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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

Melting profiles for amplicons targeting ITS-2 using primer set 5.8S-1F/28S-1R. Each profile tracing represents the result from an individual strain of the following tested species: 1. C. parapsilosis (2), 2. C. tropicalis (2), 3. C. guilliermondii (1), 4. C. glabrata (2), 5. C. dubliniensis (2), 6. C. albicans (3), 7. C. lusitaniae (2), and 8. C. krusei (2). The number in the parenthesis after the species name indicates the number of strains examined in the study.
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f2-mbi-9-2016-021: Melting profiles for amplicons targeting ITS-2 using primer set 5.8S-1F/28S-1R. Each profile tracing represents the result from an individual strain of the following tested species: 1. C. parapsilosis (2), 2. C. tropicalis (2), 3. C. guilliermondii (1), 4. C. glabrata (2), 5. C. dubliniensis (2), 6. C. albicans (3), 7. C. lusitaniae (2), and 8. C. krusei (2). The number in the parenthesis after the species name indicates the number of strains examined in the study.

Mentions: We then evaluated the abilities of the pan-Candida primer sets to distinguish the eight medically important Candida species by their specific amplicon melting profiles. For pan-Candida primer set 5.8S-1F/28S-1R targeting ITS-2, most of the species displayed a single major peak, whereas C. parapsilosis appears to have several domains when melted, with a major peak at 79.5°C (Fig. 2). The melting peaks for C. glabrata, C. dubliniensis, and C. guilliermondii seem to be clearly separated from one another; however, there are slight variations from different strains within each species that may be too close to make reliable distinction for species differentiation among themselves. For C. tropicalis, C. albicans, C. lusitaniae, and C. krusei, their unique melting profile allows species-specific differentiation with the main peak at 79.0, 81.3, 81.9 and 85.1°C, respectively.


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)

Melting profiles for amplicons targeting ITS-2 using primer set 5.8S-1F/28S-1R. Each profile tracing represents the result from an individual strain of the following tested species: 1. C. parapsilosis (2), 2. C. tropicalis (2), 3. C. guilliermondii (1), 4. C. glabrata (2), 5. C. dubliniensis (2), 6. C. albicans (3), 7. C. lusitaniae (2), and 8. C. krusei (2). The number in the parenthesis after the species name indicates the number of strains examined in the study.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4836890&req=5

f2-mbi-9-2016-021: Melting profiles for amplicons targeting ITS-2 using primer set 5.8S-1F/28S-1R. Each profile tracing represents the result from an individual strain of the following tested species: 1. C. parapsilosis (2), 2. C. tropicalis (2), 3. C. guilliermondii (1), 4. C. glabrata (2), 5. C. dubliniensis (2), 6. C. albicans (3), 7. C. lusitaniae (2), and 8. C. krusei (2). The number in the parenthesis after the species name indicates the number of strains examined in the study.
Mentions: We then evaluated the abilities of the pan-Candida primer sets to distinguish the eight medically important Candida species by their specific amplicon melting profiles. For pan-Candida primer set 5.8S-1F/28S-1R targeting ITS-2, most of the species displayed a single major peak, whereas C. parapsilosis appears to have several domains when melted, with a major peak at 79.5°C (Fig. 2). The melting peaks for C. glabrata, C. dubliniensis, and C. guilliermondii seem to be clearly separated from one another; however, there are slight variations from different strains within each species that may be too close to make reliable distinction for species differentiation among themselves. For C. tropicalis, C. albicans, C. lusitaniae, and C. krusei, their unique melting profile allows species-specific differentiation with the main peak at 79.0, 81.3, 81.9 and 85.1°C, respectively.

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