ISOLATION AND CHARACTERIZATION OF A SPECIES-SPECIFIC DNA-PROBE FOR THE DETECTION OF CANDIDA-KRUSEI

In an attempt to design species-specific primers for the detection of Candida krusei by polymerase chain reaction, a partial genomic DNA lib rary from Candida krusei was screened for hybridization with radiolabe led genomic probes from a broad variety of fungal and bacterial specie s and from human. Species-specific candidate DNA inserts were then tes ted for hybridization with dot blots of DNA from various organisms. On e 570-basepair insert from Candida krusei DNA that hybridized under st ringent conditions only with DNA from Candida krusei and human was seq uenced. It revealed considerable homology with the gene for the mitoch ondrial inner membrane protease I of Saccharomyces cerevisiae, and the 147 amino acid residues deduced from an open reading frame showed con siderable homology with the N-termial portion of the enzyme from Sacch aromyces cerevisiae. From the sequence of the Candida krusei DNA fragm ent, a pair of 21-base oligonucleotide primers enclosing a 501-basepai r sequence was designed for polymerase chain reaction. When these prim ers were tested with a broad range of genomic DNAs, the expected ampli fication was obtained only with Candida krusei DNA and not with DNA fr om any other source, including human. Experiments with DNA from mixed cultures of Candida krusei and other yeasts and bacteria showed that t he polymerase chain reaction was specific for Candida krusei and that as few as ten cells could be detected.