Detection and differentiation of fungi in clinical specimens using polymerase chain reaction (PCR) amplification and restriction enzyme analysis

Hopfer, Roy L.; Walden, Paul D.; Setterquist, S; Highsmith, W. Edward

doi:10.1080/02681219380000071

Cited by 128 publications

(80 citation statements)

References 13 publications

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“…Although there have been studies that apply PCR assays to detection of deeply invasive candidiasis (2,5,12,(24)(25)(26)(27), this to our knowledge is the first report that applies qPCR to investigate the kinetics of DNA release from C. albicans into the extracellular environment and, in addition, describes the role that H-MNCs play in Candida albicans DNA kinetics by increasing the release of fungal cell-free DNA. Further understanding of DNA kinetics of pathogens and the mechanisms by which DNA is released, especially those causing bloodstream infections, should improve strategies in designing and implementing molecular-based diagnostics for fungal pathogens.…”

Section: Discussionmentioning

confidence: 99%

“…As with previous studies (2,17,29), use of the rRNA gene complex and, more specifically, the ITS region allowed for the design of a species-specific assay. Additionally, targeting a multicopy gene such as the rRNA gene increased the number of potential targets of the assay (12). Equally as important as sequence selection of the primers and probes for specificity were the cycling parameters used for amplification.…”

Section: Discussionmentioning

confidence: 99%

“…Since the earliest introduction of PCR for the detection of C. albicans in blood (3,12), this technology has been increasingly refined for molecular detection of deeply invasive candidiasis. Real-time PCR is currently one of the promising methods to detect this infectious pathogen in clinical samples (6,21,26,27).…”

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confidence: 99%

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Use of Quantitative Real-Time PCR To Study the Kinetics of Extracellular DNA Released from Candida albicans , with Implications for Diagnosis of Invasive Candidiasis

et al. 2006

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Quantitative real-time PCR (qPCR) is considered one of the most sensitive methods to detect low levels of DNA from pathogens in clinical samples. To improve the design of qPCR for the detection of deeply invasive candidiasis, we sought to develop a more comprehensive understanding of the kinetics of DNA released from Candida albicans in vitro and in vivo. We developed a C. albicans-specific assay targeting the rRNA gene complex and studied the kinetics of DNA released from C. albicans alone, in the presence of human blood monocytes (H-MNCs), and in the bloodstream of rabbits with experimental disseminated candidiasis. The analytical qPCR assay was highly specific and sensitive (10 fg). Cells of C. albicans incubated in Hanks balanced salt solution (؎10% bovine serum albumin [BSA]) or RPMI (؎10% BSA) showed a significant release of DNA at T equal to 24 h compared to T equal to 0 h (P < 0.01). C. albicans incubated with H-MNCs exhibited a greater release of DNA than C. albicans cells alone over 24 h (P ‫؍‬ 0.0001). Rabbits with disseminated candidiasis showed a steady increase of detectable DNA levels in plasma as disease progressed. Plasma cultures showed minimal growth of C. albicans, demonstrating that DNA extracted from plasma reflected fungal cell-free DNA. In summary, these studies of the kinetics of DNA release by C. albicans collectively demonstrate that cell-free fungal DNA is released into the bloodstream of hosts with disseminated candidiasis, that phagocytic cells may play an active role in increasing this release over time, and that plasma is a suitable blood fraction for the detection of C. albicans DNA.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Use of Quantitative Real-Time PCR To Study the Kinetics of Extracellular DNA Released from Candida albicans , with Implications for Diagnosis of Invasive Candidiasis

et al. 2006

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“…Two different PCR methods capable of detecting a wide range of medically important fungi from clinical specimens have been developed by Hopfer et al (14) and by us (15). None of these methods, however, can differentiate aspergilli from other fungi contaminating the upper respiratory tract or causing pulmonary infections.…”

Section: *Present Address: Teikyo University Research Center For Medicalmentioning

confidence: 99%

Specific Detection of Aspergillus and Penicillium Species From Respiratory Specimens by Polymerase Chain Reaction (Pcr)

Makimura

Murayama

Yamaguchi

1994

JJMSB

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SUMMARY: A polymerase chain reaction (PCR) method capable of detecting both Aspergillus fumigatus infections, pulmonary non-fumigatus Aspergillus species (spp.) and Penicillium spp. from clinical specimens was established.The primer pair was designed on the basis of the sequence of the 18S-ribosomal RNA gene of A. fumigatus and P. notatum. A 385 by product was successfully amplified by this PCR method from all of 12 medically important Aspergillus spp. and Penicillium spp. (38 strains), but not from human, calf, Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA), any of 14 medically important yeastlike fungal species tested (32 strains) including Candida al bicans, several non-al bicans Candida and Saccharomyces cerevisiae, Cryptococcus neo formans, Mucor spp. or Pneumocystis carinii. This specificity was subsequently confirmed by Southern hybridization analysis. The established PCR can detect such a small amount as 1 pg of A, fumigatus DNA by staining the PCR product with ethidium bromide. With sputum specimens from clinically diagnosed aspergilloma patients, this PCR technique was demonstrated to be a more sensitive diagnostic method for Aspergillus infections than conventional culture techniques.

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“…Compared with species-specific structural genes, rRNA genes are attractive targets for amplification-based detection assays, since these genes are present at a high copy number, thus increasing the sensitivity of the PCR (15). Furthermore, rRNA genes are composed of regions of higher and lower evolutionary conservation, thereby enabling amplification at different taxonomic levels.…”

mentioning

confidence: 99%

Isolation and Characterization of a Species-Specific DNA Fragment for Identification of Candida ( Torulopsis ) glabrata by PCR

Becker

Badehorn

Keller³

et al. 2001

J Clin Microbiol

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Detection and differentiation of fungi in clinical specimens using polymerase chain reaction (PCR) amplification and restriction enzyme analysis

Cited by 128 publications

References 13 publications

Use of Quantitative Real-Time PCR To Study the Kinetics of Extracellular DNA Released from Candida albicans , with Implications for Diagnosis of Invasive Candidiasis

Use of Quantitative Real-Time PCR To Study the Kinetics of Extracellular DNA Released from Candida albicans , with Implications for Diagnosis of Invasive Candidiasis

Specific Detection of Aspergillus and Penicillium Species From Respiratory Specimens by Polymerase Chain Reaction (Pcr)

Isolation and Characterization of a Species-Specific DNA Fragment for Identification of Candida ( Torulopsis ) glabrata by PCR

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