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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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Multiplex polymerase chain reaction (PCR) for pheromone fragment analysis. A) Multiplex PCR with 4 sets of primers comprising MFα1 (V190/V191) and MFa1 (V290/V291, V1311/V1312, V1313/V1314) genes were carried out as described in Materials and Methods. Approximately 100-bp MFα1 and 117-bp MFa1 PCR amlicons were detected on 3.5% MetaPhor agarose in Tris-borate-EDTA buffer for MATα and MATa strains comprising Cryptococcus neoformans var. grubii (CnVG), Cryptococcus neoformans var. neoformans (CnVN), and Cryptococcus gattii (Cg). Lanes 1 and 7, molecular mass marker. B) Multiplex PCR depicting MFα1 and MFa1 PCR amplicons from the 8 known hybrid (A/D) isolates. Lane 1, molecular mass marker.
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Figure 2: Multiplex polymerase chain reaction (PCR) for pheromone fragment analysis. A) Multiplex PCR with 4 sets of primers comprising MFα1 (V190/V191) and MFa1 (V290/V291, V1311/V1312, V1313/V1314) genes were carried out as described in Materials and Methods. Approximately 100-bp MFα1 and 117-bp MFa1 PCR amlicons were detected on 3.5% MetaPhor agarose in Tris-borate-EDTA buffer for MATα and MATa strains comprising Cryptococcus neoformans var. grubii (CnVG), Cryptococcus neoformans var. neoformans (CnVN), and Cryptococcus gattii (Cg). Lanes 1 and 7, molecular mass marker. B) Multiplex PCR depicting MFα1 and MFa1 PCR amplicons from the 8 known hybrid (A/D) isolates. Lane 1, molecular mass marker.

Mentions: The 4 sets of primers (MFα1/MFa1) produced reproducible results for control CnVG, CnVN, Cg haploids (Figure 2A), and A/D hybrid strains (Figure 2B). These results validated the robustness of the primers, which had been designed from well within the open reading frames of 2 pheromone genes, to prevent amplification of any nontarget DNA. The latter objective also informed the decision to anchor the 3´ ends of all PCR primers within the characteristic Cys-Val-Ile-Ala (CVIA) motifs; this eliminated any possible amplification of other pheromone genes since this is the only sequence shared among fungal pheromones (18). Even though multiple copies of MFα and MFa genes have been reported in C. gattii and C. neoformans by Southern hybridization and whole genome-sequencing, PCR primers only amplify single amplicons because these genes have identical nucleotide sequences (18,23,24). Although this multiplex method was well suited for identifying mating types and hybrids, further delineation of species and varieties would require restriction digestion with several unique enzymes as we stated previously (21). Therefore, we decided to use CE-FLA and CE-SSCP to further characterize pheromone gene amplicons. These techniques have been successfully used to delineate fragment length as well gene mutations for characterizing various fungal and bacterial isolates (25–27). The SSCP analysis displays migration of the amplified DNA fragment as a function of that fragment's structural conformation. Given that the tertiary structure of a fragment is sensitive to single nucleotide substitutions, this method was shown to be suitable for detecting single nucleotide changes when 100-bp to 300-bp DNA fragments were analyzed (28). Since amplified pheromone fragments yield ≈100- to 120-bp products, they were an ideal substrate for this method of mutant detection.


Cryptococcus gattii in AIDS patients, southern California.

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

Multiplex polymerase chain reaction (PCR) for pheromone fragment analysis. A) Multiplex PCR with 4 sets of primers comprising MFα1 (V190/V191) and MFa1 (V290/V291, V1311/V1312, V1313/V1314) genes were carried out as described in Materials and Methods. Approximately 100-bp MFα1 and 117-bp MFa1 PCR amlicons were detected on 3.5% MetaPhor agarose in Tris-borate-EDTA buffer for MATα and MATa strains comprising Cryptococcus neoformans var. grubii (CnVG), Cryptococcus neoformans var. neoformans (CnVN), and Cryptococcus gattii (Cg). Lanes 1 and 7, molecular mass marker. B) Multiplex PCR depicting MFα1 and MFa1 PCR amplicons from the 8 known hybrid (A/D) isolates. Lane 1, molecular mass marker.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Multiplex polymerase chain reaction (PCR) for pheromone fragment analysis. A) Multiplex PCR with 4 sets of primers comprising MFα1 (V190/V191) and MFa1 (V290/V291, V1311/V1312, V1313/V1314) genes were carried out as described in Materials and Methods. Approximately 100-bp MFα1 and 117-bp MFa1 PCR amlicons were detected on 3.5% MetaPhor agarose in Tris-borate-EDTA buffer for MATα and MATa strains comprising Cryptococcus neoformans var. grubii (CnVG), Cryptococcus neoformans var. neoformans (CnVN), and Cryptococcus gattii (Cg). Lanes 1 and 7, molecular mass marker. B) Multiplex PCR depicting MFα1 and MFa1 PCR amplicons from the 8 known hybrid (A/D) isolates. Lane 1, molecular mass marker.
Mentions: The 4 sets of primers (MFα1/MFa1) produced reproducible results for control CnVG, CnVN, Cg haploids (Figure 2A), and A/D hybrid strains (Figure 2B). These results validated the robustness of the primers, which had been designed from well within the open reading frames of 2 pheromone genes, to prevent amplification of any nontarget DNA. The latter objective also informed the decision to anchor the 3´ ends of all PCR primers within the characteristic Cys-Val-Ile-Ala (CVIA) motifs; this eliminated any possible amplification of other pheromone genes since this is the only sequence shared among fungal pheromones (18). Even though multiple copies of MFα and MFa genes have been reported in C. gattii and C. neoformans by Southern hybridization and whole genome-sequencing, PCR primers only amplify single amplicons because these genes have identical nucleotide sequences (18,23,24). Although this multiplex method was well suited for identifying mating types and hybrids, further delineation of species and varieties would require restriction digestion with several unique enzymes as we stated previously (21). Therefore, we decided to use CE-FLA and CE-SSCP to further characterize pheromone gene amplicons. These techniques have been successfully used to delineate fragment length as well gene mutations for characterizing various fungal and bacterial isolates (25–27). The SSCP analysis displays migration of the amplified DNA fragment as a function of that fragment's structural conformation. Given that the tertiary structure of a fragment is sensitive to single nucleotide substitutions, this method was shown to be suitable for detecting single nucleotide changes when 100-bp to 300-bp DNA fragments were analyzed (28). Since amplified pheromone fragments yield ≈100- to 120-bp products, they were an ideal substrate for this method of mutant detection.

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.

Show MeSH
Related in: MedlinePlus