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the precise identification of some fungal species is often very difficult when using only biochemical or phenotypic methods ( dooley et al . , 1994 ; fenn et al . , 1994
however , the advent of dna - based methods largely overcame the limitations of traditional methods and studies using molecular approaches revealed a greater diversity in fungi ( odds et al . , 1998 ; chen et al . , 2000 ) .
some closely related species are often misidentified because of the great similarity in their biochemical and morphological characteristics .
( 2008 ) reported the misidentification of pichia guilliermondii ( teleomorph candida guilliermondii ) , debaryomyces hansenii ( teleomorph candida famata ) and candida palmioleophila .
their results showed that only 23 of 36 isolates identified as p. guilliermondii and three of 26 identified as d. hansenii were confirmed by sequencing the its1 - 5.8s - its2 and d1/d2 regions of the ribosomal cistron .
2001 ) and candida parapsilosis ( lasker et al . , 2006 ) show the same problem . in our laboratory , we have had problems differentiating ( 1 ) p. guilliermondii , c. palmioleophila and d. hansenii , ( 2 ) candida krusei and candida inconspicua , and ( 3 ) candida pelliculosa and candida subpelliculosa when using the commercial kit api 20 c aux ( biomrieux , france ) ( table s1 ) . the vitek yeast biochemical card and the i d 32c , two widely used methods for yeast identification , were tested by different groups ( dooley et al . , 1994 ; lo et al . , 2001 ;
burton et al . , 2010 ) and their findings confirmed the problem of incorrect or inconsistent identification .
lo et al . ( 2001 ) suggested that the laboratory routine should include at least two methods for yeast identification .
misidentification of the fungal species can compromise epidemiological or antibiotic susceptibility studies and over- or underestimate the species abundance .
precise identification is therefore necessary and molecular approaches can provide the tools for a fast method . in this study , we developed a real - time pcr method that can differentiate / identify p. guilliermondii , d. hansenii and c. palmioleophila , and sequencing the its 1 region of these species confirmed our results .
dna was quantified with a qubit fluorometer and a quant - it picogreen dsdna br assay kit ( invitrogen , life technologies , eugene , or , usa ) according to the manufacturers recommendations .
a go taq flexi dna polymerase kit ( promega , madison , wi , usa ) was used to amplify the its 1 region with its1/its2 primers ( white and lee , 1990 ) and a topota cloning kit ( invitrogen ) was used for the constructions .
the sequences were obtained using a big dye terminator v3.1 cycle sequencing kit followed by automatic sequencing in an abi 3100 ( applied biosystems , life technologies , foster city , ca , usa ) .
the sequences obtained from the three american tissue culture collection ( atcc ) species , d. hansenii atcc 36239 , c. famata atcc 62894 and p. guilliermondii atcc 6260 , were compared by the blastn program ( zhang et al . , 2000 ) against the genbank non - redundant database ( nr ) , embl , ddbj and pdb nucleotide collections .
the sequences obtained were aligned with clustalw software ( larkin et al . , 2007 ) and then used to create a consensus sequence for each species and to choose target regions ( data not shown ) .
the primers and taqman minor groove binder ( mgb ) probes were designed using the software primer express v. 2.0 and default parameters ( applied biosystems ) .
a real - time pcr was done using the taqman universal pcr master mix ( applied biosystems ) in the following singleplex reaction mixture : 10 ng of sample dna , 1x taqman universal pcr master mix , 200 nm of each primer , 300 nm of taqman mgb probe ( table 1 ) and water to a total volume of 25 l .
the cycling conditions were set in an abi 7300 real - time pcr cycler fitted with sds software v. 1.2.3 ( applied biosystems ) as follows : 10 min at 95 c followed by 35 cycles of 95 c for 30 s and 60 c for 1 min .
the efficiency of multiplex reactions was tested by mixing all of the probes together in the same concentrations as shown above .
the sequences obtained from the atcc strains ( data not shown ) were matched in a search against the ( nr ) nucleotide bank using the blast program ( the alignment files are provided in supplementary material figure s2 ) in order to choose the potential target region for genotyping by real - time pcr ( figure 1 ) .
comparison of the two methods of identification ( api 20c aux and ribotyping by sequencing ) revealed the difficulty in differentiating p. guilliermondii and d. hansenii , in the correct identification of c. parapsilosis versus d. hansenii ( burton et al . , 2010 and table s1 ) and the impossibility of differentiating c. krusei and c. insconspicua .
initially , three polymorphic domains ( its 1 , its 2 and d1/d2 ) of the ribosomal cistron from these species were aligned using clustalw ( larkin et al . , 2007 ) .
of these three regions , only the its 1 region provided suitable discrimination ( figure 1 ) .
this region is ideal because it is flanked by two conserved domains : the end of 18s rrna and the beginning of 5.8s rrna .
the amplicon is short ( about 300 bp ) and the sequence is variable among different species but conserved among strains of the same species .
figure 2 shows the amplification plot of the real - time pcr genotyping done using species - specific probes .
the region indicated as ntc ( no template control ) confirmed the specificity of each probe since only in the presence of the specific target was there amplification . to confirm this finding
, we analyzed the real - time pcr products in a 2% agarose gel stained with ethidium bromide and detected the expected amplicons ( data not shown ) .
the multiplex reaction worked as well as the singleplex test ( figure s1 ) and can be used for fungus identification , thereby reducing the costs of the assay .
the close relationship between c. palmioleophila and d. hansenii has previously been shown by a phylogenetic analysis using data from the d1/d2 and its regions ( desnos - ollivier et al . , 2008 ) .
in our study , the strain c. famata atcc 62894 , used as a positive control , was identified as c. palmioleophila by sequence analysis of the d1/d2 region of rrna ( table s1 ) and by the real - time pcr assay described here ( figure 2 ) . in conclusion ,
ribosomal rna sequencing is the gold standard for identification but is generally expensive and time consuming .
the real - time pcr assay described here is a very effective , rapid , low - cost alternative .
the method can unambiguously identify isolates and confirm the identification of strains analyzed by traditional methods , with the advantage of measuring species abundance if necessary .
the following online material is available for this article : comparison of results obtained dna sequencing .
traditional phenotypic methods and commercial kits based on carbohydrate assimilation patterns are unable to consistently distinguish among isolates of pichia guilliermondii , debaryomyces hansenii and candida palmioleophila . as result , these species are often misidentified . in this work 
 , we established a reliable method for the identification / differentiation of these species . 
 our assay was validated by dna sequencing of the polymorphic region used in a real - time pcr assay driven by species - specific probes targeted to the fungal its 1 region . 
 this assay provides a new tool for pathogen identification and for epidemiological , drug resistance and virulence studies of these organisms .