WO2018060451A1

WO2018060451A1 - Primers and probes for diagnosis of dermatophyte infection

Info

Publication number: WO2018060451A1
Application number: PCT/EP2017/074822
Authority: WO
Inventors: Philipp BOSSHARD; Michael Walser
Original assignee: Universität Zürich
Priority date: 2016-09-30
Filing date: 2017-09-29
Publication date: 2018-04-05

Abstract

The invention relates to a method of detection and species identification of dermatophytes in a patient sample. The method comprises a PCR step using a pair of consensus primers; a probe annealing step, in which a plurality of oligonucleotide probes is brought in contact with the product of the PCR step and a detectable fluorescent signal is obtained if the probes hybridize; a temperature ramping step determining the denaturation temperature of the probes from the PCR product; and an evaluation step in which the denaturation temperatures are used to identify the dermatophyte species present in the sample. The invention further relates to a method of detection of non-dermatophyte nail-infection causing fungal species.

Description

Primers and Probes for Diagnosis of Dermatophyte Infection Background

Dermatophytes are fungi that colonize keratin-containing tissues of animals and humans, causing infections of the skin, hair and nails.

More than 80 dermatophyte species exist. In clinical samples, infections with Trichophyton (T.) rubrum, T. interdigitale, T. violaceum, T. anamorph of Arthroderma benhamiae, T. tonsurans, T.verrucosum, T. rubrum African population, Microsporum (M.) canis, M. gypseum, M. audouinii and Epidermophyton (E.) floccosum are most common.

Conventional diagnostics of dermatophyte infection comprise identification of the dermatophyte species via culture methods taking up to four weeks.

"Molecular diagnostics" of dermatophyte infection on the other hand refer to direct pathogen detection in a clinical sample, without the need for time-consuming culture methods. PCR- based methods usually rely on the use of probes that can detect single dermatophyte species (one species per probe).

Fungal nail infections can be caused by dermatophyte species, but also by other fungal species like Aspergillus or Candida. Apart from the detection and identification of dermatophyte species, the detection of other nail infection-causing species is thus important for a reliable diagnosis.

The problem underlying the present invention is to provide a fast and reliable method for the detection and species identification of dermatophytes and for the detection of nail-infection causing non-dermatophyte species. The problem is solved by the subject-matter of the independent claims.

Description

According to a first aspect, the invention provides a method for detecting the presence of any fungus comprised in list A in a sample, if said fungus is present in said sample. The method comprises the following steps:

a. A PCR step, wherein a PCR reaction is performed on said sample using a first pair of consensus primers. These primers are capable of amplifying a nucleic acid specific for any one of the fungal species of list A and therefore can be termed "consensus" primers with regard to these species. The PCR step is conducted in the presence of a thermostable polymerase and a buffer solution comprising the necessary cofactors and substrates required for a polymerase chain reaction as known in the art. The PCR step comprises repetitions of 3 steps: a denaturation step at a denaturation temperature (usually 94 °C), a primer annealing step at an annealing temperature, and an elongation step at an elongation temperature. The denaturation temperature is 94-89 °C. The elongation temperature is the optimum temperature of the thermostable polymerase used, usually 72 °C. The annealing temperature relates to a temperature at which the oligonucleotide primers used in the PCR step specifically anneal to the template to be amplified.

b. A probe annealing step, wherein a first plurality of oligonucleotide probes is brought in contact with the product of the PCR step. The first plurality of oligonucleotide probes comprises three different probes: a probe A, a probe B, and a probe C capable of forming a hybrid with SEQ I D NO 120 or its reverse complementary sequence under stringent hybridization conditions.

A detectable fluorescent signal is obtained if one of the oligonucleotide probes hybridizes to a PCR product obtained in the PCR step, as any of the probe molecules comprise a fluorescent dye on one end of the probe and a quencher molecule on the other end of the probe, and the probe is designed to form a stem-loop structure if the bulk of the sequence is not engaged in intermolecular hybridization. This set-up is known in the art as a "molecular beacon".

c. A temperature ramping step determining a temperature of denaturation for each oligonucleotide probe comprised in the first plurality of oligonucleotide probes from the PCR product.

d. An evaluation step in which the temperature of denaturation is used to assign to the sample a likelihood of presence or absence of the fungus.

SEQ ID NO 120 corresponds to nucleotides 650 to 767 of SEQ ID NO 19, which is the Trichophyton rubrum sequence with accession number AF170472.

This region fulfils two important preconditions:

1 ) The sequence is similar between all dermatophyte species, so that probes designed to hybridize to this region will (imperfectly) hybridize to all dermatophyte species.

2) The sequence comprises some differences between the dermatophyte species, resulting in different strengths of hybridization between a given probe and the target sequences of different dermatophyte species.

In certain embodiments, probe A, probe B, and probe C are capable of forming a hybrid with the sequence characterized by nucleotides 660 to 750 of SEQ ID NO 19, or the reverse complementary sequence thereof, under stringent hybridization conditions. In certain embodiments, probe A, probe B, and probe C are capable of forming a hybrid with the sequence characterized by nucleotides 660 to 750 of SEQ ID NO 19 under stringent hybridization conditions.

In certain embodiments, probe A, probe B, and probe C are capable of forming a hybrid with the sequence characterized by nucleotides 665 to 740 of SEQ ID NO 19, or the reverse complementary sequence thereof, under stringent hybridization conditions.

In certain embodiments, probe A, probe B, and probe C are capable of forming a hybrid with the sequence characterized by nucleotides 665 to 740 of SEQ ID NO 19 under stringent hybridization conditions.

In certain embodiments, probe A, probe B, and probe C are capable of forming a hybrid with the sequence characterized by nucleotides 672 to 728 of SEQ ID NO 19, or the reverse complementary sequence thereof, under stringent hybridization conditions.

In certain embodiments, probe A, probe B, and probe C are capable of forming a hybrid with the sequence characterized by nucleotides 672 to 728 of SEQ ID NO 19 under stringent hybridization conditions.

Within the context of the present specification, the expression "capable of forming a hybrid under stringent hybridization conditions" relates to sequences that are able to bind selectively to their target sequence. Typical temperature ranges and salt concentrations for assessing stringent hybridization of probes of different length and compositions are known to the person skilled in the art and are defined in "Molecular Cloning: a laboratory manual (J. Sambrook et al., 1988, 2nd edition, Cold Spring Harbor Laboratory, CSH, New York). The hybridizing sequences may be contiguously reverse-complementary to the target sequence, or may comprise gaps, mismatches or additional non-matching nucleotides. The minimal length for a sequence to be capable of forming a hybrid depends on its composition, with C or G nucleotides contributing more to the energy of binding than A or T nucleotides, and the backbone chemistry. The oligonucleotide probes according to the invention have a length of 25-47 nucleotides. The oligonucleotide probes according to the invention are able to bind selectively to their target sequence at temperatures of between 37°C and 45-75°C. Even more specifically, the oligonucleotide probes according to the invention are able to bind selectively to their target sequence at a temperature of 37°C to 45°C under the conditions specified in the methods section of this application, particularly at a salt concentration of 50 mM KCI, 15 mM TrisHCI and 2.5 mM MgCI₂. At increasing temperatures (45°C - 99°C), the oligonucleotide probes according to the invention will detach from their target sequence, depending on their degree of reverse complementarity with the target sequence. Probes which bind to their target sequence with no or few mismatches will detach at higher melting temperatures than probes which bind to their target sequence with more mismatches. In certain embodiments, the first plurality of oligonucleotide probes comprises

- a probe A selected from probe A^* (SEQ ID NO 01 ), probe A3 (SEQ ID NO 66), probe A2 (SEQ ID NO 67) and probe A1 (SEQ ID NO 68), and

- a probe B selected from probe B^* (SEQ ID NO 02), probe B1 (SEQ ID NO 69), probe B2 (SEQ ID NO 70), probe B6 (SEQ ID NO 71 ), probe B7 (SEQ ID NO 72), probe B8

(SEQ ID NO 73), probe B3 (SEQ ID NO 74), probe B4 (SEQ ID NO 75) and probe B5 (SEQ ID NO 76), and

- a probe C selected from probe C^* (SEQ ID NO 03), probe C^** (SEQ ID NO 15), probe C^**** (SEQ ID NO 162), (SEQ ID NO 121 ), probe C1 (SEQ ID NO 77), probe C2 (SEQ ID NO 78), probe C3 (SEQ ID NO 79), probe C4 (SEQ ID NO 80), probe C5

(SEQ ID NO 81 ) and probe C6 (SEQ ID NO 82).

Within the context of the present specification, the expression "detecting the presence of a fungus selected from list A" refers to obtaining the information whether a specific fungus selected from list A is present in the sample, and which one it is. It does not refer to obtaining the information whether any fungus selected from list A (without knowing which one) is present in the sample.

The first plurality of oligonucleotide probes enables the detection of any fungus comprised in list A.

Epidermophyton floccosum, Microsporum (M.) audouinii, M. canis, M. fulvum, M. gypseum (A. gypseum), M. gypseum (A. incurvatum), M. persicolor, M. praecox, M. audouinii, M. canis, Trichophyton (T.) equinum, T. erinacei, T. mentagrophytes, T. rubrum, T. terrestre (A. quadrifidium), T. tonsurans, T. verrucosum and T. eboreum can be unambiguously identified using the first plurality of oligonucleotide probes.

Using only the first plurality of oligonucleotide probes, it is not possible to distinguish between the T. interdigitale type I, T. interdigitale type II, T. interdigitale type III, T. interdigitale type IV, T. interdigitale type IV-like, and between the species T. rubrum african population, T. violaceum (Fig. 3). These species can be distinguished using probes D and E, which are comprised in the second plurality of oligonucleotide probes.

The skilled person is aware that the first pair of consensus primers is capable of annealing to the 5' and 3' position of the PCR product and the first plurality of oligonucleotide probes is capable of annealing to a position of the same PCR product, in between of the positions where the primers anneal.

The first pair of consensus primers used in the PCR reaction is dermatophyte-specific. It enables amplification of the internal transcribed spacer 2 (ITS2) sequence (and part of the 25S region) present on the ribosomal RNA operon of dermatophytes (Fig. 2). The sample comprises DNA extracted from a clinical nail, hair or skin sample. If a fungus is present in the clinical sample, the extracted DNA comprises fungal DNA.

The oligonucleotide probes are so-called sloppy molecular beacons (SMBs), comprising a fluorophore and a fluorophore quencher at the 5'-end and 3'-end, respectively (Fig. 1 A). SMBs form a hairpin structure, since their termini are complementary. The fluorophore and the fluorophore quencher are thus in proximity, leading to quenching of the fluorescent signal emitted by the fluorophore. If a target DNA is present, the probe region of the SMB anneals to the complementary target DNA.

Importantly, SMBs are able to bind to their complementary target sequence even if some mismatches between the nucleotide sequences are present. Binding of the SMB to the target DNA leads to separation of fluorophore and fluorophore quencher and thus to a detectable fluorescent signal. At a certain temperature (denaturation temperature) the probe separates from the target DNA, resulting in formation of the hairpin structure and quenching of the fluorescent signal.

In the context of the present specification the expression "denaturation temperature" or melting temperature" of a probe refers to the temperature at which a probe separates from its target DNA. At temperatures lower than the denaturation temperature, probe and target form a hybrid. At temperatures higher than the denaturation temperature, probe and target are separated.

The amount of mismatches between SMB and target sequence determines the strength of the hybridization and the denaturation temperature. Less mismatches result in a higher denaturation temperature (Fig. 1 B, C). The information on the denaturation temperature of the probes comprised in the first plurality of probes enables the detection and/or identification of all relevant dermatophyte species.

The first plurality of probes further enables the detection of all dermatophyte species comprised in list A and the species identification of all dermatophyte species comprised in list A apart from T. violaceum and T. rubrum African population, which cannot be distinguished by the first plurality of probes, and T. interdigitale type l/l I, T. interdigitale type IV-like and T. interdigitale type IV, which cannot be distinguished by the first plurality of probes (Fig. 3). In order to achieve this, the probes comprised in the first plurality of probes were carefully selected to enable a robust and clear-cut result for each of the dermatophyte species of list A. Such a result can be obtained if the combination of the denaturation temperatures of each probe is different for each species. The length of the probes, their secondary structure, the number of mismatches and the length of the complementary probe termini were taken into account and adjusted when possible. All oligonucleotide probes comprised in the first plurality of oligonucleotide probes anneal to the ITS2 region on the ribosomal RNA operon of dermatophytes, particularly to the region between about bp 583 and bp 768, more particularly to the region between about bp 650 and bp 767, more particularly to the region between about bp 672 and bp 728 of the Trichophyton rubrum sequence with accession number AF170472 (SEQ ID NO 19). The probes anneal to either of the two complementary strands. The region where the probes anneal was carefully selected to comprise a certain, defined degree of homology between the different dermatophyte species. The homology had to be high enough to enable the use of single probes able to bind to a PCR product obtained from a sample comprising DNA of any of the species of list A, and low enough to enable species differentiation via the different denaturation temperatures.

The skilled person is aware that any probe comprised in the first plurality of oligonucleotide probes can be replaced by an alternative probe comprising modifications that do not affect the denaturation temperature of the probes, i.e. modifications that do not change the temperature at which the probes separate from their target DNA(s).

Also within the scope of the present invention are alternative first pluralities of probes that anneal to the ITS2 region on the ribosomal RNA operon of dermatophytes, particularly to the region between about bp 583 and bp 768, more particularly to the region between about bp 650 and bp 767, more particularly between about bp 672 and bp 728 of the Trichophyton rubrum sequence with accession number AF170472 (SEQ ID NO 19) and enable differentiation between the dermatophyte species of list A. Differentiation is enabled if the combination of probes exhibits a combination of denaturation temperatures that is unique for each of the different dermatophyte species of list A.

In certain embodiments, probe A is selected from probe A^* (SEQ ID NO 01 ), probe A2 (SEQ ID NO 67) and probe A1 (SEQ ID NO 68).

In certain embodiments, probe A is selected from probe A^* (SEQ ID NO 01 ), probe A3 (SEQ ID NO 66), and probe A1 (SEQ ID NO 68).

In certain embodiments, probe A is selected from probe A^* (SEQ ID NO 01 ), probe A3 (SEQ ID NO 66) and probe A2 (SEQ ID NO 67).

In certain embodiments, probe B is selected from probe B^* (SEQ ID NO 02), probe B2 (SEQ ID NO 70), probe B6 (SEQ ID NO 71 ), probe B7 (SEQ ID NO 72), probe B8 (SEQ ID NO 73), probe B3 (SEQ ID NO 74), probe B4 (SEQ ID NO 75) and probe B5 (SEQ ID NO 76).

In certain embodiments, probe B is selected from probe B^* (SEQ ID NO 02), probe B1 (SEQ ID NO 69), probe B6 (SEQ ID NO 71 ), probe B7 (SEQ ID NO 72), probe B8 (SEQ ID NO 73), probe B3 (SEQ ID NO 74), probe B4 (SEQ ID NO 75) and probe B5 (SEQ ID NO 76). In certain embodiments, probe B is selected from probe B^* (SEQ ID NO 02), probe B1 (SEQ ID NO 69), probe B2 (SEQ ID NO 70), probe B7 (SEQ ID NO 72), probe B8 (SEQ ID NO 73), probe B3 (SEQ ID NO 74), probe B4 (SEQ ID NO 75) and probe B5 (SEQ ID NO 76).

In certain embodiments, probe B is selected from probe B^* (SEQ ID NO 02), probe B1 (SEQ ID NO 69), probe B2 (SEQ ID NO 70), probe B6 (SEQ ID NO 71 ), probe B8 (SEQ ID NO 73), probe B3 (SEQ ID NO 74), probe B4 (SEQ ID NO 75) and probe B5 (SEQ ID NO 76).

In certain embodiments, probe B is selected from probe B^* (SEQ ID NO 02), probe B1 (SEQ ID NO 69), probe B2 (SEQ ID NO 70), probe B6 (SEQ ID NO 71 ), probe B7 (SEQ ID NO 72), probe B3 (SEQ ID NO 74), probe B4 (SEQ ID NO 75) and probe B5 (SEQ ID NO 76).

In certain embodiments, probe B is selected from probe B^* (SEQ ID NO 02), probe B1 (SEQ ID NO 69), probe B2 (SEQ ID NO 70), probe B6 (SEQ ID NO 71 ), probe B7 (SEQ ID NO 72), probe B8 (SEQ ID NO 73), and probe B5 (SEQ ID NO 76).

In certain embodiments, probe B is selected from probe B^* (SEQ ID NO 02), probe B1 (SEQ ID NO 69), probe B2 (SEQ ID NO 70), probe B6 (SEQ ID NO 71 ), probe B7 (SEQ ID NO 72), probe B8 (SEQ ID NO 73), probe B3 (SEQ ID NO 74) and probe B4 (SEQ ID NO 75).

In certain embodiments, probe C is selected from probe C^* (SEQ ID NO 03), probe C^** (SEQ ID NO 15), probe C^**** (SEQ ID NO 162), probe C1 (SEQ ID NO 77) and probe C2 (SEQ ID NO 78).

In certain embodiments, probe C is selected from probe C^* (SEQ ID NO 03), probe C^** (SEQ ID NO 15), probe C^**** (SEQ ID NO 162), probe C3 (SEQ ID NO 79), probe C4 (SEQ ID NO 80), probe C5 (SEQ ID NO 81 ) and probe C6 (SEQ ID NO 82).

In certain embodiments, the first plurality of oligonucleotide probes comprises probe A^* (SEQ ID NO 01 ), probe B^* (SEQ ID NO 02) and a probe selected from probe C^* (SEQ ID NO 03), probe C^** (SEQ ID NO 15), probe C^*** (SEQ ID NO 16) and probe C^**** (SEQ ID NO 162). In certain embodiments, the first plurality of oligonucleotide probes comprises probe A^* (SEQ ID NO 01 ), probe B^* (SEQ ID NO 02) and a probe selected from probe C^* (SEQ ID NO 03), probe C^** (SEQ ID NO 15) and probe C^*** (SEQ ID NO 16).

In certain embodiments, the first plurality of oligonucleotide probes comprises probe A^* (SEQ ID NO 01 ), probe B^* (SEQ ID NO 02) and a probe selected from probe C^* (SEQ ID NO 03), probe C^** (SEQ ID NO 15) and probe C^**** (SEQ ID NO 162).

In certain embodiments, the first pair of consensus primers comprises

- a first forward primer sequence comprising or consisting of a primer sequence selected from DP1 -Fs (SEQ ID NO 63), DP1 -F (SEQ ID NO 08), DP1 -F_DX10.3 (SEQ ID NO 96), DP1 -F_DX10.4 (SEQ ID NO 97), DP1 -F_DX10.5 (SEQ ID NO 98), DP1 -F_DX10.6 (SEQ ID NO 99), DP1 -F_DX10.7 (SEQ ID NO 100) and DP1 - F_DX10.8 (SEQ ID NO 101 ), and

- a first reverse primer sequence comprising or consisting of a primer sequence selected from DP1 -R (SEQ ID NO 09), DP1 -R_DX3 (SEQ ID NO 102), DP1 -R_DX4 (SEQ ID NO 103), DP1 -R_DX5 (SEQ ID NO 104) and DP1 -R_DX6 (SEQ ID NO

105).

In certain embodiments, the first forward primer sequence comprises or consists of DP1 -Fs (SEQ ID NO 63).

In certain embodiments, the first forward primer sequence comprises or consists of DP1 -F (SEQ ID NO 08).

In certain embodiments, the first reverse primer sequence comprises or consists of DP1 -R (SEQ ID NO 09).

In certain embodiments, the first pair of primers comprises or consists of primer sequences DP1 -Fs (SEQ ID NO 63) and DP1 -R (SEQ ID NO 09).

In certain embodiments, the first pair of primers comprises or consists of primer sequences DP1 -F (SEQ ID NO 08) and DP1 -R (SEQ ID NO 09).

The skilled person is aware that a pair of primers comprises only two primers. The expression "pair of consensus primers comprises sequences DP1 -F (SEQ ID NO 08) and DP1 -R (SEQ ID NO 09)" relates to the fact that a primer may contain the specified sequence and a few (1 -5) additional nucleotides.

In certain embodiments, the method comprises the following characteristics:

a. The PCR product comprises a sense strand and an antisense strand hybridizing to one another. The first pair of consensus primers is designed to anneal to the PCR product at an annealing temperature T(p).

b. The PCR step is conducted in presence of an asymmetric primer. The asymmetric primer is characterized by a sequence comprised in the 5' region of the first forward primer or the first reverse primer, wherein the asymmetric primer sequence has a length of approx. half of the sequence of the primer that it is comprised in. The asymmetric primer is further characterized by an annealing temperature T(a) that is at least 5 K lower than T(p).

c. The PCR step comprises an exponential phase, wherein a first annealing temperature T(p) is employed, and an asymmetric phase subsequent to said exponential phase, wherein a second annealing temperature T(a) is employed. In certain embodiments, the asymmetric primer is characterized by a sequence comprised in the 5' region of the first forward primer.

In certain embodiments, the asymmetric primer is characterized by a sequence comprised in the sense strand in 5' of the position where the first plurality of oligonucleotide probes anneal.

In certain embodiments, the asymmetric primer is able to anneal to the antisense strand in 3' position of the part that hybridizes to the part of the sense strand in which the probes anneal.

In certain embodiments, the first plurality of oligonucleotide probes is designed to anneal to a position on the sense strand.

The PCR reaction comprises a plurality of thermal cycles each comprising an annealing step conducted at an annealing temperature, a denaturation step conducted at a denaturation temperature and an elongation step conducted at an elongation temperature. The exponential phase comprises 20-35, particularly approximately 30 thermal cycles and the asymmetric phase comprises 40-80, particularly 50-70, more particularly approximately 60 thermal cycles. The PCR reaction is performed in presence of a thermostable polymerase, a suitable reaction buffer, primers, deoxyribose nucleotide triphosphates (dNTPs) and magnesium chloride (MgCI₂).

The skilled person is aware that dNTPs containing deoxyuridine triphosphate (dUTP) can be used to prevent carry-over contamination from previous amplifications.

In certain embodiments, the asymmetric primer is designed to be comprised within the sequence of the forward primer of said pair of consensus primers, particularly on the 5' terminus of said forward primer of said pair of consensus primers.

In the context of the present specification, the term "forward primer" refers to a primer annealing to the antisense strand of a DNA template.

In certain embodiments, the asymmetric primer is primer PX (SEQ ID NO 14).

In certain embodiments, the temperature ramping step comprises the following steps:(1 ) initial denaturation, (2) probe annealing and (3) stepwise increase of temperature with concomitant acquisition of fluorescent signal.

In certain embodiments, the steps a-d of the method are repeated or conducted in parallel using a second pair of consensus primers instead of the first pair of consensus primers and a second plurality of oligonucleotide probes instead of the first plurality of oligonucleotide probes. This enables the detection of any fungus selected from list A and/or list B if said fungus is present in the sample. In other words, in certain embodiments the method enables the detection of any fungus selected from list A and/or list B in a sample, if said fungus is present in the sample. In such embodiments, the method comprises steps e-f which are conducted after or in parallel to steps a -d:

e. a second PCR step, wherein a PCR reaction is performed on the sample using a second pair of consensus primers;

f. a second probe annealing step, wherein a second plurality of oligonucleotide probes is brought in contact with the product of the second PCR step; and a detectable fluorescent signal is obtained if an oligonucleotide probe comprised in the second plurality of oligonucleotide probes hybridizes to a PCR product obtained in the second PCR step;

g. a second temperature ramping step determining a temperature of denaturation for each oligonucleotide probe comprised in the second plurality of oligonucleotide probes from the PCR product obtained in the second PCR step; and

h. a second evaluation step, in which the temperature of denaturation determined for each oligonucleotide probe comprised in the first or the second plurality of oligonucleotide probes is used to assign to the sample a likelihood of presence or absence of the fungus selected from list A and/or list B in the sample .

The second pair of consensus primers is pan-fungal. It enables amplification of the internal transcribed spacer 1 (ITS1 ) sequence (and part of the 18S and 5.8S region) present on the ribosomal RNA operon of dermatophytes and other fungi.

The second plurality of oligonucleotide probes enables the detection of a fungus selected from list A or list B in a sample. The second plurality of oligonucleotide probes enables differentiation between a fungus from list A and a fungus from list B. In those instances where a fungus of list B is detected, the second plurality of oligonucleotide probes further enables assignment of said fungus of list B to a subgroup of fungi (see Fig. 3). In order to achieve this, the probes comprised in the second plurality of probes were carefully selected to enable a robust and clear-cut result for each fungus selected from list A or list B. Such a result can be obtained if the combination of the denaturation temperatures of each probe is different for each species. The length of the probes, their secondary structure, the number of mismatches and the length of the complementary probe termini were taken into account and adjusted when possible.

The use of the first and second plurality of oligonucleotide probes in the method according to the invention enables the detection of any fungus comprised in list A or list B. Epidermophyton floccosum, Microsporum (M.) audouinii, M. canis, M. fulvum, M. gypseum (A. gypseum), M. gypseum (A. incurvatum), M. persicolor, M. praecox, M. audouinii, M. canis, Trichophyton (T.) equinum, T. erinacei, T. mentagrophytes, T. rubrum, T. terrestre (A. quadrifidium), T. tonsurans, T. verrucosum, T. eboreum, T. interdigitale type I, T. interdigitale type II, T. interdigitale type III, T. interdigitale type IV, T. interdigitale type IV-like, T. rubrum african population, T. violaceum, Trichosporon spp., Chrysosporium ssp., Candida ssp. and Acremonium strictum, can be unambiguously identified.

In certain embodiments, the second plurality of oligonucleotide probes comprises

- a probe E selected from probe E^* (SEQ ID NO 05), probe E1 (SEQ ID NO 86), probe E2 (SEQ ID NO 87),

- a probe F selected from probe F^* (SEQ ID NO 06), probe F1 (SEQ ID NO 88), probe

F2 (SEQ ID NO 89), probe F3 (SEQ ID NO 90), probe F4 (SEQ ID NO 91 ), probe F7 (SEQ ID NO 92), probe F6 (SEQ ID NO 93) and probe F5 (SEQ ID NO 94), and

- a probe D selected from probe D^* (SEQ ID NO 04), probe D^** (SEQ ID NO 17), probe D^*** (SEQ ID NO 18), probe D1 (SEQ ID NO 83), probe D2 (SEQ ID NO 84) and probe D3 (SEQ ID NO 85).

All oligonucleotide probes comprised in the second plurality of oligonucleotide probes anneal to a region on the ribosomal RNA operon of fungi listed in list A comprising part of the 18S rRNA, the ITS1 and part of the 5.8S rRNA, particularly to the region between about bp 152 and bp 458 of the Trichophyton rubrum sequence with accession number AF170472 (SEQ ID NO 19).

Probe D and E are panfungal and able to anneal to a region on the ribosomal RNA operon of the species listed in list A and B.

Probes F is dermatophyte specific and able to anneal to a region on the ribosomal RNA operon of the species listed in list A.

The skilled person is aware that any probe comprised in the second plurality of oligonucleotide probes can be replaced by an alternative probe comprising modifications that do not affect the denaturation temperature of the probes, i.e. modifications that do not change the temperature at which the probes separate from their target DNA(s).

Also within the scope of the present invention are alternative second pluralities of probes that anneal to a region on the ribosomal RNA operon of fungi listed in list A comprising part of the 18S rRNA, the ITS1 and part of the 5.8S rRNA, particularly to the region between about bp 152 and bp 458 of the Trichophyton rubrum sequence with accession number AF170472 (SEQ ID NO 19), and enable differentiation between the different fungal species of list A and B. Differentiation is enabled if the combination of probes comprised in the first and second plurality of probes exhibits a combination of denaturation temperatures that is unique for each of the different fungal species of list A and B.

In combination with the first plurality of oligonucleotide probes, the second plurality of oligonucleotide probes enables the differentiation between the dermatophyte species T. violaceum and T. rubrum African population and the differentiation between zoophilic and anthropophilic T. interdigitale strains, in particular the differentiation between T. interdigitale type l/l I, T. interdigitale type IV-like and T. interdigitale type IV (Fig. 3). This differentiation is crucial for patient management. If an infection with zoophilic dermatophytes is detected, it is required that pets or farm animals are examined and/or treated, too. The inventive method thus enables the identification of 99% of the relevant dermatophyte species (list A). Importantly, the method can be conducted in less than 24 hours and does not rely on DNA sequencing.

In certain embodiments, probe D is selected from probe D^* (SEQ ID NO 04), probe D^** (SEQ ID NO 17), and probe D^*** (SEQ ID NO 18), probe D2 (SEQ ID NO 84) and probe D3 (SEQ ID NO 85).

In certain embodiments, probe D is selected from probe D^* (SEQ ID NO 04), probe D^** (SEQ ID NO 17), and probe D^*** (SEQ ID NO 18), probe D1 (SEQ ID NO 83), and probe D3 (SEQ ID NO 85).

In certain embodiments, probe D is selected from probe D^* (SEQ ID NO 04), probe D^** (SEQ ID NO 17), and probe D^*** (SEQ ID NO 18), probe D1 (SEQ ID NO 83) and probe D2 (SEQ ID NO 84).

In certain embodiments, probe E is selected from probe E^* (SEQ ID NO 05) and probe E2 (SEQ ID NO 87).

In certain embodiments, probe E is selected from probe E^* (SEQ ID NO 05) and probe E1 (SEQ ID NO 86).

In certain embodiments, probe F is selected from probe F^* (SEQ ID NO 06), probe F1 (SEQ ID NO 88), probe F4 (SEQ ID NO 91 ), probe F7 (SEQ ID NO 92), probe F6 (SEQ ID NO 93) and probe F5 (SEQ ID NO 94).

In certain embodiments, probe F is selected from probe F^* (SEQ ID NO 06), probe F1 (SEQ ID NO 88), probe F2 (SEQ I D NO 89), probe F3 (SEQ ID NO 90) and probe F7 (SEQ ID NO 92).

In certain embodiments, probe F is selected from probe F^* (SEQ ID NO 06), probe F1 (SEQ ID NO 88), probe F2 (SEQ ID NO 89), probe F3 (SEQ ID NO 90), probe F4 (SEQ ID NO 91 ), probe F6 (SEQ ID NO 93) and probe F5 (SEQ ID NO 94). In certain embodiments, the second plurality of oligonucleotide probes comprises probe E^* (SEQ ID NO 05), probe F^* (SEQ ID NO 06) and a probe selected from probe D^* (SEQ ID NO 04), probe D^** (SEQ ID NO 17), and probe D^*** (SEQ ID NO 18).

In certain embodiments, the second pair of consensus primers comprises

- a second forward primer sequence comprising or consisting of a primer sequence selected from DP2-Fs (SEQ ID NO 64), DP2-F (SEQ ID NO 10), DP2-F_DX1 (SEQ ID NO 106), DP2-F_DX9 (SEQ ID NO 107), DP2-F_DX15 (SEQ ID NO 108) and DP2-F_DX18 (SEQ ID NO 109), and

- a second reverse primer sequence comprising or consisting of a primer sequence selected from DP2-R (SEQ ID NO 1 1 ), DP2-R_DX2 (SEQ ID NO 1 10), DP2-R_DX14

(SEQ ID NO 1 1 1 ), DP2-R_DX19 (SEQ ID NO 1 12), DP2-R_DX21 (SEQ ID NO 1 13) and DP2-R_DX22 (SEQ ID NO 1 14).

In certain embodiments, the second pair of consensus primers comprises or consists of primer sequences DP2-Fs (SEQ ID NO 64) and DP2-R (SEQ ID NO 1 1 ).

In certain embodiments, the second pair of consensus primers comprises or consists of primer sequences DP2-F (SEQ ID NO 10) and DP2-R (SEQ ID NO 1 1 ).

In certain embodiments, the second PCR step is conducted in presence of an asymmetric primer characterized by a sequence comprised in the 5' region of the second forward primer or the second reverse primer and an annealing temperature T(a) at least 5 K lower than T(p).

In certain embodiments, the asymmetric primer is selected from primer PX (SEQ ID NO 14), primer PX_D7 (SEQ ID NO 160) and primer PX_D8 (SEQ ID NO 161 ).

In certain embodiments, the asymmetric primer is primer PX (SEQ ID NO 14).

In certain embodiments, the method further includes an inhibition control comprising the following steps:

a. a control PCR step, wherein a control template DNA sequence is added to said sample and a pair of control primers able to hybridize to said control template sequence is used;

b. a control probe annealing step, wherein a control oligonucleotide probe is brought in contact with the product of said control PCR step, and a detectable fluorescent signal is obtained if the control oligonucleotide probe hybridizes to a PCR product obtained in said control PCR.

The presence of a detectable fluorescent signal implies that no inhibitory molecules are present in the sample, and the results obtained for the sample using the first and the second plurality of oligonucleotide probes can be trusted. In certain embodiments,

- the pair of control primers comprises

- a control forward primer sequence comprising or consisting of a primer sequence selected from IC-Fs (SEQ ID NO 65), IC-F (SEQ ID NO 12), IC-FJC3 (SEQ ID NO 1 15), IC-FJC5 (SEQ ID NO 1 16), IC-FJC6 (SEQ ID NO 1 17) and IC-FJC7

(SEQ ID NO 1 18), and

- a control reverse primer sequence comprising or consisting of a primer sequence selected from IC-R (SEQ ID NO 13) and IC-RJC8 (SEQ ID NO 1 19);

- the control oligonucleotide probe is selected from probe IC^* (SEQ ID NO 07) and probe IC1 (SEQ ID NO 95); and

- the control template DNA sequence is a plasmid to which said SEQ ID NO 12, SEQ ID NO 13 and SEQ ID NO 07 hybridize under conditions of the control PCR step or the control probe annealing step, respectively.

In certain embodiments, the control template DNA sequence is a plasmid to which primers comprising or consisting of SEQ ID NO 12 or SEQ ID NO 13 hybridize under the conditions of the control PCR step. In certain embodiments, the control template DNA sequence is a plasmid to which a probe comprising SEQ ID NO 07 hybridizes under the conditions of the control probe annealing step.

In certain embodiments, the control template DNA sequence is plasmid IC.

In certain embodiments, the pair of control primers comprises

- a control forward primer sequence comprising or consisting of a primer sequence selected from IC-Fs (SEQ ID NO 65), IC-F (SEQ ID NO 12), IC-FJC3 (SEQ ID NO 1 15), IC-FJC5 (SEQ ID NO 1 16), IC-FJC6 (SEQ ID NO 1 17) and IC-FJC7 (SEQ ID NO 1 18), and

- a control reverse primer sequence comprising or consisting of a primer sequence selected from IC-R (SEQ ID NO 13) and IC-RJC8 (SEQ ID NO 1 19).

In certain embodiments, the pair of control primers comprises or consists of primer sequences IC-Fs (SEQ ID NO 65) and IC-R (SEQ ID NO 13).

In certain embodiments, the pair of control primers comprises or consists of primer sequences IC-F (SEQ ID NO 12) and IC-R (SEQ ID NO 13).

In certain embodiments, the control oligonucleotide probe is selected from probe IC^* (SEQ ID NO 07) and probe IC1 (SEQ ID NO 95). In certain embodiments, the control oligonucleotide probe is probe IC^* (SEQ ID NO 07). In certain embodiments, the control template DNA sequence is plasmid IC, said pair of control primers comprises or consists of primer sequences IC-Fs (SEQ ID NO 65) or IC-F (SEQ ID NO 12) and IC-R (SEQ ID NO 13) and said control oligonucleotide probe is probe IC^* (SEQ ID NO 07).

Within the context of the present specification, plasmid IC refers to plasmid pUC57 available from GenScript (Piscataway, NJ, USA).

In certain embodiments, the control PCR step is conducted in presence of an asymmetric primer characterized by a sequence comprised in the 5' region of the control forward primer or the control reverse primer and an annealing temperature T(a) at least 5 K lower than T(p).

In certain embodiments, the asymmetric primer is primer PX (SEQ ID NO 14).

In certain embodiments, the oligonucleotide probe and/or the control oligonucleotide probe comprises a 5'-modification and a 3'-modification, wherein one modification comprises a fluorophore and the other modification comprises a fluorophore quencher.

In certain embodiments, the concentration of the oligonucleotide probe and/or the control oligonucleotide probe is approximately 150 nM.

According to a second aspect of the invention, a collection of primers is provided. The collection of primers comprises or consists of

- a first forward primer sequence comprising or consisting of a primer sequence selected from DP1 -Fs (SEQ ID NO 63), DP1 -F (SEQ ID NO 08), DP1 -F_DX10.3 (SEQ ID NO 96), DP1 -F_DX10.4 (SEQ ID NO 97), DP1 -F_DX10.5 (SEQ ID NO 98), DP1 -F_DX10.6 (SEQ ID NO 99), DP1 -F_DX10.7 (SEQ ID NO 100) and DP1 - F_DX10.8 (SEQ ID NO 101 ),

- a first reverse primer sequence comprising or consisting of a primer sequence selected from DP1 -R_DX3 (SEQ ID NO 102), DP1 -R_DX4 (SEQ ID NO 103), DP1 - R_DX5 (SEQ ID NO 104) and DP1 -R_DX6 (SEQ ID NO 105),

- a second forward primer sequence comprising or consisting of a primer sequence selected from DP2-Fs (SEQ ID NO 64), DP2-F (SEQ ID NO 10), DP2-F_DX1 (SEQ ID NO 106), DP2-F_DX9 (SEQ ID NO 107), DP2-F_DX15 (SEQ ID NO 108) and

DP2-F_DX18 (SEQ ID NO 109),

- a second reverse primer sequence comprising or consisting of a primer sequence selected from DP2-R (SEQ ID NO 1 1 ), DP2-R_DX2 (SEQ ID NO 1 10), DP2-R_DX14 (SEQ ID NO 1 1 1 ), DP2-R_DX19 (SEQ ID NO 1 12), DP2-R_DX21 (SEQ ID NO 1 13) and DP2-R_DX22 (SEQ I D NO 1 14).

In certain embodiments, the collection of primers further comprises - a control forward primer selected from the group of primers comprising or consisting of IC-Fs (SEQ ID NO 65), IC-F (SEQ ID NO 12), IC-FJC3 (SEQ ID NO 1 15), IC- FJC5 (SEQ ID NO 1 16), IC-FJC6 (SEQ ID NO 1 17) and IC-FJC7 (SEQ ID NO 1 18), and

- a control reverse primer selected from the group of primers comprising or consisting of IC-R (SEQ ID NO 13) and IC-RJC8 (SEQ ID NO 1 19).

In certain embodiments, the collection of primers further comprises an asymmetric primer. In certain embodiments, the asymmetric primer is selected from primer PX (SEQ ID NO 14), primer PX_D7 (SEQ ID NO 160) and primer PX_D8 (SEQ ID NO 161 ). In certain embodiments, the asymmetric primer is primer PX (SEQ ID NO 14).

In certain embodiments, the collection of primers comprises primers selected from primer DP1 -Fs (SEQ ID NO 63), primer DP1 -R (SEQ ID NO 09), primer DP2-Fs (SEQ ID NO 64), primer DP2-R (SEQ ID NO 1 1 ), primer IC-F (SEQ ID NO 12), primer IC-R (SEQ ID NO 13) and primer PX (SEQ ID NO 14).

In certain embodiments, the collection of primers comprises primers selected from primer DP1 -F (SEQ ID NO 08), primer DP1 -R (SEQ ID NO 09), primer DP2-F (SEQ ID NO 10), primer DP2-R (SEQ ID NO 1 1 ), primer IC-F (SEQ ID NO 12), primer IC-R (SEQ ID NO 13) and primer PX (SEQ ID NO 14).

According to a third aspect of the invention, a collection of oligonucleotide probes is provided. The collection of oligonucleotide probes comprises

- a probe A selected from probe A^* (SEQ ID NO 01 ), probe A3 (SEQ ID NO 66), probe A2 (SEQ ID NO 67) and probe A1 (SEQ ID NO 68),

- a probe B selected from probe B^* (SEQ ID NO 02), probe B1 (SEQ ID NO 69), probe B2 (SEQ ID NO 70), probe B6 (SEQ ID NO 71 ), probe B7 (SEQ ID NO 72), probe B8 (SEQ ID NO 73), probe B3 (SEQ ID NO 74), probe B4 (SEQ ID NO 75) and probe B5

(SEQ ID NO 76),

- a probe C selected from probe C^* (SEQ ID NO 03), probe C^** (SEQ ID NO 15), probe C^*** (SEQ ID NO 16), probe C^**** (SEQ ID NO 162), probe C1 (SEQ ID NO 77), probe C2 (SEQ ID NO 78), probe C3 (SEQ ID NO 79), probe C4 (SEQ ID NO 80), probe C5 (SEQ ID NO 81 ) and probe C6 (SEQ ID NO 82),

- a probe D selected from probe D^* (SEQ ID NO 04), probe D^** (SEQ ID NO 17), probe D^*** (SEQ ID NO 18), ), probe D1 (SEQ ID NO 83), probe D2 (SEQ ID NO 84) and probe D3 (SEQ ID NO 85),

- a probe E selected from probe E^* (SEQ ID NO 05), probe E1 (SEQ ID NO 86) and probe E2 (SEQ ID NO 87), and - a probe F selected from probe F^* (SEQ ID NO 06) probe F1 (SEQ ID NO 88), probe F2 (SEQ ID NO 89), probe F3 (SEQ ID NO 90), probe F4 (SEQ I D NO 91 ), probe F7 (SEQ ID NO 92), probe F6 (SEQ ID NO 93) and probe F5 (SEQ ID NO 94).

In certain embodiments, the collection of oligonucleotide probes further comprises a control probe selected from probe IC^* (SEQ ID NO 07) and probe IC1 (SEQ ID NO 95).

In certain embodiments, the collection of oligonucleotide probes comprises probes selected from probe A^* (SEQ ID NO 01 ), probe B^* (SEQ ID NO 02), probe C^** (SEQ ID NO 03), probe C^** (SEQ ID NO 15), probe C^*** (SEQ ID NO 16), probe D^* (SEQ ID NO 04), probe D^** (SEQ ID NO 17), probe D^*** (SEQ ID NO 18), probe E^* (SEQ ID NO 09), probe F^* (SEQ ID NO 06) and probe IC^* (SEQ ID NO 07).

In certain embodiments of this aspect of the invention, the corresponding pairs of fluorophore / fluorophore quencher are selected from: carboxyfluorescein (FAM) / black hole quencher 1 (BHQ1 ), YYE/BHQ1 , ROX (Thermo Fisher catalogue no. 12223012) / black hole quencher 2 (BHQ2), Cy5 (cyanine 5; Sigma) /BHQ2.

Table III lists the probes used by the inventors including their 5' and 3' modifications.

According to yet another aspect of the invention, a kit for detecting the presence of DNA of a fungus selected from list A and/or list B in a sample is provided. The kit comprises a collection of primers according to the second aspect of the invention and a collection of oligonucleotide probes according to the third aspect of the invention.

List A: Epidermophyton floccosum, Microsporum (M.) audouinii, M. canis, M. fulvum, M. gypseum (A. gypseum), M. gypseum (A. incurvatum), M. persicolor, M. praecox, M. audouinii, M. canis, Trichophyton (T.) equinum, T. erinacei, T. interdigitale type I, T. interdigitale type II, T. interdigitale type III, T. interdigitale type IV, T. interdigitale type IV-like,

T. mentagrophytes, T. rubrum, T. rubrum african population, T. terrestre (A. quadrifidium), T. tonsurans, T. verrucosum, T. violaceum, T. eboreum.

List B: Trichosporon spp., Chrysosporium ssp., Candida ssp., Acremonium strictum, Aspergillus (Asp.) flavus, Asp. versicolor, Asp. terreus, Scytalidium (Sc.) dimidiatum, Sc. hyalinum, Fusarium oxysporum, Geomyces pannorum, Paecilomyces lilacinus, Asp. fumigatus, Scopulariopsis brevicaulis, Paecilomyces variotii, Fusarium solani.

Wherever alternatives for single separable features are laid out herein as "embodiments", it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein. The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.

Brief description of the figures

Fig. 1 depicts the functional principle of the use of sloppy molecular beacons (SMBs) for the identification of dermatophyte species in a patient sample. A) Operation of molecular beacons. When the probe region in the loop of a molecular beacon hybridizes with a target sequence, a conformational reorganization occurs that restores the fluorescence of a quenched fluorophore. Sequences: probe A^* (SEQ ID NO 01 ), target sequences M. canis (SEQ ID NO 20), T. interdigitale (SEQ ID NO 21 ), T. rubrum (SEQ ID NO 22) B) Thermal denaturation profiles of molecular beacons in the presence of M. canis target sequence, T. interdigitale target sequence, or T. rubrum target sequence. C) Normalized derivatives of the fluorescence intensity of the hybrids formed by the binding of the resulting amplicons to a SMB probe are plotted as a function of temperature.

Fig. 2 shows primers and probes used for dermatophyte detection and identification. A) Position of the primers and probes on the fungal ribosomal DNA. B) Multi-alignments of probes A^*, B^*, C^*, D^*, E^* and F^* with dermatophyte ribosomal DNA sequences and other fungal ribosomal DNA sequences. "Small molecular beacon" A B/C/D/E/F corresponds to probe A7B7C7D7E7F^*. C) Overview of primers and probes.

Fig. 3 shows the identification key that allows identification of dermatophyte species on the basis of the denaturation temperature of probes A^*, B^*, C^*, D^*, E^* and F^*.

Fig. 4 shows an exemplary way of labelling of PCR tube strips (A) and inserting the strips into the LightCycler® 96 Real-Time PCR system (B).

Methods

Reagents

AmpliTaq Gold® DNA Polymerase with Buffer II and MgCI2 : Life Technologies (N8080241 ) GeneAmp® dNTP Blend with dUTP (12.5mM): Life Technologies (N8080270)

ΜοΙβομΙθΓ Beacons: Microsynth AG

Oligonukleotide: Microsynth AG

Samples: DNA extracts of patient specimens or fungal culture

Equipment

Roche LightCycler® 96 Real-Time PCR system

Roche LightCycler® 8-Tube Strips (clear) (06 327 672 001 )

1 .5 ml safelock reaction tubes (Eppendorf PCR clean 0030125.215) Laminar Flow Bench SKAN

Protocol

1 . Prepare DP-Mix 1 and DP-Mix 2 (can be stored frozen).

DP-Mix 1

2. Prepare PCR mastermixes (PCR 1 , PCR 2, PCR IC) Mastermix PCR 1

Mastermix PCR 2

Reagents Stock Concentration Volume per sample (μΙ) dH₂0 10.8

Reaction buffer II 10x 2

MgCI₂ 25 mM 2 dUTP 12.5 mM 2

DP-Mix 2 2

AmpliTaq Gold 0.2

Vtotal 19

Mastermix PCR IC

Reagents Stock Concentration Volume per sample (μΙ) dH₂0 9.8

Reaction buffer II 10x 2

MgCI₂ 25 mM 2 dUTP 12.5 mM 2

IC-Mix 2

AmpliTaq Gold 0.2

Plasmid IC dilution 10^"B 1

Vtotal 19

3. Load LightCycler 8-Tube Strips with 19 μ I of PCR mastermix per tube. Strips are labeled PCR 1 , PCR 2, PCR3 (= PCR IC). If more than 8 samples are analyzed, strips are additionally labeled A, B, C etc (Fig. 4A).

4. Add 1 μΙ of sample to mastermix. Short centrifugation.

5. Insert strips into LightCycler® 96 Real-Time PCR system as shown in Fig. 4B.

6. Run PCR Program„Panfungal PCR"

7. Analyse data using LightCycler 96 Software (Tm calling Analysis); select Melting Peaks with„Area Marker" to determine denaturation temperature.

8. Identify dermatophyte species on the basis of the denaturation temperature (compare with identification key, Fig. 3). Table I. Tested dermatoph te species

51 T. rubrum z1008 930 67.24 63.5 58.93 63.69 62.1

52 T. rubrum Z1008 660 67.21 63.61 59.01 63.71 62.13

55 T. rubrum Z1008 183 67.26 63.49 58.87 63.77 62.02

59 T. rubrum african population 141 1 735 71.21 73.07 68.82 63.74 62.15

61 T. terrestre (A. quadrifidium) Z0906 291 - - 49.97 - -

62 T. terrestre (A. quadrifidium) Z1004 1096 56.73 48.73 49.29 - -

64 T. tonsurans Z1 101 432 65.18 57.87 67.46 - -

65 T. tonsurans Z1010 1001 64.96 57.95 67.24 61.28 59.54

66 T. verrucosum bM 132 73.01 72.23 63.05 63.53 60.58

67 T. verrucosum 1404 154 72.82 72.06 63.26 63.57 60.49

68 T. verrucosum 1005 388 72.95 72.17 63.07 63.97 60.77

70 T. violaceum Z1008 219 71.61 73.34 68.2 63.8 58.84

71 T. violaceum 1503 767 71.36 73.25 68.46 63.76 58.8

72 T. violaceum Z1001 853-3 71.03 72.9 68.64 63.72 58.76

73 artben tax. Entity 3 1506 334 71.6 72.83 64.66 - -

74 T. eboreum 1 1 1 1 932 56.71 50 50.22 - -

75 T. rubrum african population 1506 420 71.68 73.39 69.31 63.71 62.12

80 T. tonsurans 1412 121 65.41 58.69 67.51 61.29 59.58

81 T. tonsurans 1410 152 65.33 58.81 67.41 61.29 59.4

83 T. terrestre (A. quadrifidium) 1005 446 56.93 49.14 49.53 - -

85 T. rubrum 1506 34 67.4 63.79 - 63.78 62.03

87 T. rubrum 1408 898 67.25 63.64 59.04 63.82 62.08

88 T. rubrum 1406 975 67.29 63.68 59.05 63.95 62.37

89 T. rubrum 1402 93 67.34 63.73 59.12 63.79 62.2

91 artben am-eur race 1410 159 71.77 72.49 64.92 63.52 52.33

92 M. audouinii 1409 909 59.87 63.75 58.48 48.42 -

93 M. canis 1407 57 63.81 63.43 - 48.72 -

94 T. interdigitale type II 1409 370 65.07 63.96 67.2 61.04 59.66

95 T. interdigitale type 1 1401 1002 64.87 63.77 67.1 1 61.1 59.89

60* T. rubrum african population 1309 212 71.56 73.3 68.82 63.77 62.02

76* T. rubrum african population 1209 991 71.47 73.54 69.06 63.72 62.13

77* T. violaceum 1502 294 71.08 72.78 68.72 63.73 58.77

78* T. violaceum 1501 374 71.09 72.79 68.73 63.78 58.82

79* T. violaceum 1412 240 71.13 73 68.76 63.79 58.67

96* T. interdigitale type IV (Graser) Z83 65.59 64.67 67.67 68.9 59.83

97* T. interdigitale type IV (Graser) CBS255 65.34 64.41 67.67 68.99 59.67

98* T. interdigitale type IV (Graser) Z304 65.3 64.12 67.39 68.97 59.94

^*dilution of sample 1 :100

Avarage denaturation temperature of probes A^*, B^*, C^*, D^* and E^* is shown.

Table II. Tested non-dermatophyte species (probe F*)

# species ID Y555 min Y555 max

15 Acremonium strictum-1 1 105 1 1 1 62.09 63.57

5* Aspergillus flavus 1009 190 65.02 65.02

24* Aspergillus fumigatus LM2 68.02 68.02

4* Aspergillus terreus 1 102 37 65.29 65.29

31* Aspergillus versicolor 1507 1 170 65.04 65.04

6* Aspergillus versicolor 1408 471 65.26 65.26

32* Candida albicans 1508 0145 63.15 64.25

33* Candida albicans 1508 0146 63 63.68

28* Candida guillermondi LM12 62.05 62.05

10 Candida parapsilosis 0905 475 62 63.54

34* Candida parapsilosis 1507 0975 62.85 64.25

9** Candida tropicalis 0906 318 63.5 63.5

7* Chrysoporium spp 1402 556 61.56 61.56

20** Fusarium oxysporum 1008 891 65 66.05

30*** Fusarium oxysporum LM67 65.93 66.45 19** Geomyces pannorum 1 101 777 65.96 66.1 1

17 Paecilomyces lilacinus 1008 715 65.12 65.12

2* Paecilomyces variotii 1 103 121 68.73 68.73

26* Scytalidium dimidiatum LM76 64.8 64.8

21 Scytalidium hyalinum 1405 253 65.41 65.41

27* Trichsporum mucoides LM7 58.83 58.83

^*dilution of sample 1 :200, ^**1 :10, ^***1 :100

Minimum and maximum denaturation temperature of probe F^* are shown.

Table III. Sloppy Molecular Beacons (SMBs)

All beacons bear a quencher in 3': BHQ1 (SEQ ID NO 1 , 2, 4, 6) or BHQ2 (SEQ ID NO 3, 5, 7) and a 5' dye FAM (SEQ ID NO 1 , 4), YYE (SEQ ID NO 2, 6), ROX (SEQ ID NO 3, 5), Cy5 (SEQ ID NO 7).

Examples

Proof of principle

33 clinical samples were analyzed. Among the samples were 23 nail samples, 8 dander samples, and 2 hair samples obtained from patients of the dermatological clinic, university hospital Zurich. DNA was extracted using biorobot EZ1 and EZ1 DNA Tissue Kit (Qiagen).

PCR was performed as described above. Results from microscopy (gold standard), fungal culture and PCR (new detection method according to the invention) were compared.

Detection of dermatophytes was counted as positive; detection of yeast and mold was counted as negative.

Results

Using microscopy as detection method, 24 samples were positive and 9 were negative.

9 of the 24 samples that were evaluated positive in microscopic analysis were also evaluated positive using fungal culture (sensitivity 37.5%). One sample was positive only in the fungal culture, but not microscopically.

18 of the 24 samples that were evaluated positive in microscopic analysis were also evaluated positive using the new PCR (sensitivity 75%). Two more samples were evaluated borderline positive. If these two are counted as positive, the sensitivity of the new detection method would amount to 83%.

Altogether, 18 of 33 results (55%) were identical in microscopy and culture (Table IV a and b), while 27 of 33 results (82%) were identical in microscopy and PCR. In some instances, yeast or mold infections with unclear relevance were detected using culture or PCR.

Discussion

The results demonstrate that the new dermatophyte PCR is a superior detection method compared to culture. Sensitivity of the PCR is 75-83%, while sensitivity of the culture is only 37.5%. Consistency between microscopy and PCR is much higher than consistency between microscopy and culture. The inventors demonstrated that the new detection method according to the invention is suitable for dermatophyte detection in clinical samples. The new method is faster than the culture method and has a higher sensitivity, thus it is superior to the culture method. Additional studies have to be performed to find out whether microscopy, the current gold standard, can be replaced by the new dermatophyte PCR.

Table IVa. Proof of principle with clinical samples

Table IVb. Proof of principle with clinical samples

Claims

1 . A method for detecting the presence of any fungus comprised in list A in a sample, said method comprising the following steps:

a. a PCR step, wherein a PCR reaction is performed on said sample using a first pair of primers;

b. a probe annealing step, wherein a first plurality of oligonucleotide probes is brought in contact with a product of said PCR step,

wherein said first plurality of oligonucleotide probes comprises a probe A, a probe B, and a probe C, wherein said probe A, said probe B and said probe C are capable of forming a hybrid with SEQ ID NO 120 or its reverse complementary sequence under stringent hybridization conditions;

and wherein a detectable fluorescent signal is obtained if one of said oligonucleotide probes hybridizes to said PCR product;

c. a temperature ramping step whereby a temperature of denaturation is determined for each oligonucleotide probe comprised in said first plurality of oligonucleotide probes from said PCR product; and

d. an evaluation step, in which said temperature of denaturation is used to assign to said sample a likelihood of presence or absence of said fungus in said sample.

2. The method according to claim 1 , wherein

- said probe A is selected from probe A^* (SEQ ID NO 01 ), probe A3 (SEQ ID NO 66), probe A2 (SEQ ID NO 67) and probe A1 (SEQ ID NO 68),

- said probe B is selected from probe B^* (SEQ ID NO 02), probe B1 (SEQ ID NO 69), probe B2 (SEQ ID NO 70), probe B6 (SEQ ID NO 71 ), probe B7 (SEQ ID NO 72), probe B8 (SEQ ID NO 73), probe B3 (SEQ ID NO 74), probe B4 (SEQ ID NO 75) and probe B5 (SEQ ID NO 76), and

- said probe C is selected from probe C^* (SEQ ID NO 03), probe C^** (SEQ ID NO 15), probe C^**** (SEQ ID NO 162), probe C1 (SEQ ID NO 77), probe C2 (SEQ ID NO 78), probe C3 (SEQ ID NO 79), probe C4 (SEQ ID NO 80), probe C5 (SEQ ID NO 81 ) and probe C6 (SEQ ID NO 82).

3. The method according to claim 1 , wherein said first plurality of oligonucleotide probes comprises probe A^* (SEQ ID NO 01 ), probe B^* (SEQ ID NO 02) and a probe selected from probe C^* (SEQ ID NO 03), probe C^** (SEQ ID NO 15) and probe C^*** (SEQ ID NO 16).

4. The method according to any one of the above claims, wherein said first pair of primers comprises

- a first forward primer sequence comprising or consisting of a primer sequence selected from DP1 -Fs (SEQ ID NO 63), DP1 -F (SEQ ID NO 08), DP1 - F_DX10.3 (SEQ ID NO 96), DP1 -F_DX10.4 (SEQ ID NO 97), DP1 -F_DX10.5 (SEQ ID NO 98), DP1 -F_DX10.6 (SEQ ID NO 99), DP1 -F_DX10.7 (SEQ ID NO 100) and DP1 -F_DX10.8 (SEQ ID NO 101 ), and

- a first reverse primer sequence comprising or consisting of a primer sequence selected from DP1 -R (SEQ ID NO 09), DP1 -R_DX3 (SEQ ID NO 102), DP1 - R_DX4 (SEQ ID NO 103), DP1 -R_DX5 (SEQ ID NO 104) and DP1 -R_DX6 (SEQ ID NO 105).

5. The method according to any one of the above claims, wherein said first pair of primers comprises or consists of primer sequences DP1 -Fs (SEQ ID NO 63) or DP1 - F (SEQ ID NO 08) and DP1 -R (SEQ ID NO 09).

6. The method according to claim 4 or 5, wherein

a. said first pair of primers is designed to anneal to said PCR product at an annealing temperature T(p);

b. said PCR step is conducted in presence of an asymmetric primer characterized by

i. a sequence comprised in the 5' region of said first forward primer or said first reverse primer and

ii. an annealing temperature T(a) at least 5 K lower than T(p);

c. said PCR step comprises:

i. an exponential phase, wherein said PCR reaction employs a first annealing temperature T(p); and

ii. an asymmetric phase, wherein said PCR reaction employs a second annealing temperature T(a).

7. The method according to the preceding claim 6, wherein said asymmetric primer is designed to be comprised within the sequence of said first forward primer, particularly on the 5' terminus of said first forward primer.

8. The method according to claims 6 or 7, wherein said asymmetric primer is primer PX (SEQ ID NO 14).

9. The method according to any one of the above claims, wherein said temperature ramping step comprises the following steps: (1 ) initial denaturation, (2) probe annealing and (3) stepwise increase of temperature with concomitant acquisition of fluorescent signal.

10. The method according to any one of the above claims, wherein said method enables the detection of any fungus selected from list A and/or list B in a sample, comprising steps e-f which are conducted after or in parallel to steps a -d:

e. a second PCR step, wherein a PCR reaction is performed on said sample using a second pair of primers;

f. a second probe annealing step, wherein a second plurality of oligonucleotide probes is brought in contact with the product of said second PCR step; and a detectable fluorescent signal is obtained if an oligonucleotide probe comprised in said second plurality of oligonucleotide probes hybridizes to a PCR product obtained in said second PCR step;

g. a second temperature ramping step, whereby a temperature of denaturation is determined for each oligonucleotide probe comprised in said second plurality of oligonucleotide probes from said PCR product obtained in said second PCR step; and

h. a second evaluation step, in which said temperature of denaturation determined for each oligonucleotide probe comprised in said first or said second plurality of oligonucleotide probes is used to assign to said sample a likelihood of presence or absence of said fungus selected from list A and/or list B in said sample .

1 1 . The method according to claim 10, wherein said second plurality of oligonucleotide probes comprises

- a probe E selected from probe E^* (SEQ ID NO 05), probe E1 (SEQ ID NO 86) and probe E2 (SEQ ID NO 87),

- a probe F selected from probe F^* (SEQ ID NO 06), probe F1 (SEQ ID NO 88), probe F2 (SEQ ID NO 89), probe F3 (SEQ ID NO 90), probe F4 (SEQ ID NO 91 ), probe F7 (SEQ ID NO 92), probe F6 (SEQ ID NO 93) and probe F5 (SEQ ID NO 94), and - a probe D selected from probe D^* (SEQ ID NO 04), probe D^** (SEQ ID NO 17), and probe D^*** (SEQ ID NO 18), probe D1 (SEQ ID NO 83), probe D2 (SEQ ID NO 84) and probe D3 (SEQ ID NO 85).

12. The method according to claim 10, wherein said second plurality of oligonucleotide probes comprises probe E (SEQ ID NO 05), probe F (SEQ ID NO 06) and a probe selected from probe D^* (SEQ ID NO 04), probe D^** (SEQ ID NO 17) and probe D^*** (SEQ ID NO 18).

13. The method according to any one of claims 10 to 12, wherein said second pair of primers comprises

- a second reverse primer sequence comprising or consisting of a primer sequence selected from DP2-R (SEQ ID NO 1 1 ), DP2-R_DX2 (SEQ ID NO 1 10), DP2-R_DX14 (SEQ ID NO 1 1 1 ), DP2-R_DX19 (SEQ ID NO 1 12), DP2- R_DX21 (SEQ ID NO 1 13) and DP2-R_DX22 (SEQ ID NO 1 14).

14. The method according to any one of claims 10 to 12, wherein said second pair of primers comprises or consists of DP2-Fs (SEQ ID NO 64) or DP2-F (SEQ ID NO 10) and DP2-R (SEQ ID NO 1 1 ).

15. The method according to any one of the above claims, further including an inhibition control comprising the following steps:

a. a control PCR step, wherein a control template sequence is added to said sample and a pair of control primers able to hybridize to said control template sequence is used;

b. a control probe annealing step, wherein a control oligonucleotide probe is brought in contact with the product of said control PCR step, and a detectable fluorescent signal is obtained if said control oligonucleotide probe hybridizes to a PCR product obtained in said control PCR.

16. The method according to claim 14, wherein

- said pair of control primers comprises a control forward primer sequence comprising or consisting of a primer sequence selected from IC-Fs (SEQ ID NO 65), IC-F (SEQ ID NO 12), IC-FJC3 (SEQ ID NO 1 15), IC-FJC5 (SEQ ID NO 1 16), IC-FJC6 (SEQ ID NO 1 17) and IC-FJC7 (SEQ ID NO 1 18), and a control reverse primer sequence comprising or consisting of a primer sequence selected from IC-R (SEQ ID NO 13) and IC-RJC8 (SEQ ID NO 1 19),

- said control oligonucleotide probe is probe IC^* (SEQ ID NO 07) or probe IC1 (SEQ ID NO 95) and

- said control template sequence is a plasmid to which said SEQ ID NO 12, SEQ ID NO 13 and SEQ ID NO 07 hybridize under conditions of said control PCR step or said control probe annealing step, respectively.

17. The method according to any one of claims 14 to 16, wherein said oligonucleotide probe and/or said control oligonucleotide probe comprises a 5'-modification and a 3'- modification, wherein one modification comprises a fluorophore and the other modification comprises a fluorophore quencher.

18. A collection of primers comprising or consisting of

- a first forward primer sequence comprising or consisting of a primer sequence selected from DP1 -Fs (SEQ ID NO 63), DP1 -F (SEQ ID NO 08), DP1 - F_DX10.3 (SEQ ID NO 96), DP1 -F_DX10.4 (SEQ ID NO 97), DP1 -F_DX10.5 (SEQ ID NO 98), DP1 -F_DX10.6 (SEQ ID NO 99), DP1 -F_DX10.7 (SEQ ID NO 100) and DP1 -F_DX10.8 (SEQ ID NO 101 ),

- a first reverse primer sequence comprising or consisting of a primer sequence selected from DP1 -R_DX3 (SEQ ID NO 102), DP1 -R_DX4 (SEQ I D NO 103), DP1 -R_DX5 (SEQ ID NO 104) and DP1 -R_DX6 (SEQ ID NO 105),

- a second forward primer sequence comprising or consisting of a primer sequence selected from DP2-Fs (SEQ ID NO 64), DP2-F (SEQ ID NO 10), DP2-F_DX1 (SEQ ID NO 106), DP2-F_DX9 (SEQ ID NO 107), DP2-F_DX15 (SEQ ID NO 108) and DP2-F_DX18 (SEQ ID NO 109),

19. A collection of oligonucleotide probes comprising

- a probe B selected from probe B^* (SEQ ID NO 02), probe B1 (SEQ ID NO 69), probe B2 (SEQ ID NO 70), probe B6 (SEQ ID NO 71 ), probe B7 (SEQ ID NO 72), probe B8 (SEQ ID NO 73), probe B3 (SEQ ID NO 74), probe B4 (SEQ ID NO 75) and probe B5 (SEQ ID NO 76),

- a probe E selected from probe E^* (SEQ ID NO 05), probe E1 (SEQ ID NO 86) and probe E2 (SEQ ID NO 87), and

- a probe F selected from probe F^* (SEQ ID NO 06) probe F1 (SEQ ID NO 88), probe F2 (SEQ ID NO 89), probe F3 (SEQ ID NO 90), probe F4 (SEQ ID NO 91 ), probe F7 (SEQ ID NO 92), probe F6 (SEQ ID NO 93) and probe F5 (SEQ ID NO 94).

20. The collection of oligonucleotide probes according to claim 19, further comprising a control probe selected from probe IC^* (SEQ ID NO 07) and probe IC1 (SEQ ID NO 95).

21 . A kit for detecting the presence of DNA of a fungus selected from list A and/or list B in a sample, comprising a collection of primers according to claim 18 and a collection of oligonucleotide probes according to any one of claims 19 to 20.