Probe and method for detecting yeast of species Candida krusei

The detection and characterization of yeasts belonging to the species Candida krusei is disclosed. A specific probe, in particular a DNA probe for detecting and, in particular, identifying the species Candida krusei and typing (infraspecific characterization) Candida krusei strains, is particularly disclosed. The expected results are achieved by means of a probe selected from the following genetic (or related) tools: PA1 at least one portion of at least one DNA and/or RNA fragment (F) with a size of 7-4 kb, said fragment being specifically hybridizable with the DNA and/or RNA of Candida krusei while not coding for rRNA; PA1 at least one portion of the transcription and/or translation products of (F); PA1 and a combination of the abovementioned tools. The probe may be used in medical diagnostics and industrial therapeutical tests.

TECHNICAL FIELD 
The present invention relates to the detection and characterization of 
yeasts belonging to the species Candida krusei. In particular, it provides 
a specific probe, especially a nucleic acid probe, for the detection and 
particularly identification of the species Candida krusei and for the 
typing (infrapecific characterization) of strains of the species Candida 
krusei. 
PRIOR ART 
Yeasts are fungi, i.e. eukaryotic microorganisms, in which the unicellular 
form is predominant (Barnett J. W., R. W. Payne and D. Yarrow, Yeasts: 
characteristics and identification, 2nd Edition, Cambridge University 
Press, Cambridge, 1991). 
The genus Candida is a heterogeneous genus which groups together 
anamorphous species, i.e. species whose mode of sexual reproduction is or 
was unknown. When this mode of sexual reproduction is discovered, the 
species are generally reclassified within an already described species 
(teleomorph). Thus Issatchenkia orientalis is the teleomorph of Candida 
krusei. 
In the taxonomy of yeasts, it is therefore possible to encounter two 
different species names for describing one and the same taxon (group) of 
yeasts. 
In normal practice, the species name Candida krusei is the most commonly 
used and it is the one which will be employed indiscriminately in the 
present disclosure to denote Candida krusei or Issatchenkia orientalis. 
Candida krusei is one of the ten principal species of yeasts of the genus 
Candida which are responsible for infections in humans or animals. This 
species is sometimes responsible for the degradation of food products. It 
is also used for certain industrial biotransformations. 
Candida krusei infections are called "C. krusei candidoses" or "C. krusei 
mycoses"; they can affect practically any tissues in the human body. 
Systemic, generalized or deep infections are the most serious and can be 
fatal. The mortality rate can be high. 
Yeasts of the species Candida krusei are opportunist and ubiquitous; their 
prevalence and pathogenicity are high in immunosuppressed subjects, 
especially in patients suffering from neutropenia, AIDS, cancer etc., who 
are particularly sensitive to C. krusei infections. 
Finally, C. krusei candidoses arc obviously very contagious in the same way 
as any other fungal diseases. 
The whole difficulty of treating infections of this type arises from the 
fact that strains of Candida krusei are relatively resistant to certain 
antifungal agents and to fluconazole in particular. What is even more 
troublesome, however, is that the use of these antifungal agents in 
prophylaxis, or even in therapy, would favor colonization of the subjects 
by endogenous strains if they were present in the patient. 
Consequently, rapid detection and precise and reliable identification, as 
well as typing of the strains of this species, are increasingly necessary, 
especially for enabling an appropriate antifungal treatment to be applied. 
Moreover, there is a real need for reliable epidermiological markers for 
use in epidermiological studies or for tracing stains in the 
agri-foodstuffs or fermentation industries. 
One is forced to note, however, that the conventional methods of 
identifying yeasts are not entirely satisfactory in all these respects. 
Thus, known methods include especially those described by Barnett, J. W., 
R. W. Payne and D. Yarrow in: "Yeasts: characteristics and 
identification", 2nd edition, Cambridge University Press, Cambridge, 1991, 
which require prior isolation of the microorganism in pure culture and 
then a study of its morphological and especially physiological 
characteristics (more than 80 tests), these ions together taking from 10 
to 30 days for one identification. These methods are therefore tedious and 
particularly long. They are restricted to reference laboratories and 
cannot be used in normal practice. 
Although more rapid and more rational, the miniaturized and standardized 
methods of identifying yeasts of medical importance still include prior 
isolation and then identification of the microorganism by meals of 
physiological tests, which are performed for example by using API.RTM. 
strips. Such tests take about 5 to 7 days to achieve an identification. 
Furthermore, these techniques do not always enable the species Candida 
krusei to be identified unambiguously, there being possible or even 
frequent confusions with species such as Candida glabrata, Candida 
inconspicua, Candida lipolytica, Candida norvegensis, Candida rugosa and 
Candida valida. 
With the advances in molecular biology, a new technology utilizing the 
principle of DNA-DNA or DNA-RNA hybridization has been developed for 
perfecting rapid, sensitive and specific identification tests. The genetic 
material of the microorganisms present in the sample is detected directly 
with the aid of labeled DNA or RNA probes. Detection and identification 
can be carried out simultaneously without prior isolation. 
The rare methods described for identifying Candida krusei with the aid of 
nucleic acid probes have the disadvantage of being based on very small 
probes (30 to 35 nucleotides); these require the use of radioactive 
labeling, which is more sensitive than non-radioactive labeling but is 
also more restrictive. 
Another disadvantage of these probes associated with their small size is 
expressed in the context of their use in polymerization chain techniques 
(PCR). These consist in amplifying a given DNA sequence by multiplying it 
from a pair of double-stranded primers using a polymerase. Such 
amplifications can be employed for diagnostic purposes in order to 
facilitate detection. As these probes would be used as primers in PCR, the 
possible choices of primers would be limited to a small sequence of 30 to 
35 nucleotides. 
These probes are generally directed against target DNA sequences coding for 
the small subunits 5S or 18S of ribosomal RNA (ssu rRNA). These targets 
are conventionally chosen because the DNA sequences coding for the 
ribosomal RNAs are the first to have been determined. There is a problem 
associated with the choice of sequences coding for ssu rRNAs as targets. 
In fact, these sequences have a universal character, i.e. a large number 
of them are found in all microorganisms (yeasts, fungi, bacteria); they 
are said to be "hyperconserved". 
In addition, ssu rRNAs are present in large amounts in cells, the result of 
which is to increase the sensitivity of identification methods based on 
its detection. 
It follows that for these two reasons at least, they can be the cause of 
false positives in both PCR and detection. 
Moreover, none of the probes described in the prior art permits the typing 
of strains, i.e. differentiation between the individuals within the 
species, these individuals being assigned to only one group: the species 
Candida krusei. In fact, the previously described probes do not permit 
typing of the strains of C. krusei because they do not reveal sufficient 
polymorphism in the size of the restriction fragments among the strains of 
this species Candida krusei, so they do not constitute a reliable 
epidermiological marker. 
The following may be mentioned as illustrations of such known probes which 
imperfectly meet the technical needs existing in the art: 
the oligonucleotide probes developed by Gene-Trak Systems and directed 
against sequences of 30 nucleotides (probe 1351), 33 nucleotides (probe 
1537) and 35 nucleotides (probe 1530) of the DNA coding for 18S ribosomal 
RNA. These probes are described in European patent application no. 0 422 
869. They were tested against 4 strains of Candida krusei. It should be 
noted that the Examples in said patent application do not refer to the 
detection of Candida krusei by hybridization in specimens of human blood, 
sputum or cerebrospinal fluid. Furthermore, these probes do not make it 
possible to differentiate between strains within the species Candida 
krusei; 
the probe described by Niesters and coworkers (Niesters et al., 1993, 
Rapid, polymerase chain reaction-based identification assays for Candida 
species. J. Clin. Microbiol., 31, 904-910, 1993). This is a probe of 20 
nucleotides (oligonucleotide 705) which is directed against a specific 
sequence of the amplification product of the small subunit of ribosomal 
RNA (ssu rRNA). This study is utilized in a method of identifying the 
species of the genus Candida, said method being based on the chain 
amplification (PCR) of sequences of the gene coding for the ssu rRNA, 
followed by direct sequencing of these sequences. Once again, this 
methodology involves the ribosomal RNA. It has the disadvantage of 
requiring direct sequencing of the amplification product, a technique 
accessible to only a limited number of research laboratories and certainly 
not applicable to routine work. Furthermore, according to the authors, 
oligonucleotide 705 does not permit specific amplification of the DNA of 
Candida krusei, nor do the probes described by Niesters and coworkers make 
it possible to type strains of Candida krusei. 
BRIEF DESCRIPTION OF THE INVENTION 
In this state of the art, one of the essential objects of the present 
invention is to provide a perfectly specific probe for detecting yeasts of 
the species Candida krusei. 
A further object of the invention is to provide a probe, especially a 
nucleic acid probe, of sufficient size to allow the use of a marker other 
than a radioactive marker, which is therefore more convenient to use. 
A further object of the invention is that this nucleic acid probe be 
directed against target sequences of DNA other than that coding for small 
subunits 5S or 18S of ribosomal RNA, which are overall poorly 
representative of the species because they are very highly conserved in 
microorganisms. 
A further object of the invention is that this probe be able to be used in 
complex biological media of the type comprising human blood, sputum or 
cerebrospinal fluid. 
A further object of the invention is that the Candida krusei probe make it 
possible to go beyond simple detection and allow precise identification 
and typing of yeasts within the species Candida krusei. 
These and other objects are achieved by the present invention, which 
relates to a probe for the specific and/or infraspecific detection of 
yeasts of the species Candida krusei, characterized in that it is selected 
from the following genetic (or related) tools: 
at least part of: 
at least one DNA and/or RNA fragment F 
formed by at least one of the two fragments F.sub.1 and F.sub.2 derived 
from digestion of the total DNA of the strain LMCK 31 by the restriction 
enzyme EcoRI, 
with a size of between 7 and 4 kb, said fragment hybridizing specifically 
with DNA and/or RNA of Candida krusei 
and not coding for the synthesis of ribosomal RNA, 
and/or at least one analog Fa of this (or these) fragment(s) F resulting 
from the degeneracy of the genetic code, 
and/or at least one cDNA fragment Fc complementary with the fragment F, 
at least part of the transcription products of the abovementioned 
fragment(s) F and/or Fa and/or Fc, 
at least part of the translation products of the abovementioned fragment(s) 
F and/or Fa and/or Fc, 
and a combination of the abovementioned tools. 
DETAILED DESCRIPTION OF THE INVENTION: 
It is therefore to the credit of the Applicant on the one hand to have been 
able to isolate at least one nucleic acid fragment F with a substantial 
size greater than or equal to 4 kb as well as the MRNA and the proteins 
capable of resulting therefrom, and on the other hand to use these genetic 
or related tools for the specific identification of yeasts of the species 
Candida krusei, as well as for their infraspecific characterization. 
In a first stage, the research strategy developed by the Applicant 
initially consisted in selecting a particular strain of Candida krusei, 
namely the one called LMCK 31 hereafter. This strain originates from a 
bronchoalveolar specimen taken from a hospitalized patient suffering from 
a Candida krusei disease. 
The strain LMCK 31, which constitutes one of the subjects of the present 
invention, was deposited in the collection nationale de culture de 
microorganismes (CNCM) at the Institut Pasteur, IS, on Oct. 22, 1993 
under the reference I 1372. 
In a second stage, the Applicant isolated and characterized the probe, 
especially nucleic acid probe, according to the invention, which, in a 
preferred embodiment, consists of at least part of at least one of the two 
fragments F.sub.1 and F.sub.2 derived from digestion of the total DNA of 
the strain LMCK 31 by the restriction enzyme EcoRI. These fragments 
F.sub.1 and F.sub.2 are separated by agarose gel electrophoresis, 
extracted and then cloned by the conventional techniques of molecular 
biology. 
They were selected from the restriction profiles of the total DNA of LMCK 
31, among a multitude of other fragments, after much research and 
experimentation. 
The two fragments F.sub.1 and F.sub.2 are preferably present in the probe 
in the following relative proportions F.sub.1 /F.sub.2 : 99/1 to 1/99, 
preferably 80/20 to 20/80 and particularly preferably about 50:50 parts by 
weight. 
At least part of at least one of the two fragments F.sub.1 and F.sub.2, and 
preferably both of them in their entirety, are used as a probe according 
to the principle of DNA-DNA or DNA-RNA hybridization. This probe makes it 
possible to perform tests for the rapid, sensitive and specific detection 
and identification of Candida krusei and also constitutes an excellent 
marker for typing strains of this species, by virtue of its ability to 
reveal a polymorphism in the size of the restriction fragments of the 
total DNA of Candida krusei. This latter property constitutes one of 
several novel features of the present invention. 
It has to be considered that the hybridization of the nucleic acid probe of 
the invention with target nucleic acid sequences of C. krusei can be 
effected with the whole of the fragments F.sub.1 and F.sub.2 referred to 
above, but can also be effected with only a fraction of these fragments, 
the size of which preferably remains greater than or equal to 0.1 kb. 
According to one particularly advantageous modality of the invention, the 
probe, in its nucleic acid variant, is characterized in that each fragment 
F.sub.1 and F.sub.2 has a size of between 6.6 and 4.4 kb. Even more 
preferably, F.sub.1 and F.sub.2 have sizes of about 5.6 and 5.4 kb 
respectively. 
The nucleic acid probe according to the invention is labeled with the aid 
of labeling means capable of revealing hybridization. These labeling means 
may or may not be radioactive. Given that this probe has a relatively 
large size and that the quantity of markers fixed to the probe is 
proportional to its length, it is therefore possible to fix much more to 
the probe according to the invention (greater than or equal to 4 kb) than 
to probes made of smaller nucleotide sequences (less than or equal to 35 
bases). This makes it possible to mitigate the well-known fact that the 
sensitivity of non-radioactive labeling is lower than that of radioactive 
labeling. 
Now, non-radioactive markers are easy to manipulate and are accessible to 
all laboratories in the medical or agri-foodstuffs sector, in contrast to 
radioactive markers, the use of which is subject to strict and regulated 
conditions and is therefore strictly limited to authorized laboratories. 
.sup.32 P may be mentioned among the conventional radioactive markers. 
Examples of non-radioactive markers which may be mentioned are fixed 
enzymes such as peroxidase. 
Because of its large size, the probe according to the invention can 
advantageously be employed as a source of specific primer(s) for use in 
polymerization chain reactions (PCR). 
This (or these) primer(s) constitute a further subject of the invention. 
Another feature of these fragments F.sub.1 and F.sub.2 is that they only 
pair with the DNA and/or RNA of Candida krusei or its perfect form, namely 
Issatchenkia orientalis, and not with that of other yeasts of medical 
interest, filamentous fungi or bacteria. This specificity is further 
improved by the fact that these fragments F.sub.1 and F.sub.2 do not 
hybridize with a target DNA coding for 18S ribosomal RNA, in contrast to 
the known nucleic acid probes. 
According to another characteristic of the invention, at least one of the 
fragments F.sub.1 and F.sub.2 is capable of hybridizing with at least one 
of the fragments derived from digestion of the total DNA of the Candida 
krusei strain LMCK 31 by the restriction enzyme HinfI, the size of which 
is between 7.0 and 2.0 kb and preferably between 3.8 and 2.5 kb. 
In fact, the probe hybridizes especially with at least one of the four 
fragments derived from this digestion by HinfI, the respective sizes of 
which are as follows: about 3.4, 3.5, 3.1 and 2.9 kb. 
The sensitivity of this probe is perfectly suited to the detection and/or 
typing of Candida krusei by dot-blot or Southern blot hybridization. 
This sensitivity is verified in indirect hybridization assays (deposition, 
dot-blot or Southern blot method), but also in direct hybridization 
experiments in situ. The latter consist in making a filter blot of 
colonies in the growth phase on a solid culture medium and bringing the 
hybridization probe directly to the filter. 
Under these conditions, the nucleic acid probe according to the invention 
makes it possible to detect a colony of Candida krusei among more than 30 
colonies of different species after 12 to 24 hours of growth, without 
prior purification. 
The Candida krusei probe can be of a nucleic acid nature, i.e. it can 
consist of nucleic acids of the types comprising DNA, cDNA and mRNA 
derived from transcription of the DNA, or their variants and analogs. 
According to another aspect of the invention, however, the probe can also 
consist of the translation products of this or these DNAs and/or RNAs, 
these actually being the proteins which can be synthesized by reading the 
information coded by the mRNA. 
It is interesting to note that the probe according to the invention can be 
efficiently employed in any complex medium like the following biological 
media: blood, sputum and cerebrospinal fluid. This does not interfere in 
any way with its detection properties. 
The present invention further relates to a method for the specific 
detection of yeasts of the species Candida krusei, which consists in using 
at least one nucleic acid probe as described above. 
This method comes within the scope of the methodologies known in the field 
of the genetic detection and identification of microorganisms. 
In this method: 
the total genomic DNA is extracted from the strains to be studied, 
this total DNA is optionally subjected to enzymatic digestion by at least 
one restriction enzyme, 
the optionally digested, total DNA is denatured, 
the now denatured, total DNA is brought into contact with the probe, which 
itself has previously been denatured and provided with at least one 
marker, in order to effect hybridization, 
the DNA and the unhybridized probe are removed 
and the hybridization is revealed with the aid of the marker. 
This is a hybridization technique in which the target DNA of the 
microorganism to be studied is subjected to denaturation, with or without 
prior enzymatic digestion. The next step is that of recombining the 
separate stands of denatured DNA with the probe in order to re-form novel 
base pairings. 
During this recombination step, the single-stranded molecules of the probe 
and target are placed under conditions which are more or less favorable 
for pairing (more or less stringent). Under very stringent conditions, 
only the molecules whose sequences are complementary over a large number 
of bases hybridize to form a double-stranded molecule. The probe is then 
specific for the target. 
In the case of a probe labeled with a radioactive element such as .sup.32 P 
or with a grafted enzyme such as peroxidase, for example, the 
hybridization is easily revealed qualitatively and quantitatively. 
It is apparent from the above that the nucleic acid and/or protein probe 
according to the invention, and the method which applies them, are 
perfectly specific for Candida krusei and offer an excellent sensitivity, 
which gives them outlets of considerable interest in the applied fields of 
the identification and screening differentiation between strains of this 
species (medical diagnostics, industrial controls in foodstuffs, 
fermentation, etc.). Candida krusei is detected rapidly (24 to 48 hours) 
and unambiguously. These are yet further economic and industrial assets 
for the probe and method according to the invention. 
In addition to detection, which groups together the identification and 
typing of microorganisms of the Candida krusei type, the probe according 
to the invention could be applied in the context of a therapeutic strategy 
directed against Candida krusei diseases. More precisely, this means that 
the fragments forming the probe can be employed as a target for active 
principles against Candida species. This role of a target for drugs could 
be played by the DNA or its transcription products (MRNA) or translation 
products (proteins). 
The invention will be better understood and other advantages and practical 
variants thereof will become clearly apparent from the following 
non-limiting Examples, which describe, with reference to the attached 
drawings, the constitution of a probe according to the invention, its 
preparation and several C. krusei detection procedures which apply it.

EXAMPLES 
Example 1: Nucleic Acid Probe R.sub.1, F.sub.2 
In the present Example, the probe according to the invention consists of 
two fragments F.sub.1 and F.sub.2 derived from digestion of the total DNA 
of the strain LMCK 31 by EcoRI and having respective sizes of about 5.6 
and 5.4 kb. The fragments F.sub.1 and F.sub.2 were selected from the 
multitude of fragments produced by the abovementioned digestion. They were 
separated by agarose gel electrophoresis and then extracted and finally 
cloned by the conventional techniques of molecular biology. 
The base material which permitted this isolation, namely the Candida krusei 
strain is LMCK 31, was isolated from a bronchoalveolar specimen taken from 
a hospitalized patient. This strain was deposited on Oct. 22, 1993 in the 
CNCM at the Institut Pasteur, IS, under the reference I 1372. 
Example 2: Hybridization, By The Deposition Method, of The Probe F.sub.1, 
F.sub.2 With Strains of Various Species of Yeasts 
Hybridization analysis by the deposition method, in accordance with the 
procedures well known to those skilled in the art, requires immobilizing a 
previously denatured nucleic acid or population of nucleic acids on a 
membrane or a filter, such as a positively charged nylon membrane, a 
nitrocellulose filter or another membrane specially designed for this 
purpose, which can easily be obtained commercially. The DNA or RNA can 
easily be immobilized on such membranes or filters and can then be probed 
or tested for hybridization under multiple stringency conditions with 
nucleotide sequences or probes in question. Under stringent conditions, 
the probes whose nucleotide sequences possess the greatest complementary 
with the target show a greater hybridization level than the probes whose 
sequences have fewer homologies (are less complementary). The probe 
F.sub.1, F.sub.2 of the present invention is tested by hybridization using 
the deposition method. Thus 6 to 1.5 .mu.g of target total DNA of the test 
strains listed in Table 1 below, purified by phenol extraction, are 
denatured (100.degree. C., 5 min) and deposited on a positively charged 
nylon membrane (Appligene, Illkirch, France) with a MilliBlot apparatus 
(Millipore Corporation, Bedford, Ma.). The membrane is prehybridized for 2 
hours at a temperature of 41.5.degree. C. in ECL buffer (40 ml), in the 
presence of 5% (w/v) of a blocker (Amersham, Les Ulis, France) and at an 
NaCl concentration of 0.42 M. A quantity of 600 ng of the probe F.sub.1, 
F.sub.2, consisting of equal parts of the two fragments of about 5.6 and 
5.4 kb derived from restriction of the total DNA of LMCK 31 by EcoRl, is 
labeled with peroxidase (ECL system) (Amersham, Les Ulis, France). The 
probe is added to the reaction medium and hybridization of the probe with 
the target DNA is carried out for 6 to 18 hours at a temperature of 
41.5.degree. C. in ECL buffer (40 ml), in the presence of 5% (w/v) of a 
blocker (Amersham, Les Ulis, France) and at an NaCl concentration of 0.42 
M. The unhybridized probe is removed by two successive washes for 15 min 
at 41.5.degree. C. with solution 1 (urea 6 M, sodium dodecylsulfate 0.4% 
(w/v), 20.times.SSC 2.5% (v/v)) over 5 to 20 min, followed by two 
successive washes at room temperature with solution 2 (2.times.SSC) over 5 
to 20 min. The membrane is then immersed for 1 min in a mixture of equal 
volumes of developing reagents 1 and 2 ECL system (Amersham, Les Ulis, 
France)!, after which it is covered with a Hyperfilm-ECL photographic film 
for 5 to 30 min, in accordance with the manufacturer's recommendations. A 
hybridization signal very much stronger than the background is observed 
only with the DNA of C. krusei or I. orientalis. A complementary assay was 
able to show that quantities of 15 .mu.g of target genomic DNA belonging 
to the different species of Candida krusei mentioned in Table 1 do not 
make it possible to obtain a hybridization signal stronger than that of 
the control: 1.5 .mu.g of C. krusei. 
Example 3: Hybridization, By The Southern Method, of The Probe F.sub.1, 
F.sub.2 With EcoRi or HinfI Restriction Fragments of Different Strains of 
Candida krusei and Different Strains of Various Species 
The Southern transfer method is an essential technique in molecular biology 
and comprises the following steps: 
the total (genomic) DNA of the strains to be studied is digested by a 
restriction enzyme and the resulting restriction fragments are separated 
according to their size by electrophoresis on an agarose gel (0.8% w/v); 
the separated DNA is denatured in the gel with sodium hydroxide solution 
and then neutralized in Tris buffer; 
the DNA is transferred from the gel to the filter or positively charged 
nylon membrane by aspiration under vacuum (vacugene system, Appligene), in 
a high salt concentration; 
the DNA transferred in this way is fixed to the filter by baking at 
80.degree. C. for 20 min; 
the filter to which the DNA is fixed is subsequently prehybridized in a 
special buffer which saturates the non-specific binding sites with a 
carrier DNA or synthetic polymers, and is then hybridized in the 
hybridization buffer containing the denatured probe, which can be 
radioactively labeled (hot labeling) or non-radioactively labeled (cold 
labeling), for example by the fixing of an enzyme such as peroxidase. 
The hybridization temperature and conditions are determined so as to allow 
adequate probe-target hybridization. 
Following hybridization, the residual probe which is not specifically 
hybridized and fixed to the filter is removed by a series of washes, which 
do not detach the probe fixed specifically to the target. 
The probe F.sub.1, F.sub.2 of the present invention is tested by 
hybridization using the Southern method. 25 to 5 .mu.g of target total 
DNA, purified by phenol extraction, are digested by a restriction enzyme 
under the conditions recommended by the manufacturer (Appligene). The 
restriction fragments are separated by electrophoresis in 0.8% agarose gel 
in Tris-Borate-EDTA (TBE) buffer 1.times., at 2 V/cm, for 18 h. 
After electrophoresis, the separated fragments are transferred to a 
positively charged nylon membrane (Appligene) by the conventional 
techniques of alkaline transfer. After fixing of the DNA by baking of the 
transfer membrane at 80.degree. C. for 20 min, said membrane serves as a 
support for the hybridizations. 
The membrane is incubated for 2 h at 41.5.degree. C. in 40 ml of a 
prehybridization solution consisting of ECL buffer (Amersham) containing 
5% (w/v) of a blocker (Amersham) and 0.42 M NaCl. The incubations take 
place in a tube in an agitating hybridization oven rotating at 20 to 50 
rpm. 
After prehybridization, 600 ng of the probe CK1,2, labeled with peroxidase 
using the ECL system (Amersham, Les Ulis, France) according to the 
manufacturer's recommendations, are added to the reaction medium. 
Hybridization is carried out for 18 h at 41.5.degree. C. with agitation at 
between 20 and 50 rpm. The unhybridized fraction of the probe is 
subsequently removed by two successive washes with solution 1 urea 6 M, 
sodium dodecylsulfate 0.4% (w/v), 20.times.SSC 2.5% (v/v)! over 5 to 20 
min, followed by two successive washes at room temperature with solution 2 
(2.times.SSC) over 5 to 20 min. The membrane is then immersed for 1 min in 
a mixture of equal volumes of developing reagents 1 and 2 ECI, system 
(Amersham, Les Ulis, France)!, drained, wrapped in "saran-wrap" and then 
covered with a Hyperfilm-ECL photographic film for 5 to 30 min, according 
to the manufacturer's recommendations. 
The following were used in the assays corresponding to the hybridization 
profile of FIG. 1: 
a reference standard for the number of nucleotides obtained by HindIII 
restriction of the total DNA of phage lambda; 
a first assay in which the target is formed by the EcoRI restriction 
fragments of the total DNA of DNA of Candida krusei LMCK 31; 
a second assay in which the target is formed by the EcoRI restriction 
fragments of the total DNA of HinfI. 
In FIG. 1, the solid lines c nd to the fragments hybridizing with F.sub.1, 
F.sub.2. The broken line are the major restriction fragments which are 
readily observable directly on the restriction profiles. The sizes of the 
fragments are expressed in kilobases. 
HinfI restriction fragments of the total DNA of the following different 
strains of Candida krusei were used in the assays corresponding to the 
hybridization profiles of FIG. 2: LMCK 31, K26, K63, K62, K64, K66, K48, 
K43, K41, K55, K10, K16, K22 and K44. The strains K62, K63 and K64 
originate from the same patient. The same applies to the K41, K43 and K48. 
Each band represents a fragment hybridizing with F.sub.1, F.sub.2. 
This shows that it is possible to type the different strains within the 
species Candida krusei using one of the probes according to the invention, 
since the latter clearly reveals the polymorphism in the size of the 
restriction fragments of the DNA of these strains of Candida krusei. 
The microorganisms in question in the assays corresponding to FIGS. 3a and 
3b are as follows: 
S: Standard: phage lambda digested by HindIII 
1: Candida tropical is 1058; 2: C. parapsliosis CBS 604T; 3: C. 
guillermondii CBS 6021T; 4: C. lusitaniae H 278; 5: C. kefyr; 6: C. 
albicans serotype .beta.; 7: C. albicans serotype A; 8: C. albicans ATCC 
2091; 9: C. valida CBS 638T; 10: C. krusei CBS 573T; 11: Yarrowia 
lipolytica CBS 6124T (cf. legends to Table 1 for CBS, ATCC). 
In these assays, the fragments of total DNA (6 to 8 .mu.g), digested by 
EcoRI, of the abovementioned microorganisms are separated by 
electrophoresis, photographed (FIG. 3a) and then transferred to a nylon 
membrane by the Southern method under vacuum and hybridized with the probe 
F.sub.1, F.sub.2 under the conditions described above. FIG. 3b shows the 
hybridization profile obtained. 
Example 4: Direct Hybridization of The Colonies 
For the rapid screening of clinical or other specimens, it may be useful to 
perform hybridizations directly on the colonies of yeasts obtained after 
plating of the specimen on an appropriate culture medium and incubation, 
for example on Sabouraud's medium incubated at 28.degree. C. for 24 h. It 
is thus possible to identify a colony of C. krusei among several colonies 
with the aid of specific probes. 
The probe F.sub.1, F.sub.2 is tested under the following conditions: The 
strains of different species and of C. krusei are inoculated punctually on 
the surface of Sabouraud's medium in a Petri dish by the conventional 
methods of microbiology. Fifteen to thirty strains are studied 
simultaneously. After 24 h of incubation at between 30 and 35.degree. C., 
a blot of the colonies is made by applying a disk of positively charged 
nylon membrane (positive membrane, Appligene) to the surface of the 
culture medium in contact with the colonies, under a uniform weight (about 
10 to 50 grams). When the disk is completely moist, it was removed from 
the surface and turned over so that the surface which has taken up the 
blot of the colonies is facing upwards. The disk is then placed on the 
surface of a sheet of Whatman paper (Whatman 3MM) saturated with 0.5 M 
NaOH (sodium hydroxide). 
The membrane is then rinsed twice by immersion in 400 ml of 5.times.SSC 
with vigorous agitation in order to remove the cellular debris fixed to 
the surface of the membrane. The disk is then partially dried on Whatman 
3MM paper with its top side facing upwards. The disk is then used for the 
hybridizations under conditions identical to those described above. The 
disk of membrane is hybridized in 40 ml of ECL buffer, 5% of blocker and 
0.42 M NaCl, with 400 ng of the probe F.sub.1, F.sub.2 which has first 
been denatured and labeled with peroxidase using the ECL system according 
to the manufacturer's recommendations. After 18 h of hybridization and 
successive rinses, the only hybridization signals are recorded at the 
locations of the C. krusei and I. orientalis colonies on the blot made on 
the membrane. 
The probe F.sub.1, F.sub.2 can also be used under other hybridization 
conditions by the conventional methods of molecular biology which are well 
known to those skilled in the art. 
TABLE 1 
______________________________________ 
LIST OF THE STRAINS STUDIED BY HYBRIDIZATIONS WITH 
THE PROBE F.sub.1, F.sub.2 
STRAIN REFERENCE RESULTS 
______________________________________ 
Candida albicans ATCC 2091 - 
Candida albicans serotype A 
Labo Myco LYON 
- 
Candida albicans serotype B 
Labo Myco LYON 
- 
Candida boidinii 34 F 1 - 
Candida famata Labo Myco LYON 
- 
Candida glabrata Labo Myco LYON 
- 
Candida guillermondii 
CBS 6021T - 
Candida humicola CBS 2839T - 
Candida inconspicua 
Labo Myco LYON 
- 
Candida kefyr CBS 607T - 
Candida krusei CBS 573T + 
Candida krusei (57 strains) 
Labo Myco LYON 
+ 
Candida lambica CBS 1876T - 
Candida lusitaniae Labo Myco LYON 
- 
Candida norvegensis 
Labo Myco LYON 
- 
Candida parakrusei Labo Myco Lyon 
- 
Candida parapsilosis 
CBS 604T - 
Candida rugosa SIPHV 823 - 
Candida tropicalis CBS 617T - 
Candida valida (2) CBS 638T - 
Candida zeylanoides 
IPP 207 - 
Cryptococcus neoformans 
Labo Myco LYON 
- 
Geotrichum candidum 
Labo Myco LYON 
- 
Issatchenkia orientalis 
CBS 5147T + 
Kluyveromyces bulgaricus 
CBS 2762T - 
Kluyveromyces dobzhanskii 
CBS 2104T - 
Kluyveromyces drosophilarum 
Labo Myco LYON 
- 
Kluyveromyces fragilis 
CBS 297T - 
Kluyveromyces lactis 
CBS 683T - 
Kluyveromyces marxianus 
CBS 712T - 
Kluyveromyces vanudenii 
CBS 4372T - 
Kluyveromyces wickerhamii 
Phaff - 
Rhodotorula glutinis 
CBS 20T - 
Rhodotorula rubra CBS 17T - 
Saccharomyces cerevisiae 
CBS 11711T - 
Trichosporon cutaneum 
IPP 654 - 
Yarrowia lipolytica 
CBS 6124T - 
Zygosaccharomyces rouxii 
Labo Myco LYON 
- 
Aspergillus flavus Labo Myco LYON 
- 
Nocardia asteroides 
IPP 1750-88 - 
Rhodococcus equi Labo Myco LYON 
- 
Staphylococcus aureus 
Labo Myco LYON 
- 
Escherichia coli Labo Myco LYON 
- 
______________________________________ 
CBS = Centraal Bureau Voor Schimmelculture, BAARNS, THE NETHERLANDS, 
ATCC = American Type Culture Collection, ROCKVILLE, USA, 
IPP = Institut Pasteur, IS, FRANCE, 
Labo Myco LYON = Laboratoire de Mycologie, Faculte de Pharmacie, 
Universite Claude Bernard LYON 1, 8, avenue Rockefeller, 69373 LYON, 
FRANCE.