal and bacterial DNAs were screened with CkF1,2. The probe was cold labeled with peroxidase, and its specificity was assessed by using Southern blot, dot blot, and colony blot hybridization. Its sensitivity was determined by dot blot hybridization. The CkF1,2 probe proved to be species specific. It hybridized with DNA for the 112 C. krusei strains studied, whereas it failed to hybridize under low-stringency conditions to 37 DNAs from 27 different yeast species, including Candida albicans, Candida glabrata, Candida norvegensis, Candida inconspicua, Candida tropicalis, Candida valida, Candida zeylanoides, and Yarrowia lipolytica, as well as DNAs from the filamentous fungi and bacteria tested. However, CkF1,2 hybridized strongly with DNA of the yeast species Issatchenkia orientalis, the putative ascogenous perfect state of C. krusei. Amoun...
Colony PCR Is a Rapid and Sensitive Method for DNA Amplification in Yeasts
2007
Background: Yeast infections are increasing cause of morbidity and mortality in immunocompromised patients. In order to perform a DNA-based diagnostic test, availability of a rapid and easy-to-perform DNA extraction protocol is essential. In the present study we evaluated colony-PCR as the easiest way to amplification of target DNA. Methods: Instead of using templates of purified genomic DNA, we performed the PCR directly from yeast colonies or cultures. Serial cell dilution of three reference yeast strains including Candida albicans, Cryptococcus neoformans and Saccharomyces cerevisiae were used for determining the sensitivity of the colony-PCR. A total of one hundred yeast isolates were also tested. All reactions were performed using the universal fungal primers ITS1 and ITS4 complementary to the rDNA region. Results: The colony-PCR resulted in a single band (with different sizes) for 10 6 cells or more for all reference species. Furthermore 98 out of 100 (98%) of samples showed a relevant single band after PCR. Conclusion: Directly application of the yeast cells obtained from culture colony for PCR reaction is a fast, reliable, cost-effective and simple method for performing any PCR-based protocol including diagnostic tests.
Journal of Clinical Microbiology, 1994
The use of restriction endonuclease analysis and Southern hybridization with our new CkF1,2 DNA probe, cold labeled with peroxidase, for the typing of Candida krusei isolates has been investigated. Fifty-five clinical samples isolated from forty-five patients hospitalized in eight centers, one environmental strain, and two reference strains were evaluated. Patterns were analyzed by a computer-assisted method and compared by numerical analysis. Clearer and less ambiguous patterns were obtained by restriction with endonuclease Hinfl. It generated 9 to 14 (average, 11) well-separated fragments in the range of 6.5 to 2.0 kb. Both their numbers and sizes varied greatly among the strains studied. The CkF1,2 probe hybridized with one to seven fragments ofHinfl patterns. A total of 48 distinct types were distinguished among the 58 strains studied. Hinfl and CkF1,2 patterns showed similarities of less than 83 and 75% for unrelated strains and more than 91 and 100% for related strains, respectively. The methods showed 100% typeability, 98% reproducibility, and a discriminatory power of 1. C. krusei isolates from each patient were distinct, whether from one hospital or from different hospitals. Multiple isolates from the same patient were identical, both over time and at different anatomic sites. An endogenous origin is suggested for the colonizing and infecting isolates among the 45 patients. The CkF1,2 probe enhanced discrimination of the strains and provided a definitive comparison for strain identity. Genetic linkages between isolates were assessed at the subspecies level, and 12 clusters were delineated. A typing scheme is proposed for epidemiological studies of C. krusei.
1997
A single primer pair amplifying a cytochrome P-450 lanosterol-14␣-demethylase (L1A1) gene fragment that encodes a highly conserved region was used to detect yeast DNA in clinical specimens. Positive PCR products were obtained from genomic DNAs of Candida albicans, C. parapsilosis, C. tropicalis, C. guilliermondii, C. krusei, C. (Torulopsis) glabrata, and C. kefyr. No human, bacterial, or parasitic DNA was amplified. The sensitivity was evaluated for C. albicans genomic DNA by using various DNA concentrations (200 pg to 2 fg). The amplified DNAs of Candida species with unknown P-450 L1A1 gene sequences were subcloned and sequenced. Identification at the species level was achieved by digestion of the PCR products with different restriction enzymes. A specific restriction enzyme analysis pattern was determined for each species investigated. Subsequently, we used PCR to detect specific yeast DNA directly with clinical specimens such as blood and bronchoalveolar lavage specimens. After appropriate treatment, the specimens were processed by PCR and the results were compared with those obtained by traditional diagnostic procedures such as cultures and serology. Although preliminary, the PCR results seem to correlate well, at least for blood, with those of antigen detection assays and traditional blood cultures, with a better and earlier detection of candidemia.
Diagnostic Microbiology and Infectious Disease, 2000
Invasive candidiasis has become a major cause of morbidity and mortality in immunocompromised hosts. Here we describe a fast and reliable DNA extraction and PCR amplification method in combination with a slot blot hybridization assay. A genus-specific probe was designed that allowed to detect DNA from a broad range of Candida species and 3 other yeasts. In addition, species-specific oligonucleotides for emerging Candida and other yeast species allowed to identify DNA extracted from Candida lusitaniae, Candida humicola, Candida kefyr, Candida inconspicua, Candida solani, Malassezia furfur and Trichosporon cutaneum. A sensitivity of at least 10 1 CFU, corresponding to 100 fg of fungal DNA, was documented for all species-specific probes and the common Candida probe. In addition, the 18S rRNA genes of 7 yeast species (C. humicola, C. kefyr, C. solani, C. inconspicua, C. norvegensis, C. utilis and M. furfur) were completely sequenced. The sequencing primers described bind to highly conserved primer binding sites. Therefore, these primers would allow rapid cycle sequence of additional ribosomal genes throughout the whole kingdom of fungi.
Opportunistic fungal infections including candidiasis have increased dramatically in recent years. Most medically important fungi are Candida species. Rapid identification of Candida isolates to the species level in the clinical laboratory is necessary for more rapid and effective antifungal therapy and to facilitate hospital infection control measures. Conventional morphological methods for identification of candida species are often difficult and time consuming. Molecular DNA-based techniques provide useful alternative methods. In this study using universal primers, ITS1-ITS4 region of the fungal rRNA genes were amplified. Digestion by the restriction enzyme Mspl allowed us to identify of C. albicans, C. glabrata, C. krusei, C. tropicali, and C. guilliermondii.C. guilliermondii produces 3 bands whereas the others gave two distinctive bands after digestion.This panel of PCR- restriction enzyme could be rapid, simple and useful in diagnostic studies of candida and candidiasis.
Rapid, polymerase chain reaction-based identification assays for Candida species
Journal of clinical microbiology, 1993
Polymerase chain reaction (PCR) amplification of specific regions in the genomes of a variety of lower eukaryotes permits rapid identification of these microorganisms. First, on the basis of the presence of both constant and variable regions in the small subunit (ssu) rRNA, a nested PCR for direct identification of various Candida species can be designed. Amplification of the entire ssu rRNA gene and subsequent reamplification of variable sequences within the V4 domains of these PCR products were combined with direct sequencing. Restriction enzyme maps were made, and species-specific oligonucleotides for hybridization analysis were selected. Unequivocal discrimination of four of the major human pathogenic yeasts (Candida albicans, Candida glabrata, Candida tropicalis, and Candida krusei) is possible if a combination of these techniques is used. Second, by using oligonucleotides aimed at repeated sequences which occur at dispersed positions in the genomes of all eukaryotes, species-s...
Journal of clinical microbiology, 1997
A PCR method was developed to identify and fingerprint Candida krusei isolates simply and rapidly. The primer pair Arno1 and Arno2 was designed to amplify the polymorphic species-specific repetitive sequence CKRS-1 (C. krusei repeated sequence 1) that we identified in the nontranscribed intergenic regions (IGRs) of rRNA genes in C. krusei LMCK31. The specificity, sensitivity, reproducibility, and fingerprinting ability of the PCR assay were evaluated. Amplification products were obtained from all 131 C. krusei isolates studied. No other yeast species of medical importance (n = 26), including species similar to C. krusei, species of pathogenic filamentous fungi, or a variety of pathogenic bacteria, yielded a PCR product with these primers. This PCR assay allowed for the identification of C. krusei in less than 6 h. The PCR assay was sensitive enough to detect as little as 10 to 100 fg of C. krusei-purified DNA and proved to be reproducible. Since amplification products varied both in...