Closed-tube human leukocyte antigen DQA1∗05 genotyping assay based on switchable lanthanide luminescence probes

Genotyping in closed tube is commonly performed using polymerase chain reaction (PCR) amplification and allele-specific oligonucleotide probes using fluorescence resonance energy transfer (FRET). Here we introduce a homogeneous human leukocyte antigen (HLA)–DQA1∗05 end-point PCR assay based on switchable lanthanide luminescence probe technology and a simple dried blood sample preparation. The switchable probe technology is based on two non-luminescent oligonucleotide probes: one carrying a non-luminescent lanthanide chelate and the other carrying a light-absorbing antenna ligand. Hybridization of the probes in adjacent positions to the target DNA leads to the formation of a highly luminescent lanthanide chelate complex by self-assembly of the reporter molecules. Performance of the HLA–DQA1∗05 assay was evaluated by testing blood samples collected on sample collection cards and was prepared by lysing the punched samples (3-mm discs) using alkaline reaction conditions and high temperature. Testing of 147 blood samples yielded 100% correlation to the heterogeneous DELFIA technology-based reference assay. Genotyping requires carefully designed probe sequences able to discriminate matched and mismatched target sequences by hybridization. Furthermore, definite genotype discrimination was achieved because inherently non-luminescent switchable probes together with time-resolved measurement mode led to very low background signal level and, therefore, very high signal differences averaging 54-fold between DQA1∗05 and other alleles.     

Real-time PCR TaqMan assay for rapid screening of bloodstream infection

Background

Sepsis is one of the main causes of mortality and morbidity. The rapid detection of pathogens in blood of septic patients is essential for adequate antimicrobial therapy and better prognosis. This study aimed to accelerate the detection and discrimination of Gram-positive (GP) and Gram-negative (GN) bacteria and Candida species in blood culture samples by molecular methods.

Methods

The Real-GP®, -GN®, and -CAN® real-time PCR kit (M&D, Wonju, Republic of Korea) assays use the TaqMan probes for detecting pan-GP, pan-GN, and pan-Candida species, respectively. The diagnostic performances of the real-time PCR kits were evaluated with 115 clinical isolates, 256 positive and 200 negative blood culture bottle samples, and the data were compared to results obtained from conventional blood culture.

Results

Eighty-seven reference strains and 115 clinical isolates were correctly identified with specific probes corresponding to GP-bacteria, GN-bacteria and Candida, respectively. The overall sensitivity and specificity of the real-time PCR kit with blood culture samples were 99.6% and 89.5%, respectively.

Conclusions

The Real-GP®, -GN®, and -CAN® real-time PCR kits could be useful tools for the rapid and accurate screening of bloodstream infections (BSIs).     

Specific Detection of Arcobacter and Campylobacter Strains in Water and Sewage by PCR and Fluorescent In Situ Hybridization

The aim of this study was to evaluate PCR and fluorescent in situ hybridization (FISH) techniques for detecting Arcobacter and Campylobacter strains in river water and wastewater samples. Both 16S and 23S rRNA sequence data were used to design specific primers and oligonucleotide probes for PCR and FISH analyses, respectively. In order to assess the suitability of the methods, the assays were performed on naturally and artificially contaminated samples and compared with the isolation of cells on selective media. The detection range of PCR and FISH assays varied between 1 cell/ml (after enrichment) to 10³ cells/ml (without enrichment). According to our results, both rRNA-based techniques have the potential to be used as quick and sensitive methods for detection of campylobacters in environmental samples.     

Universal ProbeLibrary based real-time PCR for rapid detection of bacterial pathogens from positive blood culture bottles

A set of real-time PCR based assays using the locked nucleic acid probes from Roche Universal ProbeLibrary were developed for rapid detection of eight bacterial species from positive blood culture bottles. Four duplex real-time PCR reactions targeting to one Gram-positive bacterium and one Gram-negative bacterium were optimized for species identification according to Gram stain results. We also included mecA-specific primers and probes in the assays to indicate the presence of methicillin resistance in the bacterial species. The analytical sensitivity was in the range of 1–10 CFU per PCR reaction mixture. The specificity and cross reactivity of the assay was validated by 28 ATCC reference strains and 77 negative blood culture specimens. No cross-reactivity was observed in these samples thus demonstrating 100 % specificity. 72 previously characterized clinical isolates were tested by the real-time PCR assay and validated the accuracy and feasibility of the real-time PCR assay. Furthermore, 55 positive blood culture samples were tested using real-time PCR and 50 (90.9 %) of them were identified as the same species as judged by biochemical analysis. In total, real-time PCR showed 98.2 % consistent to that of traditional methods. Real-time PCR can be used as a supplement for early detection of the frequently-occurred pathogens from the positive blood cultures.     

Quantification and optimization of Candida albicans DNA in blood samples using Real- Time PCR

Background:

Candida albicans (C. albicans) is a major cause of candidaemia in people with impaired immunity. Blood culture is a “gold standard” for candidaemia detection but is time-consuming and relatively insensitive. We established a real-time PCR assay for C. albicans detection in blood by LightCycler PCR and melting curve analysis.

Methods:

Five milliliter blood samples from healthy volunteers were spiked with 10⁰-10⁶ C. albicans cells to determine the detection limit of our method. DNA was extracted from whole blood using glass beads and the QIAamp DNA Blood Mini Kit (Qiagen, Hilden Germany). DNA from C. albicans isolates were amplified with primers and inserted into Escherichia coli (E. coli) DH5α.1 cells with the TA cloning vector (Invitrogen). The plasmid was used for standardization and optimization. A quantitative PCR assay with the LightCycler amplification and detection system based on fluorescence resonance energy transfer (FRET) with two different specific probes was established. To assess the precision and reproducibility of real-time PCR the intra-assay precision was determined in six consecutive assays.

Results:

No cross-reactivity of the hybridization probes with the DNA of non-C. albicans species or human genomic DNA was observed, which confirmed its 100% specificity. The minimum limit detected was one C. albicans cell or 10⁰ CFU/ml (10 fg) per PCR reaction. The real-time PCR efficiency rate for Candida was high (E = 1.95). Melting curve analysis of C. albicans showed a specific melting peak temperature of 65.76 °C.

Conclusion:

The real-time PCR assay we developed is highly specific and sufficiently sensitive to detect the fungal load for early diagnosis of invasive candidiasis. Key Words: Invasive candidiasis, Real-time PCR, Candida albicans     

Pediatric visceral leishmaniasis diagnosis in Tunisia: comparative study between optimised PCR assays and parasitological methods

There has been a steady increase of visceral leishmaniasis during the past 20 years in Tunisia. In this study, we assess the value of two optimised PCR versus those of classical methods for the diagnosis of human visceral leishmaniasis. 106 samples were collected from 53 cases of pediatric visceral leishmaniasis. Peripheral blood and bone marrow samples were analysed both by parasitological methods (direct examination, leukocytoconcentration (LCC) and culture) and by PCR methods with two primer pair (R221/R332 and Lei 70L/Lei 70R). We diagnosed visceral leishmaniasis in all patients: 44 cases were diagnosed by culture (83%), 42 by direct examination of bone marrow (79%), 17 by LCC (32%), and 53 positive cases with both PCR assays (R221/R332 and/or Lei 70L/Lei 70R) (100 %). Regarding each PCR assay, for blood samples, the difference between the sensitivities of PCR Lei 70L/Lei 70R (86,8%) and PCR R221/R332 (17 %) is statistically significant with p-value 0.025. For bone marrow, the sensitivities of the two PCR methods were respectively 96,2% (Lei 70L/Lei 70R) and 75,5% (R221/R332). On the whole, PCR Lei 70L/Lei 70R was more effective than PCR R221/R332 and conventional methods for the two biological samples. Moreover, the requirement of less invasive sample using blood has the advantage of being repeatable for screening and for post therapeutic monitoring.     

Comparison of Pathogen DNA Isolation Methods from Large Volumes of Whole Blood to Improve Molecular Diagnosis of Bloodstream Infections

For patients suffering from bloodstream infections (BSI) molecular diagnostics from whole blood holds promise to provide fast and adequate treatment. However, this approach is hampered by the need of large blood volumes. Three methods for pathogen DNA isolation from whole blood were compared, i.e. an enzymatic method (MolYsis, 1–5 ml), the novel non-enzymatic procedure (Polaris, 1–5 ml), and a method that does not entail removal of human DNA (Triton-Tris-EDTA EasyMAG, 200 µl). These methods were evaluated by processing blood spiked with 0–1000 CFU/ml of Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. Downstream detection was performed with real-time PCR assays. Polaris and MolYsis processing followed by real-time PCRs enabled pathogen detection at clinically relevant concentrations of 1–10 CFU/ml blood. By increasing sample volumes, concurrent lower cycle threshold (Ct) values were obtained at clinically relevant pathogen concentrations, demonstrating the benefit of using larger blood volumes. A 100% detection rate at a concentration of 10 CFU/ml for all tested pathogens was obtained with the Polaris enrichment, whereas comparatively lower detection rates were measured for MolYsis (50–67%) and EasyMAG (58–79%). For the samples with a concentration of 1 CFU/ml Polaris resulted in most optimal detection rates of 70–75% (MolYsis 17–50% and TTE-EasyMAG 20–36%). The Polaris method was more reproducible, less labour intensive, and faster (45 minutes (including Qiagen DNA extraction) vs. 2 hours (MolYsis)). In conclusion, Polaris and MolYsis enrichment followed by DNA isolation and real-time PCR enables reliable and sensitive detection of bacteria and fungi from 5 ml blood. With Polaris results are available within 3 hours, showing potential for improved BSI diagnostics.     

Persistence of Microbial Contamination on Transvaginal Ultrasound Probes despite Low-Level Disinfection Procedure

Aim of the Study

In many countries, Low Level Disinfection (LLD) of covered transvaginal ultrasound probes is recommended between patients'' examinations. The aim of this study was to evaluate the antimicrobial efficacy of LLD under routine conditions on a range of microorganisms.

Materials and Methods

Samples were taken over a six month period in a private French Radiology Center. 300 specimens derived from endovaginal ultrasound probes were analyzed after disinfection of the probe with wipes impregnated with a quaternary ammonium compound and chlorhexidine. Human papillomavirus (HPV) was sought in the first set of s100 samples, Chlamydia trachomatis and mycoplasmas were searched in the second set of 100 samples, bacteria and fungi in the third 100 set samples. HPV, C. trachomatis and mycoplasmas were detected by PCR amplification. PCR positive samples were subjected to a nuclease treatment before an additional PCR assay to assess the likely viable microorganisms. Bacteria and fungi were investigated by conventional methods.

Results

A substantial persistence of microorganisms was observed on the disinfected probes: HPV DNA was found on 13% of the samples and 7% in nuclease-resistant form. C. trachomatis DNA was detected on 20% of the probes by primary PCR but only 2% after nuclease treatment, while mycoplasma DNA was amplified in 8% and 4%, respectively. Commensal and/or environmental bacterial flora was present on 86% of the probes, occasionally in mixed culture, and at various levels (10->3000 CFU/probe); Staphylococcus aureus was cultured from 4% of the probes (10-560 CFU/probe). No fungi were isolated.

Conclusion

Our findings raise concerns about the efficacy of impregnated towels as a sole mean for disinfection of ultrasound probes. Although the ultrasound probes are used with disposable covers, our results highlight the potential risk of cross contamination between patients during ultrasound examination and emphasize the need for reviewing the disinfection procedure.     

Rapid homogeneous PCR assay for the detection of Chlamydia trachomatis in urine samples

Chlamydia trachomatis infection is the most common bacterial sexually transmitted disease and a major public health problem worldwide. Fast and sensitive point-of-care diagnostics including non-invasive sample collection would be of value for the prevention of C. trachomatis transmission. The aim of this study was to develop a fast, reliable, non-invasive and easy-to-use homogenous PCR assay for the detection of C. trachomatis. Bacteria were concentrated from urine by a simple and fast centrifugation-based urine pretreatment method. Novel automated GenomEra technology was utilized for the rapid closed-tube PCR including time-resolved fluorometric detection of the target using lanthanide chelate labeled probes. We have developed a rapid C. trachomatis assay which provides qualitative results in 1 h with diagnostic sensitivity and specificity of 98.7% and 97.3%, respectively. The novel assay can be performed with minimal laboratory expertise and without sophisticated DNA-extraction devices and has performance comparable to current gold standard assays.     

Detection of Legionella species in potting mixes using fluorescent in situ hybridisation (FISH)

This study used Fluorescent in situ Hybridisation (FISH) with rRNA targeted oligonucleotide probes combined with scanning confocal laser microscopy to successfully detect Legionella spp. in commercially available potting mix. A range of techniques were explored to optimise the FISH method by reducing background fluorescence and preventing non-specific binding of probes. These techniques included the use of a blocking agent, UV light treatment, image subtraction of a nonsense probe and spectral unmixing of specific probes fluorescence and autofluorescence dependent on the specific emission spectra of probe fluorophores.Spectral unmixing was the best microscopy technique for reducing background fluorescence and non-specific binding of probes was not observed. The rapid turnaround time and increased sensitivity of the FISH provides as an alternative to traditional culture methods, which are tedious and often give varied results. FISH is also advantageous compared to PCR methods as it provides information on the structure of the microbial community the bacteria is situated in. This study demonstrates that FISH could provide an alternative method for Legionella detection and enumeration in environmental samples.     

A five-minute DNA extraction method for expedited detection of Phytophthora ramorum following prescreening using Phytophthora spp. lateral flow devices

In a direct comparison with established methods for Phytophthora ramorum detection (isolation followed by morphological identification, or conventional DNA extraction followed by TaqMan real-time PCR) a rapid, simplified detection method in which membranes of lateral flow devices (LFDs) are added directly to TaqMan real-time PCR reactions was used to test 202 plant samples collected by plant health inspectors in the field. P. ramorum prevalence within the 202 samples was approximately 40% according to routine testing by isolation or TaqMan real-time PCR. The diagnostic sensitivity and specificity of the rapid detection method were 96.3% and 91.2%, respectively. This method can be used in conjunction with Phytophthora spp. lateral flow devices to reduce the number of samples requiring testing using more laborious conventional methods. The effect of combining prescreening for Phytophthora spp. with P. ramorum-specific tests is discussed in terms of the positive and negative predictive values of species-specific detection when testing samples collected in different inspection scenarios.     

Quantitative and Sensitive Detection of GNAS Mutations Causing McCune-Albright Syndrome with Next Generation Sequencing

Somatic activating GNAS mutations cause McCune-Albright syndrome (MAS). Owing to low mutation abundance, mutant-specific enrichment procedures, such as the peptide nucleic acid (PNA) method, are required to detect mutations in peripheral blood. Next generation sequencing (NGS) can analyze millions of PCR amplicons independently, thus it is expected to detect low-abundance GNAS mutations quantitatively. In the present study, we aimed to develop an NGS-based method to detect low-abundance somatic GNAS mutations. PCR amplicons encompassing exons 8 and 9 of GNAS, in which most activating mutations occur, were sequenced on the MiSeq instrument. As expected, our NGS-based method could sequence the GNAS locus with very high read depth (approximately 100,000) and low error rate. A serial dilution study with use of cloned mutant and wildtype DNA samples showed a linear correlation between dilution and measured mutation abundance, indicating the reliability of quantification of the mutation. Using the serially diluted samples, the detection limits of three mutation detection methods (the PNA method, NGS, and combinatory use of PNA and NGS [PNA-NGS]) were determined. The lowest detectable mutation abundance was 1% for the PNA method, 0.03% for NGS and 0.01% for PNA-NGS. Finally, we analyzed 16 MAS patient-derived leukocytic DNA samples with the three methods, and compared the mutation detection rate of them. Mutation detection rate of the PNA method, NGS and PNA-NGS in 16 patient-derived peripheral blood samples were 56%, 63% and 75%, respectively. In conclusion, NGS can detect somatic activating GNAS mutations quantitatively and sensitively from peripheral blood samples. At present, the PNA-NGS method is likely the most sensitive method to detect low-abundance GNAS mutation.     

Establishing and evaluation of a polymerase chain reaction for the detection of Echinococcus multilocularis in human tissue

BackgroundAlveolar echinococcosis (AE) is caused by metacestode larva of the tapeworm Echinococcus multilocularis. AE diagnostics currently rely on imaging techniques supported by serology, but unequivocal detection of AE is difficult. Although polymerase chain reaction (PCR)-based methods to detect tapeworm DNA in biopsies have been suggested for several species, no validated protocol adhering to accepted guidelines has so far been presented for AE diagnostics. We herein established a PCR protocol for metacestode biopsies and technically evaluated the method using isolated parasite DNA and cells, biopsies of clinically relevant material, and formalin fixed paraffin-embedded (FFPE) human tissue blocks. We compared the results with an immunochemical (IHC) approach using the monoclonal antibody Em2G11 specific for the antigen Em2 of E. mulitlocularis.Methodology/Principal findingsBased on tapeworm 12S rDNA sequences we established and validated a PCR protocol for robust detection of as little as 50 parasite cells per specimen and report 127 cases of positive identification of Echinococcus species in samples from humans and animals. For further validation, we analyzed 45 liver, heart, brain, and soft tissue samples as well as cytological probes of aspirates of FFPE-material from 18 patients with clinically confirmed AE. Of each patient we analyzed (i) fully viable lesions with laminated layer; (ii) tissue with mAbEm2G11-positive small particles of E. multilocularis (spems); (iii) mAbEm2G11-negative tissue adjacent to the main lesion; and (iv) lymph node tissue with mAbEm2G11-positive spems. To identify the areas for the PCR-based approach, we performed IHC-staining with the monoclonal antibody Em2G11. Micro-dissected tissue of these areas was then used for PCR-analysis. 9 of 15 analyzed samples with viable E. multilocularis lesions with laminated layer were positive by PCR. Of this group, all samples preserved for less than 6 years (6/6) were tested positive. 11 of 15 samples of spems and 7 of 9 samples of the control group mAbEm2G11-negative tissue were negative by PCR. We further show that all probes from lymph nodes with spems are PCR negative.Conclusions/SignificanceWe present a sensitive PCR method for the detection of E. multilocularis in human tissue, particularly in fresh biopsy material and tissue blocks stored for less than 5 years. While the diagnostic sensitivity of material containing only spems was higher using IHC, PCR detection was possible in IHC negative liver tissue and in patients with negative serology. Our results support the view that spems do not contain parasitic DNA or viable cells of the parasite. spems thus most probably do not directly contribute to metastasis formation during AE.     

Quantitative assessment of faecal bifidobacterial populations by real-time PCR using lanthanide probes

AIM: To develop real-time quantitative PCR methods, based on the use of probes labelled with a stable fluorescent lanthanide chelate, for the quantification of different human faecal bifidobacterial populations. METHODS AND RESULTS: The designed quantitative PCR assays were found to be specific for the corresponding Bifidobacterium species or groups (Bifidobacterium longum group, Bifidobacterium catenulatum group, Bifidobacterium adolescentis, Bifidobacterium breve, Bifidobacterium angulatum, Bifidobacterium bifidum and Bifidobacterium dentium). The detection limits of the methodologies used ranged between 2 x 10(5) and 9 x 10(3) cells g(-1) of faeces. The applicability of the developed assays was tested by analysing 20 human faecal samples. Bif. longum group was found to be the qualitatively and quantitatively predominant bifidobacterial group. CONCLUSIONS: The real-time PCR procedures developed here are specific, accurate, rapid and easy methods for the quantification of Bifidobacterium groups or species in human faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed procedures will facilitate rapid and objective counting of large numbers of samples increasing our knowledge on the role of gut bifidobacterial microbiota in health and disease. This will contribute to the efficient use of intestinal bacterial assays in research, food and pharmaceutical development as well as in the assessment of dietary management of diseases.     

International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients

Background

A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The purpose of this study was to evaluate the performance of PCR methods in detection of Trypanosoma cruzi DNA by an external quality evaluation.

Methodology/Findings

An international collaborative study was launched by expert PCR laboratories from 16 countries. Currently used strategies were challenged against serial dilutions of purified DNA from stocks representing T. cruzi discrete typing units (DTU) I, IV and VI (set A), human blood spiked with parasite cells (set B) and Guanidine Hidrochloride-EDTA blood samples from 32 seropositive and 10 seronegative patients from Southern Cone countries (set C). Forty eight PCR tests were reported for set A and 44 for sets B and C; 28 targeted minicircle DNA (kDNA), 13 satellite DNA (Sat-DNA) and the remainder low copy number sequences. In set A, commercial master mixes and Sat-DNA Real Time PCR showed better specificity, but kDNA-PCR was more sensitive to detect DTU I DNA. In set B, commercial DNA extraction kits presented better specificity than solvent extraction protocols. Sat-DNA PCR tests had higher specificity, with sensitivities of 0.05–0.5 parasites/mL whereas specific kDNA tests detected 5.10⁻³ par/mL. Sixteen specific and coherent methods had a Good Performance in both sets A and B (10 fg/µl of DNA from all stocks, 5 par/mL spiked blood). The median values of sensitivities, specificities and accuracies obtained in testing the Set C samples with the 16 tests determined to be good performing by analyzing Sets A and B samples varied considerably. Out of them, four methods depicted the best performing parameters in all three sets of samples, detecting at least 10 fg/µl for each DNA stock, 0.5 par/mL and a sensitivity between 83.3–94.4%, specificity of 85–95%, accuracy of 86.8–89.5% and kappa index of 0.7–0.8 compared to consensus PCR reports of the 16 good performing tests and 63–69%, 100%, 71.4–76.2% and 0.4–0.5, respectively compared to serodiagnosis. Method LbD2 used solvent extraction followed by Sybr-Green based Real time PCR targeted to Sat-DNA; method LbD3 used solvent DNA extraction followed by conventional PCR targeted to Sat-DNA. The third method (LbF1) used glass fiber column based DNA extraction followed by TaqMan Real Time PCR targeted to Sat-DNA (cruzi 1/cruzi 2 and cruzi 3 TaqMan probe) and the fourth method (LbQ) used solvent DNA extraction followed by conventional hot-start PCR targeted to kDNA (primer pairs 121/122). These four methods were further evaluated at the coordinating laboratory in a subset of human blood samples, confirming the performance obtained by the participating laboratories.

Conclusion/Significance

This study represents a first crucial step towards international validation of PCR procedures for detection of T. cruzi in human blood samples.     

Quantitative detection of Proteus species by real-time polymerase chain reaction using SYBR Green I

A SYBR Green real-time polymerase chain reaction (PCR) method for rapid detection of Proteus species was developed and evaluated. Of 322 clinical and food samples tested, 75 samples were positive for Proteus species by using conventional PCR and real-time PCR assays. The results were consistent with standard culture methods and the Vitek auto-microbe system, indicating a 100 % specificity obtained by both PCR assays. For the real-time PCR method, the minimum detectable level was 10 colony forming units (CFU) /ml, which was a 10³ multiple higher than the conventional PCR method. Correlation coefficients of standard curves which were constructed using the threshold cycle (Ct) versus copy numbers of Proteus showed good linearity (R ²?=?0.997). In conclusion, several significant advantages such as higher sensitivity and rapidness were observed by using the SYBR Green real-time PCR method for identifying Proteus species.     

Selection and optimization of PCR-based methods for the detection of Histoplasma capsulatum var. capsulatum

BackgroundCurrent methods for the laboratory diagnosis of histoplasmosis are problematic in terms of their sensitivity, specificity and runtime.ObjectivesThus, in this study, we sought to select and optimize methods for the detection of Histoplasma capsulatum var. capsulatum by polymerase chain reaction (PCR).MethodsThree DNA extraction methods and three PCR methods were evaluated. We optimised the concentration of the components of this PCR reaction and determined its sensitivity and specificity using blood samples to which H. capsulatum had been added.ResultsThe DNA extraction method that yielded the highest-quality DNA used silica membranes (DNeasy Blood &; Tissue Kit, Qiagen, Hilden, Germany), and the amplification method with the best detection capacity used a target gene encoding a 100-kDa protein. Our optimisation of the PCR conditions indicated that the reaction works over a significant range of component concentrations; in addition, it was able to detect H. capsulatum better than traditional culture techniques, with a detection limit of only 10 pg of DNA.ConclusionsIn our experimental conditions, the PCR method selected in this work (instead of nested-PCR) is a tool sensitive enough for the diagnosis of histoplasmosis.     

Multi-probe Real-Time PCR Identification of Four Common Candida Species in Blood Culture Broth

We developed a single-tube real-time polymerase chain reaction (PCR) assay with multiple hybridization probes for detecting Candida albicans, C. tropicalis, C. glabrata, and C. parapsilosis. Primers were designed to amplify 18S rRNA gene of the genus Candida, and DNA probes were designed to hybridize two areas of the amplicons. The amplification curves and specific melting peaks of the probes hybridized with PCR product were used for definite species identifications. The reaction specificity was 100 % when evaluating the assay using DNA samples from 21 isolates of fungal and bacterial species. The assay was further evaluated in 129 fungal blood culture broth samples which were culture positive for fungus. Of the 129 samples, 119 were positively identified as: C. albicans (39), C. tropicalis (30), C. parapsilosis (23), C. glabrata (20), Candida spp. (5), and two samples containing mixed C. glabrata/C. albicans and C. glabrata/C. tropicalis. The five Candida spp. were identified by sequencing analysis as C. krusei, C. dubliniensis, C. aquaetextoris, and two isolates of C. athensensis. Of the ten samples which showed negative PCR results, six were Cryptococcus neoformans, and the others were Trichosporon sp., Rhodotorula sp., Fusarium sp., and Penicillium marneffei. Our findings show that the assay was highly effective in identifying the four medically important Candida species. The results can be available within 3 h after positivity of a blood culture broth sample.