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Development of Candida-Specific Real-Time PCR Assays for the Detection and Identification of Eight Medically Important Candida Species.

Zhang J, Hung GC, Nagamine K, Li B, Tsai S, Lo SC - Microbiol Insights (2016)

Bottom Line: In this study, we designed pan-Candida primer sets based on the ribosomal DNA-coding regions conserved within Candida but distinct from those of Aspergillus and Penicillium.We demonstrate that the final two selected pan-Candida primer sets would not amplify Aspergillus DNA and could be used to differentiate eight medically important Candida pathogens in real-time PCR assays based on their melting profiles, with a sensitivity of detection as low as 10 fg of Candida genomic DNA.These real-time PCR primer sets could potentially be assembled into a single PCR array for the rapid detection of Candida species in various clinical settings, such as corneal transplantation.

View Article: PubMed Central - PubMed

Affiliation: Tissue Microbiology Laboratory, Division of Cellular and Gene Therapies, Office of Cellular, Tissue and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration, Silver Spring, MD, USA.

ABSTRACT
Culture-based identification methods have been the gold standard for the diagnosis of fungal infection. Currently, molecular technologies such as real-time PCR assays with short turnaround time can provide desirable alternatives for the rapid detection of Candida microbes. However, most of the published PCR primer sets are not Candida specific and likely to amplify DNA from common environmental contaminants, such as Aspergillus microbes. In this study, we designed pan-Candida primer sets based on the ribosomal DNA-coding regions conserved within Candida but distinct from those of Aspergillus and Penicillium. We demonstrate that the final two selected pan-Candida primer sets would not amplify Aspergillus DNA and could be used to differentiate eight medically important Candida pathogens in real-time PCR assays based on their melting profiles, with a sensitivity of detection as low as 10 fg of Candida genomic DNA. Moreover, we further evaluated and selected species-specific primer sets covering Candida albicans, Candida glabrata, Candida tropicalis, and Candida dubliniensis and show that they had high sensitivity and specificity. These real-time PCR primer sets could potentially be assembled into a single PCR array for the rapid detection of Candida species in various clinical settings, such as corneal transplantation.

No MeSH data available.


Related in: MedlinePlus

Schematic representation of rDNA transcriptional unit, including the 18S, 5.8S, and 28S genes and the first and second internal transcribed spacers (ITS-1 and ITS-2, respectively), showing the relative positions of the primer sets used in PCR. Bases identical to those of C. albicans are represented by a dot (.). The primer sequences are listed in Table 1.
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f1-mbi-9-2016-021: Schematic representation of rDNA transcriptional unit, including the 18S, 5.8S, and 28S genes and the first and second internal transcribed spacers (ITS-1 and ITS-2, respectively), showing the relative positions of the primer sets used in PCR. Bases identical to those of C. albicans are represented by a dot (.). The primer sequences are listed in Table 1.

Mentions: The sequences of 18S, 5.8S, and 28S rDNA of C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, C. dubliniensis, C. krusei, C. lusitaniae, C. guilliermondii, A. flavus, A. fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus penicillioides, Aspergillus terreus, and Penicillium chrysogenum were downloaded from the National Center for Biotechnology Information and aligned. Regions of DNA sequences that are conserved within Candida species but distinct from Aspergillus and Penicillium species were identified. A total of four pan-Candida primer sets were designed: two primer sets (18S-1F/5.8S-1R and 18S-2F/5.8S-1R) for amplifying ITS-1, one primer set (5.8S-1F/28S-1R) for amplifying ITS-2, and one primer set (28S-2F/28S-2R) for amplifying a variable segment of 28S ribosomal gene were designed. Nucleotide sequences of four in-house designed primer sets, and the published primer sets used in this study are listed in Table 1. A schematic representation of the relative positions of 18S-1F, 5.8S-1R, 5.8S-1F, and 28S-1R primers and two previously published primer sets (Fungal-7a,7b/RT1 and UNF1/UNF2)13,14 in the rDNA transcriptional unit is shown in Figure 1.


Development of Candida-Specific Real-Time PCR Assays for the Detection and Identification of Eight Medically Important Candida Species.

Zhang J, Hung GC, Nagamine K, Li B, Tsai S, Lo SC - Microbiol Insights (2016)

Schematic representation of rDNA transcriptional unit, including the 18S, 5.8S, and 28S genes and the first and second internal transcribed spacers (ITS-1 and ITS-2, respectively), showing the relative positions of the primer sets used in PCR. Bases identical to those of C. albicans are represented by a dot (.). The primer sequences are listed in Table 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4836890&req=5

f1-mbi-9-2016-021: Schematic representation of rDNA transcriptional unit, including the 18S, 5.8S, and 28S genes and the first and second internal transcribed spacers (ITS-1 and ITS-2, respectively), showing the relative positions of the primer sets used in PCR. Bases identical to those of C. albicans are represented by a dot (.). The primer sequences are listed in Table 1.
Mentions: The sequences of 18S, 5.8S, and 28S rDNA of C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, C. dubliniensis, C. krusei, C. lusitaniae, C. guilliermondii, A. flavus, A. fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus penicillioides, Aspergillus terreus, and Penicillium chrysogenum were downloaded from the National Center for Biotechnology Information and aligned. Regions of DNA sequences that are conserved within Candida species but distinct from Aspergillus and Penicillium species were identified. A total of four pan-Candida primer sets were designed: two primer sets (18S-1F/5.8S-1R and 18S-2F/5.8S-1R) for amplifying ITS-1, one primer set (5.8S-1F/28S-1R) for amplifying ITS-2, and one primer set (28S-2F/28S-2R) for amplifying a variable segment of 28S ribosomal gene were designed. Nucleotide sequences of four in-house designed primer sets, and the published primer sets used in this study are listed in Table 1. A schematic representation of the relative positions of 18S-1F, 5.8S-1R, 5.8S-1F, and 28S-1R primers and two previously published primer sets (Fungal-7a,7b/RT1 and UNF1/UNF2)13,14 in the rDNA transcriptional unit is shown in Figure 1.

Bottom Line: In this study, we designed pan-Candida primer sets based on the ribosomal DNA-coding regions conserved within Candida but distinct from those of Aspergillus and Penicillium.We demonstrate that the final two selected pan-Candida primer sets would not amplify Aspergillus DNA and could be used to differentiate eight medically important Candida pathogens in real-time PCR assays based on their melting profiles, with a sensitivity of detection as low as 10 fg of Candida genomic DNA.These real-time PCR primer sets could potentially be assembled into a single PCR array for the rapid detection of Candida species in various clinical settings, such as corneal transplantation.

View Article: PubMed Central - PubMed

Affiliation: Tissue Microbiology Laboratory, Division of Cellular and Gene Therapies, Office of Cellular, Tissue and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration, Silver Spring, MD, USA.

ABSTRACT
Culture-based identification methods have been the gold standard for the diagnosis of fungal infection. Currently, molecular technologies such as real-time PCR assays with short turnaround time can provide desirable alternatives for the rapid detection of Candida microbes. However, most of the published PCR primer sets are not Candida specific and likely to amplify DNA from common environmental contaminants, such as Aspergillus microbes. In this study, we designed pan-Candida primer sets based on the ribosomal DNA-coding regions conserved within Candida but distinct from those of Aspergillus and Penicillium. We demonstrate that the final two selected pan-Candida primer sets would not amplify Aspergillus DNA and could be used to differentiate eight medically important Candida pathogens in real-time PCR assays based on their melting profiles, with a sensitivity of detection as low as 10 fg of Candida genomic DNA. Moreover, we further evaluated and selected species-specific primer sets covering Candida albicans, Candida glabrata, Candida tropicalis, and Candida dubliniensis and show that they had high sensitivity and specificity. These real-time PCR primer sets could potentially be assembled into a single PCR array for the rapid detection of Candida species in various clinical settings, such as corneal transplantation.

No MeSH data available.


Related in: MedlinePlus