Novel lysine biosynthetic gene sequences (LYS1 and LYS5) used as PCR targets for the detection of the pathogenic Candida yeast

We report here a sensitive and specific polymerase chain reaction (PCR) detection assay for the pathogenic Candida yeast based on the novel LYS1 [encoding saccharopine dehydrogenase (SDH)] and LYS5 [encoding phosphopantetheinyl transferase (PPTase)] gene sequences of the fungal unique lysine biosynthetic pathway. Both LYS1 and LYS5 DNA-specific PCR primers SG1, SG2 and SG3, SG4, respectively, amplified predicted 483 and 648-bp fragments from Candida albicans genomic DNA but not from other selected fungal, bacterial, or human DNA. The 18S rDNA control primers exhibited positive amplifications in all PCR assays. The LYS1-and LYS5-specific primers strongly amplified C. albicans and Candida tropicalis target sequences; however, the LYS1 primers also weakly amplified fragments from Candida kefyr and Candida lusitaniae DNA. Both sets of primers amplified target sequences from less than 10 pg of serially diluted C. albicans DNA, and the LYS1 specific primers also detected DNA isolated from serially diluted 50 C. albicans cells. The PCR primers reported here are sufficiently sensitive and specific for the potential early detection of Candida infections with no possibility of false positive results from cross-contamination with bacterial or human DNA.     

Improvements of PCR-based identification targeting the DNA topoisomerase II gene to determine major species of the opportunistic fungi Candida and Aspergillus fumigatus

For the simple and rapid detection/identification of major pathogenic fungal species such as Candida albicans, C. tropicalis, C. parapsilosis, C. glabrata and Aspergillus fumigatus, common primers for these species and specific primers for each species, designed on the basis on the genomic nucleotide sequences of the DNA topoisomerase II genes, were prepared and tested for their specificities in PCR amplifications. Twelve specific primers were pooled and designated PsVI. Genomic DNAs were amplified by the common primer pair, and followed by PCR amplification using PsVI. Using PsVI, six unique DNA fragments, all of which corresponded to a Candida or A. fumigatus species, were specifically and acceptably amplified from each template DNA even in the presence of other DNAs. Similarly, the results of identification of clinical samples based on the PCR amplification coincided with those of conventional identification techniques. The sensitivities of the direct PCR and the nested PCR using PsVI were found to be 1,000 and 50 yeast cells, respectively.     

Comparison of PCR-RFLP with 21-plex PCR and rDNA Sequencing for Identification of Clinical Yeast Isolates

Non-albicans Candida species and other rare yeasts have emerged as major opportunistic pathogens in fungal infections. Identification of opportunistic yeasts in developing countries is mainly performed by phenotypic assay, which are time-consuming and prone to errors. The aim of the present study was to evaluate PCR-RFLP as a routinely used identification technique for the most clinically important Candida species in Iran and make a comparison with a novel multiplex PCR, called 21-plex PCR. One hundred and seventy-three yeast isolates from clinical sources were selected and identified with sequence analysis of the D1/D2 domains of rDNA (LSU rDNA) sequencing as the gold standard method. The results were compared with those obtained by PCR-RFLP using MspI restriction enzyme and the 21-plex PCR. PCR-RFLP correctly identified 93.4% of common pathogenic Candida species (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and P. kudriavsevii (=?C. krusei)) and was able to identify 45.5% of isolates of the uncommon yeast species compared to the D1/D2 rDNA sequencing. Compared with PCR-RFLP, all common Candida species and 72.7% of uncommon yeast species were correctly identified by the 21-plex PCR. The application of the 21-plex PCR assay as a non-sequence-based molecular method for the identification of common and rare yeasts can reduce turnaround time and costs for the identification of clinically important yeasts and can be applied in resource-limited settings.

     

Differentiation of sourdough yeast species by a novel species-specific PCR assay

The objective of this study was to develop species-specific primer pairs based on the internal transcribed spacer region (ITS) of the ribosome DNA for species identification of the frequently found sourdough yeast species. Species-specific primers were designed by the alignment of sourdough yeast ITS sequences, which were then employed for PCR using the template DNA of sourdough yeast strains. PCR primers were shown to be specific for the following species: Issatchenkia orientalis (Candida krusei), C. humilis, Kazachstania exigua (C. holmii), Pichia anomala and P. subpelliculosa. Therefore, we conclude that our novel species-specific primers could be used to rapidly and accurately identify the most frequent sourdough yeast species using a PCR-based assay.     

Molecular Identification of <Emphasis Type="Italic">Candida orthopsilosis</Emphasis> Isolated from Blood Culture

The incidence of candidemia and invasive candidiasis have increased markedly due to the increasing number of immunocompromised patients. There are five major medically important species of Candida with their frequency of isolation in the diminishing order namely Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata and Candida krusei. In addition, there are numerous other species of Candida which differ in their genetic makeup, virulence properties, drug susceptibilities and sugar assimilation capabilities. In this report, an unusual Candida species was isolated from the blood of two leukaemic patients. Conventional culture and biochemical tests identified the Candida species as C. parapsilosis. Using fungal-specific oligonucleotide primers ITS1 and ITS4, we managed to amplify the ribosomal RNA gene and its internal transcribed spacer region from the genomic DNA of these isolates. The PCR products were then purified and subjected to automated DNA sequencing using BLAST and CLUSTAL sequence analysis identified these isolates to be Candida orthopsilosis. Candida orthopsilosis is a new species recently identified in 2005, being morphologically indistinguishable from C. parapsilosis and was previously classified as a subspecies of C. parapsilosis. This report highlights the importance of complementing traditional culture and biochemical-based identification methods with DNA-based molecular assays such as PCR as the latter is more superior in terms of its discriminatory power and speed.     

A real‐time PCR assay for the differentiation of Candida species

Aims: We established a real‐time PCR assay for the detection and strain identification of Candida species and demonstrated the ability to differentiate between Candida albicans the most common species, and also Candida parapsilosis, Candida glabrata, Candida tropicalis and Candida dubliniensis by LightCycler PCR and melting curve analysis. Methods and Results: The DNA isolation from cultures and serum was established using the QIAmp Tissue Kit. The sensitivity of the assay was ≥ 2 genome equivalents/assay. It was possible to differentiate all investigated Candida species by melting curve analysis, and no cross‐reaction to human DNA or Aspergillus species could be observed. Conclusions: The established real‐time PCR assay is a useful tool for the rapid identification of Candida species and a base technology for more complex PCR assays. Significance and Impact of the Study: We carried out initial steps in validation of a PCR assay for the detection and differentiation of medically relevant Candida species. The PCR was improved by generating PCR standards, additional generation of melting curves for species identification and the possibility to investigate different specimens simultaneously.     

PCR primed with minisatellite core sequences yields species-specific patterns and assessment of population variability in fishes of the genus Brycon

Minisatellite core sequences were used as single primers in polymerase chain reaction (PCR) to amplify genomic DNA in a way similar to the random amplified polymorphic DNA methodology. This technique, known as Directed Amplification of Minisatellite‐region DNA, was applied in order to differentiate three neotropical fish species (Brycon orbignyanus, B. microlepis and B. lundii) and to detect possible genetic variations among samples of the threatened species, B. lundii, collected in two regions with distinct environmental conditions in the area of influence of a hydroelectric dam. Most primers generated species‐specific banding patterns and high levels of intraspecific polymorphism. The genetic variation observed between the two sampling regions of B. lundii was also high enough to suggest the presence of distinct stocks of this species along the same river basin. The results demonstrated that minisatellite core sequences are potentially useful as single primers in PCR to assist in species and population identification. The observed genetic stock differentiation in B. lundii associated with ecological and demographic data constitute a crucial task to develop efficient conservation strategies in order to preserve the genetic diversity of this endangered fish species.     

Development of species-specific primers for rapid identification by PCR of the ecologically important pathogen Fusarium keratoplasticum from isolated and environmental samples

The genus Fusarium contains many fungal species known to be pathogenic to animals and plants alike. One species complex within this genus, the Fusarium solani species complex (FSSC), is of particular concern due to its high numbers of pathogenic members. FSSC members are known to contribute significantly to plant, human and other animal fungal disease. One member of the FSSC, Fusarium keratoplasticum, is of particular ecological concern and has been implicated in low hatching success of endangered sea turtle eggs, as well as contribute to human and other animal Fusarium pathogenesis. Species-specific primers for molecular identification of F. keratoplasticum currently do not exist to our knowledge, making rapid identification, tracking and quantitation of this pathogenic fungus difficult. The objective of this study was to develop primers specific to F. keratoplasticum that could be applied to DNA from isolated cultures as well as total (mixed) DNA from environmental samples. RPB2 sequence from 109 Fusarium isolates was aligned and analysed to determine nucleotide polymorphisms specific to F. keratoplasticum useful for primer design. A set of primers were generated and found to be effective for identification of F. keratoplasticum from total DNA extracted from sand surrounding sea turtle nesting sites.     

The first clinical isolates of <Emphasis Type="Italic">Candida dubliniensis</Emphasis> in Slovakia

Candida dubliniensis, yeast closely related to Candida albicans, is a new pathogen associated mainly with infections of immunocompromised hosts. In this study, we report the first isolation of three isolates of C. dubliniensis in Slovakia. The first selection of both C. albicans and C. dubliniensis from the other Candida species was done on the basis of specific green color of primoculture grown on CHROMagar Candida. The presumptive identification was completed by supplemental tests: germ-tube formation, production of chlamydospores, ability or inability to grow at 42 and 45,°C and by commercial set API 20C AUX. Parallely, the discrimination between both species was performed by PCR assay using primers specific for Candida dubliniensis     

Comparison of Detection Methods for Legionella Species in Environmental Water by Colony Isolation,Fluorescent Antibody Staining,and Polymerase Chain Reaction

Three detection methods for Legionella species in water samples from cooling towers and a river were examined. Direct counting of bacteria stained with fluorescent antibody (FA) for L. pneumophila (serogroups 1 to 6) could detect the cell of 10⁴ to 10⁶ cell/100 ml in all 14 samples, while colony counting method detected 10 to 10³ CFU/100 ml only in 8 samples from cooling towers. Polymerase chain reaction (PCR) assay with primers to amplify 16S ribosomal DNA sequence of most Legionella species (LEG primer) detected legionellae in 13 samples, while species-specific primers for L. pneumophila detected the DNAs from 3 samples. In laboratory examination, LEG primers could amplify DNAs of 29 species of genus Legionella with high sensitivity, even from 1 cell of L. pneumophila GIFU 9134. The PCR assay with LEG primers was specific and sensitive methods to be satisfied the survey of legionellae. Thus, PCR assay is a suitable method to detect and monitor Legionella species in an environment.     

A method for taxonomic determination ofCandida albicans with DNA probes

Determination ofCandida species represents an important problem derived from the clinical implications of the species belonging to this genus. DNA probes have already been used for the epidemiology ofCandida albicans, as well as for taxonomic analysis ofCandida and other genera, although these probes are based on non-species-specific DNA sequences. In this work we carried out a 48-h assay, allowing the identification ofC. albicans from clinical isolates, using DNA probes based onC. albicans LEU2 andURA3 genes. Another probe related toC. albicans SEC18 gene was shown not to beC. albicans specific.     

The use of the GDH gene for molecular identification and phylogenetic analysis of the yeast Kluyveromyces marxianus

Summary Our previous work showed that NADP⁺-dependent glutamate dehydrogenase from K. marxianus behaves similarly to its counterpart in S. cerevisiae. It suggested that the ammonia assimilation pathway might be different between K. marxianus and the genetic closed species K. lactis. In the present work, we analyzed the genetic similarity among the GDH gene family in K. marxianus and closed yeasts. Specific primers for GDH genes were designed based on the K. marxianus sequences deposited in the Génolevures Database. One of them, for the KmGDH2 gene, proved to be specific for K. marxianus DNA samples, which confirmed the molecular identification of environmental yeast isolates, and can be proposed for rapid screening of this yeast from environmental samples. The nucleotide sequence revealed that KmGDH2 belongs to the S. cerevisiae GDH1 gene family together with KlGDH gene.     

Phylogenetic Diversity of Natural Populations of Ammonia Oxidizers Investigated by Specific PCR Amplification

Abstract The species composition of ammonia-oxidizing bacteria in aquatic environments was investigated using PCR primers for 16S rRNA genes to amplify specific subsets of the total ammonia-oxidizer population. The specificity of the amplification reactions was determined using total genomic DNA from known nitrifying strains and non-nitrifying strains identified as having similar rDNA sequences. Specificity of amplification was determined both for direct amplification, using the nitrifier specific primers, and with nested amplification, in which the nitrifier primers were used to reamplify a fragment obtained from direct amplification with Eubacterial universal primers. The present level of specificity allows the distinction between Nitrosomonas europaea, Nitrosomonas sp. (marine) and the other known ammonia-oxidizers in the beta subclass of the Proteobacteria. Using total DNA extracted from natural samples, we used direct amplification to determine presence/absence of different species groups. Species composition was found to differ among depths in vertical profiles of lake samples and among samples and enrichments from various other aquatic environments. Nested PCR yielded several more positive reactions, which implies that nitrifier DNA was present in most samples, but often at very low levels. Received: 25 September 1995; Revised: 15 January 1996; Accepted: 20 February 1996     

Multiplex PCR assay for rapid identification of three endangered snake species of India

Species identification has been the core issue in all approaches of conservation of endangered wild life. In this regard molecular techniques for species authentication have proved indispensable. A novel multiplex PCR assay for the identification of three Indian snake species Python morulus, Ptyas mucosus, and Naja naja is successfully demonstrated using 16S rRNA gene. Three reverse primers and a common forward primer were designed to generate three different size species-specific PCR fragments. Absence of any PCR amplification in non-target species proves the specificity of the primers. These four primers were combined in a multiplex assay to enable identification of three snake species in a single reaction. The assay described here shows its utility in identifying unknown snake specimen and in case of samples yielding low quality DNA. This multiplex PCR technique using novel primers is an unprecedented approach offered for forensic identification of exhibits originating from three Indian snake species. It is expected that this endeavor will help strengthening conservation efforts for these species.     

Use of variability of the fungal ITS2 intergenic region for the identification of medically important yeast species

Yeasts are common fungal opportunistic pathogens in humans playing a significant role in the morbidity and mortality of immunocompromised patients. Number of resistant yeast species is responsible for infections and consequent infectious complications, but the final microbiological diagnosis can be affected by variability of their phenotype and may result in incorrect identification. For the purposes of this study, advanced applications of molecular genetic methods based on certain up-to-date knowledge of fungal internal transcribed spacer (ITS) regions have been employed, which could support a possibility of universal application of such methods for identification of any pure yeast culture. In this study, the targeted DNA was amplified by a couple of primers, and the products of PCR reaction were divided by capillary electrophoresis. In the cases, in which the measured sizes of fragments did not correspond with the anticipated sizes, fragments were used for the sequencing analysis and compared to the nucleotide databases using the BLAST tool. Out of 208 isolates, 7.2% (n = 16) of cases occurred to be incorrectly determined, particularly in the group of non-albicans Candida species accounting for as many as 21.7%.     

A Molecular Method for Detection of <Emphasis Type="Italic">Aspergillus carbonarius</Emphasis> in Coffee Beans

Ochratoxin A (OTA) is a carcinogenic and nephrotoxic mycotoxin that has been detected in a variety of food products, including green coffee beans. About 80% of Aspergillus carbonarius strains collected from coffee beans are able to produce OTA on this substrate. The rapid identification of this fungal species would be desirable. RAPD assays were applied to identify amplification products specific for A. carbonarius. One selected fragment, denoted OPX7₈₀₉, was cloned and sequenced. Based on the nucleotide sequence obtained, specific oligonucleotides (OPX7F₈₀₉ and OPX7R₈₀₉) were designed and used as primers for DNA amplification. One amplified band of 809 bp was obtained from A. carbonarius genomic DNA, whereas no amplified fragment from DNA of other Aspergillus species was detected. This PCR analysis was also successfully employed to detect A. carbonarius in coffee beans. This PCR assay could contribute to the early and rapid detection of the potential presence of OTA in coffee samples.     

Identification of the pathogenic Aspergillus species by nested PCR using a mixture of specific primers to DNA topoisomerase II gene

For PCR-based identification of Aspergillus species, a common primer of the DNA topoisomerase II genes of Candida, Aspergillus and Penicillium, and species-specific primers of the genomic sequences of DNA topoisomerase II of A. fumigatus, A. niger, A. flavus (A. oryzae), A. nidulans and A. terreus were tested for their specificities in PCR amplifications. The method consisted of amplification of the genomic DNA topoisomerase II gene by a common primer set, followed by a second PCR with a primer mix consisting of 5 species-specific primer pairs for each Aspergillus species. By using the common primer pair, a DNA fragment of approximately 1,200 bp was amplified from the Aspergillus and Penicillium genomic DNAs. Using each species-specific primer pair, unique sizes of PCR products were amplified, all of which corresponded to a species of Aspergillus even in the presence of DNAs of several fungal species. The sensitivity of A. fumigatus to the nested PCR was found to be 100 fg of DNA in the reaction mixture. In the nested PCR obtained by using the primer mix (PsIV), the specific DNA fragment of A. fumigatus was amplified from clinical specimens. These results suggest that this nested PCR method is rapid, simple and available as a tool for identification of pathogenic Aspergillus to a species level.     

Detection of section-specific random amplified polymorphic DNA (RAPD) markers in Lilium

Random amplified polymorphic DNA (RAPD) markers were utilized for the identification of Lilium species and inter-specific hybrids. The optimum annealing temperature of the polymerase chain reaction (PCR) for the RAPD assay in Lilium was 54 °C, which is relatively higher than the temperature used for other genera reported by previous researchers. Among 76 primers used to amplify genomic DNA by PCR, 18 primers (24%) generated polymorphic DNA fragments in Lilium species and hybrids. Cultivars were also identified by RAPD markers. Some amplified fragments were unique to species of each section and to hybrids derived from these species; that is, they were the section-specific DNA markers. Sections, Sinomartagon, Leucolirion b, Leucolirion a and Archelirion could be identified by 6 section-specific markers amplified with five primers. Seven inter-section hybrids showed the section-specific bands of both parental sections, indicating that these markers would be useful for identifying the parental sections of inter-section hybrids.     

Cultivation,identification and quantification of one species of yeast‐like symbiotes,Candida, in the rice brown planthopper,Nilaparvata lugens

Abstract The brown planthopper (BPH), Nilaparvata lugens Stål, which is one of the most destructive pests of rice, has been confirmed to harbor yeast‐like symbiotes (YLS) in the fat body. Several morphologically different YLS have been previously isolated and cultured in vitro from BPH, but direct evidence is lacking to further clarify whether the cultured YLS were from BPH. In this study, one species of YLS was successfully cultured in vitro and simultaneously verified to exist in the fat body of BPH by sequence analysis and nested polymerase chain reaction (PCR). The cultured YLS isolate in vitro was identified as a member of the genus Candida on the basis of 18S rDNA (ribosomal DNA) and 5.8S‐ITS (internal transcribed spacer) rDNA sequence and a phylogenetic analysis of ITS sequences from yeast. Therefore, this yeast isolate was named as Candida‐like symbiotes. Candida‐like symbiotes was found to exist in fat bodies, ovaries and newly laid eggs of the BPH, but not in the heads, thoraxes and mid‐guts. In addition, the number of Candida‐like symbiotes in 1 × 10⁶ of purified YLS from BPH fat bodies was speculated to be (5.32 ± 0.22) × 10⁴ on the basis of a quantitative PCR analysis.     

Simultaneous detection of major blackleg and soft rot bacterial pathogens in potato by multiplex polymerase chain reaction

A multiplex polymerase chain reaction (PCR) assay for simultaneous, fast and reliable detection of the main soft rot and blackleg potato pathogens in Europe has been developed. It utilises three pairs of primers and enables detection of three groups of pectinolytic bacteria frequently found in potato, namely: Pectobacterium atrosepticum, Pectobacterium carotovorum subsp. carotovorum together with Pectobacterium wasabiae and Dickeya spp. in a multiplex PCR assay. In studies with axenic cultures of bacteria, the multiplex assay was specific as it gave positive results only with strains of the target species and negative results with 18 non‐target species of bacteria that can possibly coexist with pectinolytic bacteria in a potato ecosystem. The developed assay could detect as little as 0.01 ng µL^–1 of Dickeya sp. genomic DNA, and down to 0.1 ng µL^–1 of P. atrosepticum and P. carotovorum subsp. carotovorum genomic DNA in vitro. In the presence of competitor genomic DNA, isolated from Pseudomonas fluorescens cells, the sensitivity of the multiplex PCR decreased tenfold for P. atrosepticum and Dickeya sp., while no change was observed for P. carotovorum subsp. carotovorum and P. wasabiae. In spiked potato haulm and tuber samples, the threshold level for target bacteria was 10¹ cfu mL^–1 plant extract (10² cfu g^–1 plant tissue), 10² cfu mL^–1 plant extract (10³ cfu g^–1 plant tissue), 10³ cfu mL^–1 plant extract (10⁴ cfu g^–1 plant tissue), for Dickeya spp., P. atrosepticum and P. carotovorum subsp. carotovorum/P. wasabiae, respectively. Most of all, this assay allowed reliable detection and identification of soft rot and blackleg pathogens in naturally infected symptomatic and asymptomatic potato stem and progeny tuber samples collected from potato fields all over Poland.