Identification of fungemia agents using the polymerase chain reaction and restriction fragment length polymorphism analysis

Santos, Maria Cecília Loschiavo dos; Souza, Edna Santos de; S., R. M.; Talhari, Sinésio; Souza, João Vicente Braga de

doi:10.1590/s0100-879x2010007500065

Cited by 11 publications

(14 citation statements)

References 21 publications

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“…PCR-RFLP is a simple molecular assay, requires only standard equipment used for molecular biology and provides results suitable for the simultaneous analysis of several species [14,15].…”

Section: Discussionmentioning

confidence: 99%

“…Generation of PCR products and subsequent digestion was performed as described by Santos et al [14]. DNA was extracted from samples (200 μL of fungal biomass or biological sample) using the QIAamp Blood and Tissue kit (Qiagen, Hilden, Germany) following the recommendations of the manufacturer.…”

Section: Detection and Identification By Pcr-rflpmentioning

confidence: 99%

“…Santos et al [14] presented a PCR-RFLP assay targeting the ITS region from the fungi rDNA that was able to differentiate the most important agents from invasive mycosis. The present work is the continuation of the assessment of this study by applying it in the routine of a hospital with patients at risk of being affected by invasive mycosis.…”

Section: Introductionmentioning

confidence: 99%

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PCR-RFLP as a useful tool for diagnosis of invasive mycoses in a healthcare facility in the North of Brazil

Sousa

Santos

Wanke

et al. 2015

Electronic Journal of Biotechnology

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Background: The incidence of invasive mycoses is increasing worldwide. PCR-RFLP was applied to the identification of 10 reference strains and 90 cultures of agents of invasive mycoses. In addition, the new approach was applied to detect fungal agents in 120 biological samples (blood, cerebrospinal fluid and bone marrow). PCR-RFLP results were compared with the ones obtained with conventional methods (culture, microscopy, and biochemical testing). Results: The assays carried out with the reference strains (Candida albicans, Candida parapsilosis, Candida tropicalis, Candida krusei, Candida guilliermondii, Cryptococcus neoformans, Cryptococcus gattii and Histoplasma capsulatum), demonstrated that the RFLP profiles were correctly predicted by the in silico investigation and allowed unequivocal identification of all chosen reference strains. The PCR-RFLP also identified 90 cultures of agents of invasive mycoses correctly, 2.5 times faster than the conventional assays. Evaluating PCR-RFLP with biological samples it was observed that the PCR was found to be 100% accurate and the RFLP profiles allowed the identification of the etiological agents: C. neoformans (n = 3) and C. gattii (n = 1) in CSF samples, H. capsulatum (n = 1) in bone marrow and C. albicans (n = 2) in blood cultures. The detection and identification by PCR-RFLP were found to be between two to ten times faster than the conventional assays. Conclusion: The results showed that PCR-RFLP is a valuable tool for the identification of invasive mycoses that can be implemented in hospital laboratories, allowing for a high number of clinical analyses per day.

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“…PCR-RFLP is a simple molecular assay, requires only standard equipment used for molecular biology and provides results suitable for the simultaneous analysis of several species [14,15].…”

Section: Discussionmentioning

confidence: 99%

Section: Detection and Identification By Pcr-rflpmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

PCR-RFLP as a useful tool for diagnosis of invasive mycoses in a healthcare facility in the North of Brazil

Sousa

Santos

Wanke

et al. 2015

Electronic Journal of Biotechnology

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“…A negative control containing sterile deionized water and a positive control of C. albicans DNA (ATCC 10231) were included in each PCR run. Reaction mixtures were subjected to an initial denaturation step at 94°C for seven minutes followed by 30 cycles of denaturation at 94°C for 45 seconds, annealing at 56°C for one minute, and extension at 72°C for one minute, with a final extension at 72°C for seven minutes (1,2,8) in Veriti Thermal Cycler ABI (applied biosystems, Foster City, CA, USA).…”

Section: Polymerase Chain Reactionmentioning

confidence: 99%

Detection of Candida vulvovaginitis in Clinical Samples ; Using Direct Polymerase Chain Reaction Without DNA Extraction

Sarookhani¹,

Sohrabi²,

Ezani³

2014

Biotech Health Sci

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Background: Vulvovaginal candidiasis (VVC) is a common disease which infects women. The current study investigated the performance of direct sample polymerase chain reaction (DS-PCR) method to detect Candida spp. in clinical samples of vulvovaginitisto compare the results to those of standard microbiological laboratory methods. Objectives: The current study aimed to further simplify the DNA extraction procedure, and shorten the time required for isolation and identification by using direct PCR to identify Candida vulvovaginitis without DNA extraction from samples or colonies. Patients and Methods: In the current study, totally 150 sexually active women participated. Vaginal discharge samples were collected using two sterile Dacron swabs that were immediately placed in two tubes each containing 1 mL of distilled water. One of the tubes was used for conventional culture methods whereas the other one was used for DS-PCR without DNA extraction. The number of yeast cells in each sample was counted. Results:The results showed that out of the 150 samples, 55 were positive and 63 samples were negative by both methods, and 32 samples were positive using the culture method, but negative by DS-PCR. All positive DS-PCR samples had > 10 7 yeast or conidia cells/mL. The sensitivity and specificity of DS-PCR were calculated as 63.2% and 100%, respectively. Conclusions: Direct sample PCR has the potential to rapidly and accurately diagnose Candida vulvovaginitis in patients, especially if sufficient samples are obtained.

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“…The high discriminatory power of the m olecular tools like polymerase chain reaction (PCR) PCR, PCR-RFLP, RAPD-PCR, as well as sequencing, have provided change barter fast, relatively simple to perform, precise and reliable methods for the diagnosis of the Candida spp. (Mirhendi et al, 2005;Santos et al, 2010;Mijiti et al, 2010;Shokohi et al, 2010). McCullough et al (1999) utilized CABF59F and CADBR125R primers designed to span the region that includes the site of the transposable intron of the 25S rRNA gene (rDNA).…”

Section: Introductionmentioning

confidence: 99%

Candida albicans ssp. dubliniensis stat.et comb. nov., a new combination for Candida dubliniensis based on genetic criteria

Imran¹

2015

Afr. J. Microbiol. Res.

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Identification of fungemia agents using the polymerase chain reaction and restriction fragment length polymorphism analysis

Cited by 11 publications

References 21 publications

PCR-RFLP as a useful tool for diagnosis of invasive mycoses in a healthcare facility in the North of Brazil

PCR-RFLP as a useful tool for diagnosis of invasive mycoses in a healthcare facility in the North of Brazil

Detection of Candida vulvovaginitis in Clinical Samples ; Using Direct Polymerase Chain Reaction Without DNA Extraction

Candida albicans ssp. dubliniensis stat.et comb. nov., a new combination for Candida dubliniensis based on genetic criteria

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