Identification of clinically relevant yeasts by PCR/RFLP. 2004

Anja Trost, and Barbara Graf, and Jan Eucker, and Orhan Sezer, and Kurt Possinger, and Ulf B Göbel, and Thomas Adam
Institute for Microbiology and Hygiene, Medical Faculty of Humboldt University, Charité, Dorotheenstr. 96, 10117 Berlin, Germany. thomas.adam@charite.de

For molecular diagnosis of fungal disease using DNA amplification procedures in the routine laboratory, choice of appropriate target structures and rapid and inexpensive identification of amplification products are important prerequisites. Most diagnostic procedures described thus far are characterized by limited applicability, considerable cost for laboratory equipment or low power of discrimination between species. This study aimed at identification of a PCR target appropriate for diagnosis of clinically relevant yeasts and an affordable procedure for characterization of the PCR products to the species level. Here, we describe a PCR-based system using amplification of intergenic spacers ITS1 and ITS2 and restriction length polymorphism of PCR products after sequence-specific enzymatic cleavage. We show the evaluation of the system for clinically relevant Candida species. The simple and inexpensive procedure should be instrumental for rapid identification of medically important yeasts.

UI MeSH Term Description Entries
D012150 Polymorphism, Restriction Fragment Length Variation occurring within a species in the presence or length of DNA fragment generated by a specific endonuclease at a specific site in the genome. Such variations are generated by mutations that create or abolish recognition sites for these enzymes or change the length of the fragment. RFLP,Restriction Fragment Length Polymorphism,RFLPs,Restriction Fragment Length Polymorphisms
D002175 Candida A genus of yeast-like mitosporic Saccharomycetales fungi characterized by producing yeast cells, mycelia, pseudomycelia, and blastophores. It is commonly part of the normal flora of the skin, mouth, intestinal tract, and vagina, but can cause a variety of infections, including CANDIDIASIS; ONYCHOMYCOSIS; VULVOVAGINAL CANDIDIASIS; and CANDIDIASIS, ORAL (THRUSH). Candida guilliermondii var. nitratophila,Candida utilis,Cyberlindnera jadinii,Hansenula jadinii,Lindnera jadinii,Monilia,Pichia jadinii,Saccharomyces jadinii,Torula utilis,Torulopsis utilis,Monilias
D002177 Candidiasis Infection with a fungus of the genus CANDIDA. It is usually a superficial infection of the moist areas of the body and is generally caused by CANDIDA ALBICANS. (Dorland, 27th ed) Candida Infection,Moniliasis,Candida Infections,Candidiases,Infection, Candida,Moniliases
D004271 DNA, Fungal Deoxyribonucleic acid that makes up the genetic material of fungi. Fungal DNA
D012340 RNA, Ribosomal, 5.8S Constituent of the 60S subunit of eukaryotic ribosomes. 5.8S rRNA is involved in the initiation of polypeptide synthesis in eukaryotes. 5.8S Ribosomal RNA,5.8S RRNA,RNA, 5.8S Ribosomal,Ribosomal RNA, 5.8S
D016133 Polymerase Chain Reaction In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships. Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain
D017931 DNA Primers Short sequences (generally about 10 base pairs) of DNA that are complementary to sequences of messenger RNA and allow reverse transcriptases to start copying the adjacent sequences of mRNA. Primers are used extensively in genetic and molecular biology techniques. DNA Primer,Oligodeoxyribonucleotide Primer,Oligodeoxyribonucleotide Primers,Oligonucleotide Primer,Oligonucleotide Primers,Primer, DNA,Primer, Oligodeoxyribonucleotide,Primer, Oligonucleotide,Primers, DNA,Primers, Oligodeoxyribonucleotide,Primers, Oligonucleotide
D021901 DNA, Intergenic Any of the DNA in between gene-coding DNA, including untranslated regions, 5' and 3' flanking regions, INTRONS, non-functional pseudogenes, and non-functional repetitive sequences. This DNA may or may not encode regulatory functions. DNA, Junk,DNA, Spacer,Intergenic DNA,Junk DNA,Spacer DNA,Intercistronic Region,Intercistronic Sequence,Intergenic Region,Intergenic Sequence,Sequence, Intergenic,DNAs, Intergenic,DNAs, Junk,DNAs, Spacer,Intercistronic Regions,Intercistronic Sequences,Intergenic DNAs,Intergenic Regions,Intergenic Sequences,Junk DNAs,Region, Intercistronic,Region, Intergenic,Regions, Intercistronic,Regions, Intergenic,Sequence, Intercistronic,Sequences, Intercistronic,Sequences, Intergenic,Spacer DNAs

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