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Cryptococcus gattii in AIDS patients, southern California.

Chaturvedi S, Dyavaiah M, Larsen RA, Chaturvedi V - Emerging Infect. Dis. (2005)

Bottom Line: CE-SSCP analyses of MFalpha1 showed 219 (79.3%) CnVG, 23 (8.3%) CnVN, and 34 (12.3%) Cg isolates.CE-FLA and CE-SSCP are promising tools for high-throughput screening of Cryptococcus isolates.The high prevalence of Cg was noteworthy, in view of its sporadic reports from AIDS patients in North America and its recent emergence as a primary pathogen on Vancouver Island, Canada.

View Article: PubMed Central - PubMed

Affiliation: Mycology Laboratory, Wadsworth Center, 120 New Scotland Avenue, Albany, NY 12208-2002, USA.

ABSTRACT
Cryptococcus isolates from AIDS patients in southern California were characterized by molecular analyses. Pheromone MFalpha1 and MFa1 gene fragments were polymerase chain reaction-amplified with fluorescently labeled primers and analyzed by capillary electrophoresis (CE) on DNA analyzer. CE-fragment-length analyses (CE-FLAs) and CE-single-strand conformation polymorphisms (CE-SSCPs) were used to determine Cryptococcus gattii (Cg), C. neoformans (Cn) varieties neoformans (CnVN) and grubii (CnVG), mating types, and hybrids. Corroborative tests carried out in parallel included growth on specialized media and serotyping with a commercial kit. All 276 clinical strains tested as haploid MATalpha by CE-FLA. CE-SSCP analyses of MFalpha1 showed 219 (79.3%) CnVG, 23 (8.3%) CnVN, and 34 (12.3%) Cg isolates. CE-FLA and CE-SSCP are promising tools for high-throughput screening of Cryptococcus isolates. The high prevalence of Cg was noteworthy, in view of its sporadic reports from AIDS patients in North America and its recent emergence as a primary pathogen on Vancouver Island, Canada.

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Capillary electrophoresis fragment-length analyses (CE-FLA) for the identification of mating types and hybrids. The ABI PRISM 310 Genetic Analyzer and GeneScan analysis software were used for the fragment length analysis of the pheromone genes. Sense strands of MFα1 and MFa1 were labeled with fluorescent probes TET (green) and HEX (black), respectively, and polymerase chain reaction amplicons were analyzed with POP-4 polymer under denaturing conditions at 60°C. Green peak, MFα1; black peak, MFa1. These peaks were aligned by using an internal size standard, GeneScan-500 TAMRA (red peaks).
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Figure 3: Capillary electrophoresis fragment-length analyses (CE-FLA) for the identification of mating types and hybrids. The ABI PRISM 310 Genetic Analyzer and GeneScan analysis software were used for the fragment length analysis of the pheromone genes. Sense strands of MFα1 and MFa1 were labeled with fluorescent probes TET (green) and HEX (black), respectively, and polymerase chain reaction amplicons were analyzed with POP-4 polymer under denaturing conditions at 60°C. Green peak, MFα1; black peak, MFa1. These peaks were aligned by using an internal size standard, GeneScan-500 TAMRA (red peaks).

Mentions: The 16 reference strains of known Cryptococcus species, varieties, mating types, and hybrids were used to establish a robust CE-FLA protocol with denaturing POP-4 polymer at 60°C. The electrophoretic runs with POP-4 polymer produced a 112-bp DNA fragment for MFa1 and 97-bp fragment for MFα1, which were easily distinguished with the GeneScan software by the characteristic peak sizes (Figure 3). CE-FLA allowed Cryptococcus mating types and hybrids to be identified, but not CnVG, CnVN, and Cg.


Cryptococcus gattii in AIDS patients, southern California.

Chaturvedi S, Dyavaiah M, Larsen RA, Chaturvedi V - Emerging Infect. Dis. (2005)

Capillary electrophoresis fragment-length analyses (CE-FLA) for the identification of mating types and hybrids. The ABI PRISM 310 Genetic Analyzer and GeneScan analysis software were used for the fragment length analysis of the pheromone genes. Sense strands of MFα1 and MFa1 were labeled with fluorescent probes TET (green) and HEX (black), respectively, and polymerase chain reaction amplicons were analyzed with POP-4 polymer under denaturing conditions at 60°C. Green peak, MFα1; black peak, MFa1. These peaks were aligned by using an internal size standard, GeneScan-500 TAMRA (red peaks).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3367345&req=5

Figure 3: Capillary electrophoresis fragment-length analyses (CE-FLA) for the identification of mating types and hybrids. The ABI PRISM 310 Genetic Analyzer and GeneScan analysis software were used for the fragment length analysis of the pheromone genes. Sense strands of MFα1 and MFa1 were labeled with fluorescent probes TET (green) and HEX (black), respectively, and polymerase chain reaction amplicons were analyzed with POP-4 polymer under denaturing conditions at 60°C. Green peak, MFα1; black peak, MFa1. These peaks were aligned by using an internal size standard, GeneScan-500 TAMRA (red peaks).
Mentions: The 16 reference strains of known Cryptococcus species, varieties, mating types, and hybrids were used to establish a robust CE-FLA protocol with denaturing POP-4 polymer at 60°C. The electrophoretic runs with POP-4 polymer produced a 112-bp DNA fragment for MFa1 and 97-bp fragment for MFα1, which were easily distinguished with the GeneScan software by the characteristic peak sizes (Figure 3). CE-FLA allowed Cryptococcus mating types and hybrids to be identified, but not CnVG, CnVN, and Cg.

Bottom Line: CE-SSCP analyses of MFalpha1 showed 219 (79.3%) CnVG, 23 (8.3%) CnVN, and 34 (12.3%) Cg isolates.CE-FLA and CE-SSCP are promising tools for high-throughput screening of Cryptococcus isolates.The high prevalence of Cg was noteworthy, in view of its sporadic reports from AIDS patients in North America and its recent emergence as a primary pathogen on Vancouver Island, Canada.

View Article: PubMed Central - PubMed

Affiliation: Mycology Laboratory, Wadsworth Center, 120 New Scotland Avenue, Albany, NY 12208-2002, USA.

ABSTRACT
Cryptococcus isolates from AIDS patients in southern California were characterized by molecular analyses. Pheromone MFalpha1 and MFa1 gene fragments were polymerase chain reaction-amplified with fluorescently labeled primers and analyzed by capillary electrophoresis (CE) on DNA analyzer. CE-fragment-length analyses (CE-FLAs) and CE-single-strand conformation polymorphisms (CE-SSCPs) were used to determine Cryptococcus gattii (Cg), C. neoformans (Cn) varieties neoformans (CnVN) and grubii (CnVG), mating types, and hybrids. Corroborative tests carried out in parallel included growth on specialized media and serotyping with a commercial kit. All 276 clinical strains tested as haploid MATalpha by CE-FLA. CE-SSCP analyses of MFalpha1 showed 219 (79.3%) CnVG, 23 (8.3%) CnVN, and 34 (12.3%) Cg isolates. CE-FLA and CE-SSCP are promising tools for high-throughput screening of Cryptococcus isolates. The high prevalence of Cg was noteworthy, in view of its sporadic reports from AIDS patients in North America and its recent emergence as a primary pathogen on Vancouver Island, Canada.

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