WO2024043830A1 - Methods for the detection and identification of fungal species - Google Patents
Methods for the detection and identification of fungal species Download PDFInfo
- Publication number
- WO2024043830A1 WO2024043830A1 PCT/SG2023/050556 SG2023050556W WO2024043830A1 WO 2024043830 A1 WO2024043830 A1 WO 2024043830A1 SG 2023050556 W SG2023050556 W SG 2023050556W WO 2024043830 A1 WO2024043830 A1 WO 2024043830A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- candida
- nucleotide sequence
- primer set
- seq
- probe
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 80
- 238000001514 detection method Methods 0.000 title claims abstract description 36
- 230000002538 fungal effect Effects 0.000 title abstract description 14
- 239000000523 sample Substances 0.000 claims abstract description 357
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 58
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 51
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 51
- 206010058872 Fungal sepsis Diseases 0.000 claims abstract description 38
- 238000003745 diagnosis Methods 0.000 claims abstract description 19
- 239000013615 primer Substances 0.000 claims description 461
- 239000002773 nucleotide Substances 0.000 claims description 343
- 125000003729 nucleotide group Chemical group 0.000 claims description 343
- 230000000295 complement effect Effects 0.000 claims description 123
- 241000222120 Candida <Saccharomycetales> Species 0.000 claims description 109
- 230000008685 targeting Effects 0.000 claims description 70
- 241000222122 Candida albicans Species 0.000 claims description 69
- 229940095731 candida albicans Drugs 0.000 claims description 66
- 241000222178 Candida tropicalis Species 0.000 claims description 60
- 241000235645 Pichia kudriavzevii Species 0.000 claims description 57
- 241000221204 Cryptococcus neoformans Species 0.000 claims description 52
- 201000007336 Cryptococcosis Diseases 0.000 claims description 51
- 241000645784 [Candida] auris Species 0.000 claims description 50
- 241000123365 Cryptococcus neoformans var. neoformans Species 0.000 claims description 49
- 241000222173 Candida parapsilosis Species 0.000 claims description 45
- 241000223218 Fusarium Species 0.000 claims description 45
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 claims description 44
- 241001522864 Cryptococcus gattii VGI Species 0.000 claims description 44
- 229940055022 candida parapsilosis Drugs 0.000 claims description 43
- 241000223230 Trichosporon Species 0.000 claims description 40
- 241000235048 Meyerozyma guilliermondii Species 0.000 claims description 39
- 241000222126 [Candida] glabrata Species 0.000 claims description 33
- 208000032343 candida glabrata infection Diseases 0.000 claims description 32
- 241000235347 Schizosaccharomyces pombe Species 0.000 claims description 27
- 230000003321 amplification Effects 0.000 claims description 26
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 26
- 108020004414 DNA Proteins 0.000 claims description 23
- 244000053095 fungal pathogen Species 0.000 claims description 20
- 101150052672 IGS1 gene Proteins 0.000 claims description 17
- 238000009396 hybridization Methods 0.000 claims description 17
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 14
- 108091093088 Amplicon Proteins 0.000 claims description 12
- 241001527609 Cryptococcus Species 0.000 claims description 10
- 108091034117 Oligonucleotide Proteins 0.000 claims description 10
- 102000018832 Cytochromes Human genes 0.000 claims description 8
- 108010052832 Cytochromes Proteins 0.000 claims description 8
- 101150076489 B gene Proteins 0.000 claims description 7
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical group C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 6
- 108091023242 Internal transcribed spacer Proteins 0.000 claims description 5
- 239000003155 DNA primer Substances 0.000 claims description 4
- 241000894007 species Species 0.000 description 33
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 8
- 230000009260 cross reactivity Effects 0.000 description 8
- 206010040047 Sepsis Diseases 0.000 description 7
- 244000052769 pathogen Species 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 6
- 230000003993 interaction Effects 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 206010017533 Fungal infection Diseases 0.000 description 5
- 208000031888 Mycoses Diseases 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 230000001717 pathogenic effect Effects 0.000 description 5
- 241000233866 Fungi Species 0.000 description 4
- 229940121375 antifungal agent Drugs 0.000 description 4
- 229960002685 biotin Drugs 0.000 description 4
- 235000020958 biotin Nutrition 0.000 description 4
- 239000011616 biotin Substances 0.000 description 4
- 230000008878 coupling Effects 0.000 description 4
- 238000010168 coupling process Methods 0.000 description 4
- 238000005859 coupling reaction Methods 0.000 description 4
- 210000002381 plasma Anatomy 0.000 description 4
- 239000002987 primer (paints) Substances 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 238000011895 specific detection Methods 0.000 description 4
- 238000011282 treatment Methods 0.000 description 4
- 239000003429 antifungal agent Substances 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 206010040070 Septic Shock Diseases 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 210000001124 body fluid Anatomy 0.000 description 2
- 239000010839 body fluid Substances 0.000 description 2
- 238000004590 computer program Methods 0.000 description 2
- 230000037029 cross reaction Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000002887 multiple sequence alignment Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 230000036303 septic shock Effects 0.000 description 2
- 125000006850 spacer group Chemical group 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 241000228212 Aspergillus Species 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- BQENDLAVTKRQMS-SBBGFIFASA-L Carbenoxolone sodium Chemical compound [Na+].[Na+].C([C@H]1C2=CC(=O)[C@H]34)[C@@](C)(C([O-])=O)CC[C@]1(C)CC[C@@]2(C)[C@]4(C)CC[C@@H]1[C@]3(C)CC[C@H](OC(=O)CCC([O-])=O)C1(C)C BQENDLAVTKRQMS-SBBGFIFASA-L 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 108010049047 Echinocandins Proteins 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 208000037026 Invasive Fungal Infections Diseases 0.000 description 1
- 102000006404 Mitochondrial Proteins Human genes 0.000 description 1
- 108010058682 Mitochondrial Proteins Proteins 0.000 description 1
- 241000142787 Pneumocystis jirovecii Species 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 108020001027 Ribosomal DNA Proteins 0.000 description 1
- 241000132889 Scedosporium Species 0.000 description 1
- 206010042938 Systemic candida Diseases 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000843 anti-fungal effect Effects 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000011203 antimicrobial therapy Methods 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 238000002869 basic local alignment search tool Methods 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000009640 blood culture Methods 0.000 description 1
- 208000037815 bloodstream infection Diseases 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 208000017773 candidemia Diseases 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 231100000676 disease causative agent Toxicity 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000009112 empiric therapy Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000005923 long-lasting effect Effects 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 150000003852 triazoles Chemical class 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Definitions
- the present invention is generally in the field of the detection and identification nucleic acids in a sample. More specifically, it relates to the detection and identification of fungal species in a sample by detecting and identifying genomic DNA of said fungal species, in particular of fungal species that are involved in fungal sepsis. The detection and identification of such fungal nucleic acids in the sample then allows the diagnosis of fungal sepsis in a subject from which said sample has been obtained.
- a further complication is that the current gold standard methods for the detection of fungal sepsis pathogens (blood culture and microbiology tests) and the following diagnosis take 3 to 7 days. It has been estimated that the mortality rate among septic shock patients increases by 7.6% for each hour appropriate antimicrobial treatment is delayed. In many cases of suspected sepsis, empirical treatment with broad-spectrum antibiotics is therefore immediately started without waiting for the result of pathogen identification. In some of these cases, inappropriate antibiotic selection does not only annul the effects of chemotherapy but also promotes the emergence of drug-resistant bacteria. This is highly undesired and has long-lasting consequences in that it promotes the occurrence of multi -resista nt bacteria and fungi strains.
- Candida species While invasive fungal infections are predominantly caused by Candida species, with candidemia being a highly common health care-associated bloodstream infection, increasingly immunocompromised patient populations have given rise to the emergence of fungal infections with other fungal species such as Aspergillus species, Pneumocystis jiroveci, Cryptococcus, Zygomycetes, Fusarium species, and Scedosporium species. At least 17 Candida species have been reported to cause severe infections in humans, but 5 species (C. albicans, C. giabrata, C. parapsilosis, C. tropicalis. and C. krusei) represent more than 90%. C. albicans has historically been the predominant pathogen, but to date other Candida species, such as C.
- the present invention meets this need by providing a method and kit that allow for the rapid detection and identification of a variety of fungal species in a sample simultaneously and thus allow avoiding the current laborious and time-consuming detection methods.
- the present invention is thus directed to a method for the detection of the presence and/or amount of two or more target nucleic acids in a sample, wherein the two or more target nucleic acids are of fungal pathogens involved in fungal sepsis, the method comprising: i) contacting the sample with a combination of two or more oligonucleotide amplification primer sets under conditions that allow hybridization of the oligonucleotide primers to the target nucleic acids of the fungal pathogens, wherein the two or more primer sets are directed to different target nucleic acids of at least two different fungal pathogen species involved in fungal sepsis and are independently selected from the group consisting of:
- Candida albicans primer set targeting a nucleotide sequence in the rDNA of Candida albicans
- Candida parapsilosis primer set targeting a nucleotide sequence in the rDNA of Candida parapsilosis
- Candida auris primer set targeting a nucleotide sequence in the rDNA of Candida auris
- a Candida spp. primer set targeting a nucleotide sequence in the rDNA of Candida spp/, and ii) amplifying the target nucleic acid(s) hybridized to any one or more of the oligonucleotide primers by PCR; and ill) detecting the presence and/or amount of the target nucleic acid(s) in the sample by contacting the PCR amplified sample of step ii) with combination of oligonucleotide detection probes comprising at least one probe specific for each of the amplicons produced by the amplification of the target nucleic acids in step ii) under conditions that allow the hybridization of the probes to their specific target nucleic acid sequences and determining the presence and/or amount of probe:target hybrids.
- the target region of rDNA of Candida albicans is of ITS2 region thereof;
- the target region of rDNA of Candida tropicalis is of ITS2 region thereof;
- the target region of rDNA of Candida glabrata is of ITS1 region thereof;
- the target region of rDNA of Candida parapsilosis is of ITS2 region thereof
- the target region of rDNA of Candida krusei is of ITS2 region thereof;
- the target region of rDNA of Candida guilliermondii is of ITS2 region thereof;
- the target region of rDNA of Candida auris is of ITS2 region thereof;
- the target region of rDNA of Cryptococcus neoformans is of IGS1 region thereof;
- the target region of rDNA of Cryptococcus deneoformans is of IGS1 region thereof;
- the target region of rDNA of Cryptococcus gatiii is of IGS1 region thereof;
- the target region of rDNA of Fusarium spp. is of 26S region thereof;
- the target region of rDNA of Tnchosporon spp. is of 18S region thereof;
- the target region of genomic DNA of Schizosaccharomyces pombe is of Cytochrome B gene region;
- the target region of rDNA of Candida spp. is of 18S region thereof.
- At least one primer of the Candida albicans primer set, the Candida tropicalis primer set, the Candida parapsilosis primer set, the Candida krusei primer set, the Candida guilliermondii primer set, and the Candida auris primer set comprises the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 1 over its entire length, or a complement thereof.
- At least one primer of the Candida albicans primer set comprises the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 2 over its entire length, or a complement thereof;
- At least one primer of the Candida tropicalis primer set comprises the nucleotide sequence of SEQ ID NO: 3 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 3 over its entire length, or a complement thereof;
- At least one primer of the Candida parapsilosis primer set comprises the nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 4 over its entire length, or a complement thereof;
- At least one primer of the Candida krusei primer set comprises the nucleotide sequence of SEQ ID NO: 5 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 5 over its entire length, or a complement thereof;
- At least one primer of the Candida guilliermondii primer set comprises the nucleotide sequence of SEQ ID NO: 6 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 6 over its entire length, or a complement thereof:
- At least one primer of the Candida auris primer set comprises the nucleotide sequence of SEQ ID NO: 7 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 7 over its entire length, or a complement thereof.
- the other primer of the primer pair may a primer that comprises the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 1 over its entire length, or a complement thereof.
- the Candida glabrata primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 8 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 8 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 9 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 9 over its entire length, or a complement thereof.
- the Cryptococcus neoformans primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 10 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 10 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 11 over its entire length, or a complement thereof.
- the CryptoGoccus deneoformans primer set comprises a primer having a nucleotide sequence of SEQ ID NO: 12 or the nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 12 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 13 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 13 over its entire length, or a complement thereof
- the Cryptococcus gattii primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 14 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 15 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 15 over its entire length, or a complement thereof.
- the Fusarium spp. primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 16 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 16 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 17 over its entire length, or a complement thereof.
- the Trichosporon spp. primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 18 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 18 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 19 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 19 over its entire length, or a complement thereof.
- the Schizosaccharomyces pombe primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 20 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 20 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 21 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 21 over its entire length, or a complement thereof.
- the Candida spp. primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 22 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 22 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 23 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 23 over its entire length, or a complement thereof.
- any two or more of the primer sets described herein above may be used in a single multiplexing method, in various embodiments, the methods of the invention thus use 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14 primer sets as described herein.
- These primer sets are preferably different in that they are directed against different species or families of organisms and allow specific detection and identification thereof.
- the more general primer sets disclosed herein namely those directed to Candida spp., Trichosporon spp., and Fusarium spp., are specific for the respective family of fungi, but cannot distinguish between the different species belonging to that family. However, their inclusion may still be preferable, as a positive signal with a Candida spp. primer set, while all specific Candida primer sets are negative indicates that the sample comprises a Candida species other than those specifically tested.
- the combination of oligonucleotide detection probes comprises two or more probes selected from:
- a Candida aibicans probe having a nucleotide sequence specific to a target region in the rDNA of Candida albicans'
- a Candida tropicalis probe having a nucleotide sequence specific to a target region in the rDNA of Candida tropicalis
- Candida giabrata probe having a nucleotide sequence specific to a target region in the rDNA of Candida giabrata
- Candida parapsilosis probe having a nucleotide sequence specific to a target region in the rDNA of Candida parapsilosis
- Candida krusei probe having a nucleotide sequence specific to a target region in the rDNA of Candida krusei;
- Candida guilliermondii probe having a nucleotide sequence specific to a target region in the rDNA of Candida guilliermondii;
- Candida auris probe having a nucleotide sequence specific to a target region in the rDNA of Candida auris
- a Cryptococcus neoformans probe having a nucleotide sequence specific to a target region in the rDNA of Cryptococcus neoformans
- a Fusarium spp. probe having a nucleotide sequence specific to a target region in the rDNA of Fusarium spp. ;
- Trichosporon spp. probe having a nucleotide sequence specific to a target region in the rDNA of Trichosporon spp. ;
- a Candida spp. probe having a nucleotide sequence specific to a target region in the rDNA of Candida spp.
- the target region of rDNA of Candida albicans is in the ITS2 region thereof;
- the target region of rDNA of Candida tropicalis is in the ITS2 region thereof;
- the target region of rDNA of Candida giabrata is in the ITS 1 region thereof;
- the target region of rDNA of Candida parapsilosis is in the ITS2 region thereof
- the target region of rDNA of Candida krusei is in the ITS2 region thereof;
- the target region of rDNA of Candida guilliermondii is in the ITS2 region thereof;
- the target region of rDNA of Candida auris is in the ITS2 region thereof;
- the target region of rDNA of Cryptococcus neoformans is in the IGS1 region thereof;
- the target region of rDNA of Cryptococcus deneoformans is in the IGS1 region thereof;
- the target region of rDNA of Cryptococcus gattii is in the IGS1 region thereof;
- the target region of rDNA of Fusarium spp. is in the 26S region thereof;
- the target region of rDNA of Trichosporon spp. is in the 18S region thereof (13) the target region of genomic DNA of Schizosaccharomyces pombe is in the Cytochrome B gene region;
- the target region of rDNA of Candida spp. is in the 18S region thereof. in various embodiments,
- the Candida albicans probe comprises the nucleotide sequence of SEQ ID NO: 24 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 24 over its entire length, or a complement thereof;
- the Candida tropicalis probe comprises the nucleotide sequence of SEQ ID NO: 25 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 25 over its entire length, or a complement thereof;
- the Candida giabrata probe comprises the nucleotide sequence of SEQ ID NO: 30 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 30 over its entire length, or a complement thereof;
- Candida parapsilosis probe comprises the nucleotide sequence of SEQ ID NO: 26 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 26 over its entire length, or a complement thereof;
- the Candida krusei probe comprises the nucleotide sequence of SEQ ID NO: 27 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 27 over its entire length, or a complement thereof;
- the Candida guilliermondii probe comprises the nucleotide sequence of SEQ ID NO: 28 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 28 over its entire length, or a complement thereof;
- the Candida auris probe comprises the nucleotide sequence of SEQ ID NO: 29 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 29 over its entire length, or a complement thereof;
- the CryptococGus neoformans probe comprises the nucleotide sequence of SEQ ID NO: 32 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 32 over its entire length, or a complement thereof;
- the Cryptococcus deneoformans probe comprises the nucleotide sequence of SEQ ID NO: 33 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 33 over its entire length, or a complement thereof;
- the Cryptococcus gattii probe comprises the nucleotide sequence of SEQ ID NO: 34 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 34 over its entire length, or a complement thereof;
- the Fusarium spp. probe comprises the nucleotide sequence of SEQ ID NO: 35 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 35 over its entire length, or a complement thereof;
- the Trichosporon spp. probe comprises the nucleotide sequence of SEQ ID NO: 36 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 36 over its entire length, or a complement thereof;
- the Schizosaccharomyces pombe probe comprises the nucleotide sequence of SEQ ID NO: 37 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 37 over its entire length, or a complement thereof;
- the Candida spp. probe comprises the nucleotide sequence of SEQ ID NO: 31 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 31 over its entire length, or a complement thereof.
- the combination of primer sets comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13 or at least 14 different primer sets.
- the combination of primer sets may be selected from the group consisting of:
- Candida albicans primer set Candida tropicaiis primer set, Candida giabrata primer set, Candida parapsiiosis primer set, Candida krusei primer set, Candida guilliermondii primer set, Candida auris primer set, Cryptococcus neoformans primer set, Cryptococcus deneoformans primer set, Cryptococcus gattii primer set, Fusarium spp. primer set and Trichosporon spp. primer set;
- Candida albicans primer set Candida tropicaiis primer set, Candida giabrata primer set, Candida parapsiiosis primer set, Candida krusei primer set, Candida guilliermondii primer set and Candida auris primer set;
- Candida albicans primer set Candida tropicaiis primer set, Candida giabrata primer set, Candida parapsiiosis primer set and Candida krusei primer set;
- Candida albicans primer set Candida tropicaiis primer set and Candida giabrata primer set
- Candida albicans primer set Candida tropicaiis primer set, Candida giabrata primer set, Candida parapsiiosis primer set, Candida krusei primer set, Candida auris primer set, Cryptococcus neoformans primer set, Cryptococcus deneoformans primer set, Cryptococcus gattii primer set;
- Cryptococcus neoformans primer set Cryptococcus deneoformans primer set and Cryptococcus gattii primer set.
- the combination of probes may comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13 or at least 14 different probes.
- the different probes are each specific for a different pathogenic fungi species.
- the combination of probes may be selected from the group consisting of:
- Candida tropicaiis probe Candida giabrata probe, Candida parapsiiosis probe, Candida krusei probe, Candida guilliermondii probe, Candida auris probe, Cryptococcus neoformans probe, Cryptococcus deneoformans probe, Cryptococcus gattii probe, Fusarium spp. probe and Trichosporon spp. probe;
- Candida albicans probe Candida tropicaiis probe, Candida giabrata probe, Candida parapsiiosis probe, Candida krusei probe, Candida guilliermondii probe and Candida auris probe;
- Candida albicans probe Candida tropicalis probe, Candida glabrata probe, Candida parapsitosis probe and Candida krusei probe;
- Candida albicans probe, Candida tropicalis probe and Candida glabrata probe (4) Candida albicans probe, Candida tropicalis probe and Candida glabrata probe;
- Candida albicans probe Candida tropicalis probe, Candida glabrata probe, Candida parapsitosis probe, Candida krusei probe, Candida auris probe, Cryptococcus neoformans probe, Cryptococcus deneoformans probe, Cryptococcus gattii probe;
- Cryptococcus neoformans probe Cryptococcus deneoformans probe and Cryptococcus gattii probe.
- the method of the invention may be a method forthe diagnosis of fungal sepsis in a subject.
- the subject may be a human subject.
- the sample is a blood, serum or plasma sample.
- the subject may be diagnosed with fungal sepsis, if one or more of the target nucleic acids are detected in the sample.
- an appropriate therapy may be selected, typically an antifungal agent that is effective against the identified pathogen.
- kits for carrying out the method of the invention.
- kits typically comprise two or more amplification primer sets and two or more detection probes, wherein (A) the two or more amplification primer sets are directed to different target nucleic acids of at least two different fungal pathogen species involved in fungal sepsis and are independently selected from the group consisting of:
- Candida albicans primer set targeting a nucleotide sequence in the rDNA of Candida albicans
- Candida parapsitosis primer set targeting a nucleotide sequence in the rDNA of Candida parapsitosis
- Candida auris primer set targeting a nucleotide sequence in the rDNA of Candida auris
- Trichosporon spp. primer set targeting a nucleotide sequence in the rDNA of Trichosporon spp. ;
- a Candida spp. primer set targeting a nucleotide sequence in the rDNA of Candida spp.; and (B) the two or more detection probes comprise at least one probe specific for each of the amplicons produced by amplification of the target nucleic acids by each of the two or more primer pairs.
- the primer sets and probes are defined as described above in relation to the described methods.
- the invention further features the use of the kit of the invention for the diagnosis of fungal sepsis in a subject.
- the subject and the sample analyzed may be as described above for the methods of the invention.
- Also encompassed are methods for the diagnosis of fungal sepsis in a subject, wherein said kits are used, preferably to allow practicing the methods of the invention.
- Figure 1 shows the test results of a test panel of 14 primer set/probe combinations for 14 different pathogenic fungi. In these experiments, PCR was used for amplification of genomic DNA of the respective target.
- the present invention is based on the inventors’ surprising finding that pathogens that are involved in and responsible for fungal infection and fungal sepsis and human patients can be rapidly detected and identified using a multiplex diagnostic assay that uses a panel of primers and probes that allow simultaneous detection and identification of fungal pathogens in a given sample. It has been found that the developed methods and assays allow specific detection of numerous fungal pathogens in a sample without suffering from high background signals, cross-reactivity or any other interference that may hamper nucleic acid amplification and detection.
- the methods of the invention are therefore for the detection of the presence and/or amount of two or more target nucleic acids in a sample, wherein the two or more target nucleic acids are of fungal pathogens involved in fungal sepsis. While the described methods may qualitatively detect the presence or absence of a given target, with the presence being confirmed if the detected signal exceeds a predetermined threshold value, they are similarly suitable to quantitatively determine the target in a sample and thus provide information on the concentration levels of the target in the sample. Accordingly, the methods can detect either only the presence or additionally also the amount of two or more target nucleic acids in sample.
- Target nucleic acids refer to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or more. It is understood that it used in relation to a specific target nucleic acid, it relates not to the absolute number of identical target molecules but rather to different types of target molecules.
- target nucleic acids of at least two different fungal pathogens may comprise target nucleic acids of C. albicans and C. glabrata. The same applies if this term is used in relation to amplification primer sets and probes.
- the sample used in the methods of the invention may be any type of sample and includes tissue and body fluid samples, such as those obtained from a subject, as well as cultured samples.
- the sample may thus be a blood, serum or plasma sample or a cultured blood sample or any other cultured sample.
- the sample may be subjected to various processing steps before being used in the methods of the invention, including common isolation and/or purification steps, for examples steps to extract DNA from cells that may be present in the sample.
- the methods of the invention generally comprise the steps of amplification of the target nucleic acid(s) and the subsequent detection of the amplicons.
- the two or more oligonucleotide amplification primer sets are contacted with the sample, (suspected of) containing the target nucleic acid(s) under conditions that allow the hybridization of the respective primers to their target.
- the hybridization conditions are selected such that they allow the specific hybridization of the primer to its complementary target sequence.
- the hybridization conditions are typically selected such that they are strict enough to avoid unspecific interactions or interactions with nucleic acids that are not sufficiently, preferably not fully, complementary to the target-specific region of the primer.
- hybridization to nucleic acids that are not fully complementary but comprise one or more mismatches does not occur or does at least not stably occur under the selected hybridization conditions. These hybridization conditions therefore allow to minimize unspecific interaction and thus serve to reduce background signals in the following amplification.
- the primers generally comprise a target-specific region, i.e. a sequence stretch that is fully complementary to the intended target sequence.
- the primers consist of such a target-complementary region, i.e. the full length of the primer is designed and intended to hybridize to the target.
- the primers are typically used in pairs. Such primer pair generally comprises a forward and a reverse primer that are designed such that they flank the sequence of the target to be amplified.
- “Complementary”, as used herein in relation to nucleic acid interaction, relates to conventional Watson- Crick base pairing.
- the primer pairs are selected from the following primers pairs:
- Candida albicans primer set targeting a nucleotide sequence in the rDNA of Candida albicans
- Candida parapsilosis primer set targeting a nucleotide sequence in the rDNA of Candida parapsilosis
- Candida krusei primer set targeting a nucleotide sequence in the rDNA of Candida krusei
- Candida auris primer set targeting a nucleotide sequence in the rDNA of Candida auris
- Trichosporon spp. primer set targeting a nucleotide sequence in the rDNA of Trichosporon spp. ;
- a “Candida aibicans primer set” refers to an oligonucleotide amplification primer set, as defined herein, that targets a region in the nucleic acid of Candida aibicans.
- the selected target is generally selected such that it is specific for the indicated species to avoid crossreactivity with other species.
- this means that the Candida albicans primer set recognizes and binds to a Candida albicans target sequence but does not hybridize to nucleic acid regions in the DNA of other Candida species, at least no under the hybridization conditions chosen or at least with significantly lower affinity.
- rDNA refers to ribosomal DNA.
- genomic DNA refers to the general genomic DNA of an organism.
- primer pairs for which no species information is given such as Fusarium spp., Trichosporon spp. and Candida spp. are not specific for a certain species but detect to rDNA of several, but not necessarily all, members of this family of organisms, but have little or no cross-reactivity with species of other families.
- primer sets are used to detect numerous species of the respective family without discriminating such as to cover also those species for which no specific primer set is included. In various embodiments, this may either serve as a confirmation that a species of a given family is indeed present, i.e.
- the target region of rDNA of Candida albicans is of ITS2 region thereof;
- the target region of rDNA of Candida tropicalis is of ITS2 region thereof;
- the target region of rDNA of Candida glabrata is of ITS1 region thereof;
- the target region of rDNA of Candida parapsilosis is of ITS2 region thereof
- the target region of rDNA of Candida krusei is of ITS2 region thereof;
- the target region of rDNA of Candida guilliermondii is of ITS2 region thereof;
- the target region of rDNA of Candida auris is of ITS2 region thereof;
- the target region of rDNA of Cryptococcus neoformans is of IGS1 region thereof;
- the target region of rDNA of Cryptococcus deneoformans is of IGS1 region thereof;
- the target region of rDNA of Cryptococcus gattii is of IGS1 region thereof;
- the target region of rDNA of Fusarium spp. is of 26S region thereof;
- the target region of rDNA of Tnchosporon spp. is of 18S region thereof;
- the target region of genomic DNA of Schizosaccharomyces pombe is of Cytochrome B gene region;
- the target region of rDNA of Candida spp. is of 18S region thereof.
- ITS refers to the internal transcribed spacer region of rDNA.
- iGS refers to ribosomal intergenic spacer.
- the 26S and 18S regions are those regions of the rDNA that encode the 26S subunit of the ribosome and the 18S subunit of the ribosome, respectively.
- the cytochrome B gene is a gene that encodes for the mitochondrial protein cytochrome B and is found in the genomic DNA. The above target regions have been selected to ensure sufficient specificity for the respective species or family and avoid cross-reactivity with the other listed primer pairs.
- At least one primer of the Candida albicans primer set, the Candida tropicalis primer set, the Candida parapsilosis primer set, the Candida krusei primer set, the Candida guilliermondii primer set, and the Candida auris primer set comprises or consists of the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 1 over its entire length, or a complement thereof.
- the primer of SEQ ID NO:1 has sufficient complementarity to the target nucleic acids of all these species to form a target hybrid under the reaction conditions. The desired specificity of the detection is then brought about by the second primer of the pair, as described below.
- nucleotide sequences are determined by a sequence comparison.
- This comparison or alignment is typically based on the BLAST algorithm well-established and known in the art (See, e.g., Aitschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J, (1990): "Basic local alignment search tool", J. Mol. Biol. 215:403-410, and Aitschul, Stephan F., Thomas L. Madden, Alejandro A, Schaffer, Jinghui Zhang, Hheng Zhang, Webb Miller, and David J.
- sequence identities relate to the percentage over the entire length of the respective sequence, i.e. typically the reference sequence. If the reference sequence is 20 nucleotides in length, a sequence identity of 90 % means that 18 nucleotides in a query sequence are identical while 2 may differ.
- the first primer of the primer pair comprises or consists of the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 1 over its entire length, or a complement thereof,
- At least one primer of the Candida albicans primer set comprises or consists of the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 2 over its entire length, or a complement thereof;
- At least one primer of the Candida tropicalis primer set comprises or consists of the nucleotide sequence of SEQ ID NO: 3 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 3 over its entire length, or a complement thereof;
- At least one primer of the Candida parapsilosis primer set comprises or consists of the nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 4 over its entire length, or a complement thereof;
- At least one primer of the Candida krusei primer set comprises or consists of the nucleotide sequence of SEQ ID NO: 5 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 5 over its entire length, or a complement thereof;
- At least one primer of the Candida guilliermondii primer set comprises or consists of the nucleotide sequence of SEQ ID NO: 6 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 6 over its entire length, or a complement thereof; and/or
- At least one primer of the Candida auris primer set comprises or consists of the nucleotide sequence of SEQ ID NO: 7 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 7 over its entire length, or a complement thereof.
- the primer of embodiment (1) to (6) is the respective second primer of the primer pair. It is understood that if, in such embodiments, the first primer is a complement of SEQ ID NO:1 , the second primer is also the complement of the respective indicated sequence.
- the Candida glabrata primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 8 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 8 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 9 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 9 over its entire length , or a complement thereof.
- a primer having the nucleotide sequence of SEQ ID NO: 9 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 9 over its entire length , or a complement thereof As described above, if one of these primer sequences is the complement of the indicated sequence, the other is also a complement of the given sequence. This similarly applies to ail other primer pairs described below.
- the Cryptococcus neoformans primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 10 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 10 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 11 over its entire length, or a complement thereof.
- the Cryptococcus deneoformans primer set comprises a primer having a nucleotide sequence of SEQ ID NO: 12 or the nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 12 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 13 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 13 over its entire length, or a complement thereof.
- the Cryplococcus gatlii primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 14 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 15 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 15 over its entire length, or a complement thereof.
- the Fusarium spp. primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 16 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 16 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 17 over its entire length, or a complement thereof.
- the Trichosporon spp. primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 18 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 18 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 19 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 19 over its entire length, or a complement thereof.
- the Schizosaccharomyces pombe primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 20 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 20 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 21 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 21 over its entire length, or a complement thereof.
- the Candida spp. primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 22 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 22 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 23 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 23 over its entire length, or a complement thereof.
- any two or more of the primer sets described herein above may be used in a single multiplexing method.
- the methods of the invention thus use at least 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14 primer sets as described herein.
- These primer sets are preferably different in that they are directed against different species or families of organisms and allow specific detection and identification thereof.
- primer sets that are directed fo the same target but are directed against different target regions of the same target may also be included.
- the methods of the invention are preferably designed such that at least tow different pathogenic fungal species can be detected, i.e. at least two primer sets directed to different fungal pathogen species are included.
- primer sets used in the methods of the invention may be selected from the groups comprising or consisting of:
- Candida aibicans primer set Candida tropicalis primer set, Candida glabrata primer set, Candida parapsilosis primer set, Candida krusei primer set, Candida guilliermondii primer set, Candida auris primer set, Cryptococcus neoformans primer set, Cryptococcus deneoformans primer set, Cryptococcus galtii primer set, Fusarium spp. primer set and Trichosporon spp. primer set;
- Candida aibicans primer set Candida tropicalis primer set, Candida glabrata primer set, Candida parapsilosis primer set, Candida krusei primer set, Candida guilliermondii primer set and Candida auris primer set;
- Candida aibicans primer set Candida tropicalis primer set, Candida giabrata primer set, Candida parapsilosis primer set and Candida krusei primer set;
- Candida albicans primer set Candida tropicalis primer set and Candida giabrata primer set
- Candida albicans primer set Candida tropicalis primer set, Candida giabrata primer set, Candida parapsilosis primer set, Candida krusei primer set, Candida auris primer set, Cryptococcus neoformans primer set, Cryptococcus deneoforrnans primer set, Cryptococcus gattii primer set;
- Cryptococcus neoformans primer set Cryptococcus deneoforrnans primer set and Cryptococcus gattii primer set.
- primer set combinations (1) to (8) may be combined or supplemented by any one or more of the other primer sets described herein.
- These primer set combinations (1) to (8) preferably comprise the primers with the specific nucleotide sequences disclosed herein.
- the primer set for Candida albicans disclosed herein as comprising the nucleic acid sequences of SEQ ID Nos:1 and 2 may be combined with further second primers for the other Candida species, namely those of SEQ ID Nos. 3-7 as well as with the primer set for C. giabrata, i,e. the primers with the sequences set forth in SEQ ID Nos. 8 and 9. in various embodiments, the primers of SEQ ID Nos.
- primers of SEQ ID Nos. 1 and any two of 3-7 are combined, optionally further combined with those of SEQ ID Nos. 8 and 9.
- Such primer set combinations are specificaliy suited for methods for diagnosis of fungal sepsis caused by Candida species.
- the Candida spp. primer set i.e. of SEQ ID Nos, 22 and 23, may be additionally included in all aforementioned embodiments.
- the amplification reaction is carried out.
- the hybridized primer:target complexes are recognized by a polymerase and the respective target sequence amplified.
- the preferred method of amplification in the methods of the invention is PCR.
- PCR is a well-known and established technique and commonly known and practiced by those skilled in the art.
- Said amplification step generates a multitude of amplicons of all target nucleic acids present in the sample for which also a suitable primer set is included by thermocycling the reaction mixture in the presence of a thermostable DNA polymerase to allow multiple cycles of primer annealing and elongation.
- the primer sets have been designed such that they have under the conditions of the described methods no significant cross-reactivity and thus allow specific detection of one or more, preferably 2, 3, 4, 5, 6 ,7, 8, 9, 10, or 11 species for which primer sets are described herein. These primer sets may therefore be used in combination to allow multiplexing. In the multiplexing methods of the invention, in various embodiments, at least 2, preferably 3 or more of the species-specific primer sets are used. These may be further combined with 1 , 2 or 3 of the family-specific primer sets, i.e. the Candida, the Fusarium or the Trichosporon primer sets. Ail these primer sets disclosed herein below with their nucleic acid sequences have been found to allow simultaneous use without significant interference or crossreactivity.
- the primers used in the methods of the invention may comprise further portions in addition to their target-complementary nucleotide sequence necessary for target recognition and binding. These portions may include further nucleotide sequences that do not base-pair with the target and serve different purposes, for example providing ligation sites or restriction enzyme sites or for interaction with enzymes/proteins, such as the polymerase, or non-nucleotide moieties, such as labeling or coupling moieties, including biotin and various chromophores or fluorophores. in various embodiments, one of the primers of a given primer pair is biotinylated on its 5’ end.
- the presence and/or amount of the target nucleic acid(s) in the sample is detected by contacting the amplified sample / reaction mixture containing the amplicons of any target nucleic acid that may have been present in the sample with a combination of oligonucleotide detection probes comprising at least one probe specific for each of the targets of the amplification primer sets used.
- oligonucleotide detection probes comprising at least one probe specific for each of the targets of the amplification primer sets used.
- This hybridization is again carried out under conditions that are sufficiently strict to avoid unspecific hybridization or prevent hybridization to nucleic acids that carry one or more mismatches.
- the thus formed probertarget hybrids are then detected and optionally quantified to determine the presence and/or amount of the respective target nucleic acids in the sample.
- Various detection methods are known in the art and can be chosen by those skilled in the art based on preference. Common detection methods include radio-labeled, fluorophore- or chromophore-labeled probes that can then be detected by a read-out of the radioactivity, the fluorescence or color, for example by using available instruments, such as scintillation counters and spectrometers.
- probes coupled to a solid support such as a nanoparticle or bead, including magnetic beads, or so-called micro-discs.
- a solid support such as a nanoparticle or bead, including magnetic beads, or so-called micro-discs.
- One system that may advantageously used for probertarget complex detection is the rtCodeTM system of PlexBioTM.
- the combination of oligonucleotide detection probes comprises two or more probes selected from:
- Candida albicans probe having a nucleotide sequence specific to a target region in the rDNA of Candida albicans'
- Candida tropicalis probe having a nucleotide sequence specific to a target region in the rDNA of Candida tropicalis
- Candida glabrata probe having a nucleotide sequence specific to a target region in the rDNA of Candida glabrata
- Candida parapsilosis probe having a nucleotide sequence specific to a target region in the rDNA of Candida parapsilosis
- Candida krusei probe having a nucleotide sequence specific to a target region in the rDNA of Candida krusei;
- Candida guilliermondii probe having a nucleotide sequence specific to a target region in the rDNA of Candida guilliermondii;
- Candida auris probe having a nucleotide sequence specific to a target region in the rDNA of Candida auris
- a Cryptococcus neoformans probe having a nucleotide sequence specific to a target region in the rDNA of Cryptococcus neoformans
- a Fusarium spp. probe having a nucleotide sequence specific to a target region in the rDNA of Fusarium spp. ;
- Trichosporon spp. probe having a nucleotide sequence specific to a target region in the rDNA of Trichosporon spp. ;
- a Candida spp. probe having a nucleotide sequence specific to a target region in the rDNA of Candida spp.
- the probes are selected in dependence of the primer sets used for amplification such that for each potential target a probe is used. Accordingly, the method uses suitable primer pairprobe combinations.
- the target regions for the probes are generally selected such that they are within the amplified sequence stretch, i.e. lie in the region that is amplified by the use of the primers in an amplification reaction. It is preferably that the to be detected sequence does not overlap with the primer binding sites, but rather lies between the sequence stretches bound by the forward and reverse primers. As a consequence, thereof, the target region of the probes used is the same as that to which the primers are directed. Therefore, in various embodiments in accordance with the preferred primer target regions,
- the target region of rDNA of Candida albicans is in the ITS2 region thereof;
- the target region of rDNA of Candida tropicalis is in the ITS2 region thereof;
- the target region of rDNA of Candida parapsilosis is in the ITS2 region thereof
- the target region of rDNA of Candida krusei is in the ITS2 region thereof;
- the target region of rDNA of Candida guilliermondii is in the ITS2 region thereof;
- the target region of rDNA of Candida auris is in the ITS2 region thereof;
- the target region of rDNA of Cryptococcus neoformans is in the IGS1 region thereof;
- the target region of rDNA of Cryptococcus deneoformans is in the IGS1 region thereof;
- the target region of rDNA of Cryptococcus gattii is in the iGS1 region thereof;
- the target region of rDNA of Fusarium spp. is in the 26S region thereof;
- the target region of rDNA of Trichosporon spp. is in the 18S region thereof
- the target region of rDNA of Candida spp. is in the 18S region thereof.
- the probes used in the methods of the invention may comprise further portions in addition to their target- complementary nucleotide sequence necessary for target recognition and binding.
- These portions may include further nucleotide sequences that do not base-pair with the target and serve different purposes, for example providing ligation sites or restriction enzyme sites or for interaction with enzymes/proteins, such as the polymerase, or non-nucleotide moieties, such as labeling or coupling moieties, including biotin and various chromophores or fiuorophores,
- additional portions may provide for a linker function that allows coupling with a non-nucleotide moiety.
- these nucleotides or nucleotide sequences may be further modified with a functional group or a spacer including a functional group, such as an amino group. This may then allow coupling to various delectable labels, such as pi-code microdiscs (ex PlexBio, TW).
- Probe sequences may be designed in accordance with the above-described principles. It is further important that the probe sequences are again sufficiently specific to avoid cross-reactivity between different targets. Therefore, within the amplified sequence stretches sufficiently different target regions for probe binding need to be identified that allow preferential binding of a specific probe forthe respective target over binding of any other probe directed to a different target.
- probe sequences that have been designed accordingly and can be used in combination without the risk of cross-reactivity and thus false positives and undesired interference with each other.
- the Candida albicans probe comprises the nucleotide sequence of SEQ ID NO: 24 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 24 over its entire length, or a complement thereof;
- the Candida tropicalis probe comprises the nucleotide sequence of SEQ ID NO: 25 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 25 over its entire length, or a complement thereof;
- the Candida giabrata probe comprises the nucleotide sequence of SEQ ID NO: 30 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 30 over its entire length, or a complement thereof;
- Candida parapsilosis probe comprises the nucleotide sequence of SEQ ID NO: 26 or a nucleotide sequence having at ieast 90% sequence identity to SEQ ID NO: 26 over its entire length, or a complement thereof;
- the Candida krusei probe comprises the nucleotide sequence of SEQ ID NO: 27 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 27 over its entire length, or a complement thereof;
- the Candida guilliermondii probe comprises the nucleotide sequence of SEQ ID NO: 28 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 28 over its entire length, or a complement thereof;
- the Candida auris probe comprises the nucleotide sequence of SEQ ID NO: 29 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 29 over its entire length, or a complement thereof;
- the Cryptococcus neoformans probe comprises the nucleotide sequence of SEQ ID NO: 32 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 32 over its entire length, or a complement thereof;
- the Cryptococcus deneoformans probe comprises the nucleotide sequence of SEQ ID NO: 33 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 33 over its entire length, or a complement thereof;
- the Cryptococcus gattii probe comprises the nucleotide sequence of SEQ ID NO: 34 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 34 over its entire length, or a complement thereof;
- the Fusarium spp. probe comprises the nucleotide sequence of SEQ ID NO: 35 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 35 over its entire length, or a complement thereof;
- the Trichosporon spp. probe comprises the nucleotide sequence of SEQ ID NO: 36 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 36 over its entire length, or a complement thereof;
- the Schizosaccharomyces pombe probe comprises the nucleotide sequence of SEQ ID NO: 37 or a nucleotide sequence having at least 90'% sequence identity to SEQ ID NO: 37 over its entire length, or a complement thereof;
- the Candida spp. probe comprises the nucleotide sequence of SEQ ID NO: 31 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 31 over its entire length, or a complement thereof.
- the combination of probes may comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13 or at least 14 different probes.
- the different probes are each specific for a different pathogenic fungi species. As described above, the different probes are selected such that at least one probe for each potentially amplified target, i.e. those targets for which primers have been used/included, is present to ensure that each target that has successfully been amplified is subsequently detected.
- the respective preferred combinations of probes are thus equivalent to the preferred combinations of primer sets disclosed herein. Accordingly, the combination of probes may be selected from the group comprising or consisting of:
- Candida tropicalis probe Candida giabrata probe, Candida parapsilosis probe, Candida krusei probe, Candida guilliermondii probe, Candida auris probe, Cryptococcus neoformans probe, Cryptococcus deneoformans probe, Cryptococcus gattii probe, Fusarium spp. probe and Trichosporon spp. probe;
- Candida albicans probe Candida tropicaiis probe, Candida giabrata probe, Candida parapsilosis probe, Candida krusei probe, Candida guilliermondii probe and Candida auris probe;
- Candida albicans probe Candida tropicalis probe, Candida giabrata probe, Candida parapsilosis probe and Candida krusei probe;
- Candida albicans probe, Candida tropicalis probe and Candida giabrata probe (4) Candida albicans probe, Candida tropicalis probe and Candida giabrata probe;
- Candida albicans probe Candida tropicalis probe, Candida giabrata probe, Candida parapsilosis probe, Candida krusei probe, Candida auris probe, Cryptococcus neoformans probe, Cryptococcus deneoformans probe, Cryptooocous gattii probe;
- Cryptococcus neoformans probe Cryptococcus deneoformans probe and Cryptococcus gattii probe.
- the method of the invention may be a method for the diagnosis of fungal sepsis in a subject.
- the invention also covers a method for the diagnosis of fungal sepsis in a subject that comprises a first step of obtaining a sample from said subject and then subjecting said sample to the methods of the invention forthe detection of the presence and/or amount of two or more target nucleic acids in a sample, wherein the two or more target nucleic acids are of fungal pathogens involved in fungal sepsis.
- the respective analysis steps are carried out in vitro, i.e. using the sample obtained from the subject.
- the subject may be a human subject.
- the sample is a blood, serum or plasma sample.
- other sample types may be used, including tissue or other body fluid samples, although blood, plasma and serum samples are the most common.
- the subject may be diagnosed with fungal sepsis, if one or more of the target nucleic acids are detected in the sample.
- this may in a further step then lead to diagnosing the subject with fungal infection and fungal sepsis.
- other factors may also be considered, including medical conditions, such as fever and the like.
- an appropriate therapy may be selected, typically an antifungal agent that is effective against the identified pathogen.
- appropriate antifungal treatment options exist and suitable medicaments are available in the art. The skilled person can thus readily select an appropriate antifungal agent depending on the results of the inventive methods.
- kits for carrying out the methods of the invention.
- Such kits typically comprise a number of agents and reagents useful and necessary to perform the methods of the invention.
- the kits thus comprise at least the primer sets and corresponding probes disclosed herein, i.e. two or more amplification primer sets and two or more detection probes.
- the primers and probes are preferably those that have been described in relation to the methods herein.
- the kit may comprise a number of further agents that may selected from, without limitation, a polymerase, buffers, mononucleotides, and all other agents necessary for a PCR reaction and subsequent isolation/purification of the amplicons.
- kits for the diagnosis of fungal sepsis in a subject i.e. for use in the methods of diagnosis described herein above.
- kits for the diagnosis of fungal sepsis in a subject wherein said kits are used, preferably to allow practicing the methods of the invention.
- the subject and the sample analyzed may be as described above for the methods of the invention.
- Example 1 Fungal sepsis panel and testing thereof
- a panel of primer/probe combinations was tested using DNAs of different fungal species.
- genomic DNA (2 pg) of one fungus was used as a template.
- the full multiplex primer mixture was used for each conditions.
- Said primer mixture comprised primers for Candida albicans; Candida tropicalis, Candida giabrata, Candida parapsiiosis, Candida krusei, Candida guilliermondii, Candida auris, Cryptococcus neoformans, Cryptococcus deneoformans, Cryptococcus gattii, Fusarium spp., Trichosporon spp., Schizosaccharomyces pombe. and Candida spp. having the nucleic acid sequences set forth in SEQ ID Nos. 1 -23. PCR conditions were 94°C for 1 min; 40x cycles of 94°C for 30 sec, 60°C for 1 min; and 72°C for 10 min.
- one of the primers for each target is conjugated with biotin at the 5’ end as shown in the list below (5Biosg):
- the probes (SEQ ID NO 24-37) of individual targets were modified with adding a short oligo (dT) fragment at the 5’ end.
- the short oligo (dT) fragment serves as a linker between the pi-Code microdiscs (ex PlexBio, TW) and the probe sequence.
- the 5’ end of the oligo (dT) fragment was modified with an amino group with a (C6) spacer arm as shown below (5AmMC6).
- multiplex nCodeTM probes (ex PlexBioTM) were prepared in advance.
- the probe mixture comprised probes for each of the targets of the primer sets having the nucleotide sequences set forth in SEQ ID Nos. 24-37.
- the mixture was added into each well of a 96-well plate for later use.
- Each sample was detected specifically by the corresponding probe(s), for example, Candida albicans gDNA was detected by the specific probe Probe_C.aibi_JTS2 and the pan-Candida probe Probe_Candida_spp_18S.
- the strain of Cryptococcus neoformans is a hybrid strain, it contains chromosomes of both C. neoformans and C.deneoformans, therefore, both probes (Probe_Cr.neof_.IGS1 and Probe_Cr.deneofo_.IGS1) were positive. There is no cross-reaction among different fungal species.
- SAFE Control probe is a probe with random artificial sequence conjugated with Biotin at the 3’ end, which is supposed to be positive after the test. Blank Control probe is with the same artificial sequence which is supposed to be negative since no amplicons are supposed to hybridize on it.
- the primers used in the panel were found not to interfere with each other. The same applies to the probes used. Each sample only needed one round of PCR to obtain the amplicon.
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Botany (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to methods for the detection of the presence and/or amount of target nucleic acids in a sample, wherein said target nucleic acids are those of two or more fungal species involved in fungal sepsis. The methods include the use of primers to amplify the targets and probes for the subsequent detection. Also encompassed are methods for the diagnosis of fungal sepsis in a subject, kits for practicing the methods of the invention and various uses thereof.
Description
METHODS FOR THE DETECTION AND IDENTIFICATION OF FUNGAL SPECIES
Field of the Invention
The present invention is generally in the field of the detection and identification nucleic acids in a sample. More specifically, it relates to the detection and identification of fungal species in a sample by detecting and identifying genomic DNA of said fungal species, in particular of fungal species that are involved in fungal sepsis. The detection and identification of such fungal nucleic acids in the sample then allows the diagnosis of fungal sepsis in a subject from which said sample has been obtained.
Background of the invention
Sepsis is a serious medical condition with high mortalities that requires rapid treatment. Each year, according to the Centers for Disease Control and Prevention (CDC), at least 1.7 million adults in the U.S. develop sepsis, and nearly 270,000 die as a result. While conventionally sepsis is associated with bacterial infection, over the recent years, fungal infections and fungal sepsis have been reported to be increasing and are associated with considerable morbidity and mortality. Particularly problematic is that many fungal infections go undiagnosed. Fungal sepsis is therefore often diagnosed too late or not at all to the detriment of the patients. It is estimated that the risk of death from sepsis is as high as 30% and increases in cases of severe sepsis up to 50% and septic shock up to 80%.
A further complication is that the current gold standard methods for the detection of fungal sepsis pathogens (blood culture and microbiology tests) and the following diagnosis take 3 to 7 days. It has been estimated that the mortality rate among septic shock patients increases by 7.6% for each hour appropriate antimicrobial treatment is delayed. In many cases of suspected sepsis, empirical treatment with broad-spectrum antibiotics is therefore immediately started without waiting for the result of pathogen identification. In some of these cases, inappropriate antibiotic selection does not only annul the effects of chemotherapy but also promotes the emergence of drug-resistant bacteria. This is highly undesired and has long-lasting consequences in that it promotes the occurrence of multi -resista nt bacteria and fungi strains.
While invasive fungal infections are predominantly caused by Candida species, with candidemia being a highly common health care-associated bloodstream infection, increasingly immunocompromised patient populations have given rise to the emergence of fungal infections with other fungal species such as Aspergillus species, Pneumocystis jiroveci, Cryptococcus, Zygomycetes, Fusarium species, and Scedosporium species. At least 17 Candida species have been reported to cause severe infections in humans, but 5 species (C. albicans, C. giabrata, C. parapsilosis, C. tropicalis. and C. krusei) represent more than 90%. C. albicans has historically been the predominant pathogen, but to date other Candida species, such as C. giabrata, C. parapsilosis and C. tropicalis become more and more common in septic infections. As these species differ significantly in their susceptibility to certain compounds, such as triazoles and echinocandins, knowledge of the epidemiology is imperative for selection of appropriate empirical therapy.
There is thus need in the art for methods that allow rapid detection and identification of causative agents for fungal sepsis which allow the distinction between common and related fungal species involved in fungal sepsis such as to allow appropriate antimicrobial therapy without delay.
Summary of the Invention
The present invention meets this need by providing a method and kit that allow for the rapid detection and identification of a variety of fungal species in a sample simultaneously and thus allow avoiding the current laborious and time-consuming detection methods.
In a first aspect, the present invention is thus directed to a method for the detection of the presence and/or amount of two or more target nucleic acids in a sample, wherein the two or more target nucleic acids are of fungal pathogens involved in fungal sepsis, the method comprising: i) contacting the sample with a combination of two or more oligonucleotide amplification primer sets under conditions that allow hybridization of the oligonucleotide primers to the target nucleic acids of the fungal pathogens, wherein the two or more primer sets are directed to different target nucleic acids of at least two different fungal pathogen species involved in fungal sepsis and are independently selected from the group consisting of:
(1) a Candida albicans primer set targeting a nucleotide sequence in the rDNA of Candida albicans;
(2) a Candida tropicalis primer set targeting a nucleotide sequence in the rDNA of Candida tropicalis;
(3) a Candida glabrata primer set targeting a nucleotide sequence in the rDNA of Candida glabrata;
(4) a Candida parapsilosis primer set targeting a nucleotide sequence in the rDNA of Candida parapsilosis;
(5) a Candida krusei primer set targeting a nucleotide sequence in the rDNA of Candida krusei;
(6) a Candida guilliermondii primer set targeting a nucleotide sequence in the rDNA of Candida guilliermondii;
(7) a Candida auris primer set targeting a nucleotide sequence in the rDNA of Candida auris;
(8) a Cryptococcus neoformans primer set targeting a nucleotide sequence in the rDNA of Cryptococcus neoformans;
(9) a Cryptococcus deneoformans primer set targeting a nucleotide sequence in the rDNA of Cryptococcus deneoformans;
(10) a Cryptococcus gattii primer set targeting a nucleotide sequence in the rDNA of Cryptococcus gattii;
(11) a Fusarium spp. primer set targeting a nucleotide sequence in the rDNA of Fusarium spp.;
(12) a Trichosporon spp. primer set targeting a nucleotide sequence in the rDNA of
Thchosporon spp. ;
(13) a Schizosaccharomyces pombe primer set targeting a nucleotide sequences in the genomic DNA of Schizosaccharomyces pombe', and
(14) a Candida spp. primer set targeting a nucleotide sequence in the rDNA of Candida spp/, and ii) amplifying the target nucleic acid(s) hybridized to any one or more of the oligonucleotide primers by PCR; and ill) detecting the presence and/or amount of the target nucleic acid(s) in the sample by contacting the PCR amplified sample of step ii) with combination of oligonucleotide detection probes comprising at least one probe specific for each of the amplicons produced by the amplification of the target nucleic acids in step ii) under conditions that allow the hybridization of the probes to their specific target nucleic acid sequences and determining the presence and/or amount of probe:target hybrids.
In various embodiments of these methods according to the invention,
(1) the target region of rDNA of Candida albicans is of ITS2 region thereof;
(2) the target region of rDNA of Candida tropicalis is of ITS2 region thereof;
(3) the target region of rDNA of Candida glabrata is of ITS1 region thereof;
(4) the target region of rDNA of Candida parapsilosis is of ITS2 region thereof,
(5) the target region of rDNA of Candida krusei is of ITS2 region thereof;
(6) the target region of rDNA of Candida guilliermondii is of ITS2 region thereof;
(7) the target region of rDNA of Candida auris is of ITS2 region thereof;
(8) the target region of rDNA of Cryptococcus neoformans is of IGS1 region thereof;
(9) the target region of rDNA of Cryptococcus deneoformans is of IGS1 region thereof;
(10) the target region of rDNA of Cryptococcus gatiii is of IGS1 region thereof;
(11) the target region of rDNA of Fusarium spp. is of 26S region thereof;
(12) the target region of rDNA of Tnchosporon spp. is of 18S region thereof;
(13) the target region of genomic DNA of Schizosaccharomyces pombe is of Cytochrome B gene region; and/or
(14) the target region of rDNA of Candida spp. is of 18S region thereof.
In various embodiments, at least one primer of the Candida albicans primer set, the Candida tropicalis primer set, the Candida parapsilosis primer set, the Candida krusei primer set, the Candida guilliermondii primer set, and the Candida auris primer set comprises the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 1 over its entire length, or a complement thereof. In various embodiments,
(1) at least one primer of the Candida albicans primer set comprises the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 2 over its entire length, or a complement thereof;
(2) at least one primer of the Candida tropicalis primer set comprises the nucleotide sequence of SEQ ID NO: 3 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 3 over
its entire length, or a complement thereof;
(3) at least one primer of the Candida parapsilosis primer set comprises the nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 4 over its entire length, or a complement thereof;
(4) at least one primer of the Candida krusei primer set comprises the nucleotide sequence of SEQ ID NO: 5 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 5 over its entire length, or a complement thereof;
(5) at least one primer of the Candida guilliermondii primer set comprises the nucleotide sequence of SEQ ID NO: 6 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 6 over its entire length, or a complement thereof: and
(6) at least one primer of the Candida auris primer set comprises the nucleotide sequence of SEQ ID NO: 7 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 7 over its entire length, or a complement thereof. In these embodiments (1) to (6) the other primer of the primer pair may a primer that comprises the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 1 over its entire length, or a complement thereof.
In various embodiments, the Candida glabrata primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 8 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 8 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 9 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 9 over its entire length, or a complement thereof.
In various embodiments, the Cryptococcus neoformans primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 10 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 10 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 11 over its entire length, or a complement thereof.
In various embodiments, the CryptoGoccus deneoformans primer set comprises a primer having a nucleotide sequence of SEQ ID NO: 12 or the nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 12 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 13 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 13 over its entire length, or a complement thereof, in various embodiments, the Cryptococcus gattii primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 14 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 15 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 15 over its entire length, or a complement thereof.
In various embodiments, the Fusarium spp. primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 16 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 16 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 17 over its entire length, or a complement thereof.
In various embodiments, the Trichosporon spp. primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 18 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 18 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 19 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 19 over its entire length, or a complement thereof.
In various embodiments, the Schizosaccharomyces pombe primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 20 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 20 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 21 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 21 over its entire length, or a complement thereof.
In various embodiments the Candida spp. primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 22 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 22 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 23 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 23 over its entire length, or a complement thereof.
In various embodiments, any two or more of the primer sets described herein above may be used in a single multiplexing method, in various embodiments, the methods of the invention thus use 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14 primer sets as described herein. These primer sets are preferably different in that they are directed against different species or families of organisms and allow specific detection and identification thereof.
It is understood that the more general primer sets disclosed herein, namely those directed to Candida spp., Trichosporon spp., and Fusarium spp., are specific for the respective family of fungi, but cannot distinguish between the different species belonging to that family. However, their inclusion may still be preferable, as a positive signal with a Candida spp. primer set, while all specific Candida primer sets are negative indicates that the sample comprises a Candida species other than those specifically tested. in various embodiments, the combination of oligonucleotide detection probes comprises two or more probes selected from:
(1) a Candida aibicans probe having a nucleotide sequence specific to a target region in the rDNA of Candida albicans',
(2) a Candida tropicalis probe having a nucleotide sequence specific to a target region in the rDNA of Candida tropicalis;
(3) a Candida giabrata probe having a nucleotide sequence specific to a target region in the rDNA of Candida giabrata;
(4) a Candida parapsilosis probe having a nucleotide sequence specific to a target region in the rDNA of Candida parapsilosis;
(5) a Candida krusei probe having a nucleotide sequence specific to a target region in the rDNA of Candida krusei;
(6) a Candida guilliermondii probe having a nucleotide sequence specific to a target region in the rDNA of Candida guilliermondii;
(7) a Candida auris probe having a nucleotide sequence specific to a target region in the rDNA of Candida auris;
(8) a Cryptococcus neoformans probe having a nucleotide sequence specific to a target region in the rDNA of Cryptococcus neoformans;
(9) a Cryptococcus deneoformans probe having a nucleotide sequence specific to a target region in the rDNA of Cryptococcus deneoformans;
(10) a Cryptococcus gattii probe having a nucieotide sequence specific to a target region in the rDNA of Cryptococcus gattii;
(11) a Fusarium spp. probe having a nucleotide sequence specific to a target region in the rDNA of Fusarium spp. ;
(12) a Trichosporon spp. probe having a nucleotide sequence specific to a target region in the rDNA of Trichosporon spp. ;
(13) a Schizosaccharomyces pombe probe having a nucieotide sequence specific to a target region in the genomic DNA of Schizosaccharomyces pombe; and
(14) a Candida spp. probe having a nucleotide sequence specific to a target region in the rDNA of Candida spp.
In various embodiments,
(1) the target region of rDNA of Candida albicans is in the ITS2 region thereof;
(2) the target region of rDNA of Candida tropicalis is in the ITS2 region thereof;
(3) the target region of rDNA of Candida giabrata is in the ITS 1 region thereof;
(4) the target region of rDNA of Candida parapsilosis is in the ITS2 region thereof,
(5) the target region of rDNA of Candida krusei is in the ITS2 region thereof;
(6) the target region of rDNA of Candida guilliermondii is in the ITS2 region thereof;
(7) the target region of rDNA of Candida auris is in the ITS2 region thereof;
(8) the target region of rDNA of Cryptococcus neoformans is in the IGS1 region thereof;
(9) the target region of rDNA of Cryptococcus deneoformans is in the IGS1 region thereof;
(10) the target region of rDNA of Cryptococcus gattii is in the IGS1 region thereof;
(11) the target region of rDNA of Fusarium spp. is in the 26S region thereof;
(12) the target region of rDNA of Trichosporon spp. is in the 18S region thereof
(13) the target region of genomic DNA of Schizosaccharomyces pombe is in the Cytochrome B gene region; and
(14) the target region of rDNA of Candida spp. is in the 18S region thereof. in various embodiments,
(1) the Candida albicans probe comprises the nucleotide sequence of SEQ ID NO: 24 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 24 over its entire length, or a complement thereof;
(2) the Candida tropicalis probe comprises the nucleotide sequence of SEQ ID NO: 25 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 25 over its entire length, or a complement thereof;
(3) the Candida giabrata probe comprises the nucleotide sequence of SEQ ID NO: 30 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 30 over its entire length, or a complement thereof;
(4) the Candida parapsilosis probe comprises the nucleotide sequence of SEQ ID NO: 26 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 26 over its entire length, or a complement thereof;
(5) the Candida krusei probe comprises the nucleotide sequence of SEQ ID NO: 27 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 27 over its entire length, or a complement thereof;
(6) the Candida guilliermondii probe comprises the nucleotide sequence of SEQ ID NO: 28 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 28 over its entire length, or a complement thereof;
(7) the Candida auris probe comprises the nucleotide sequence of SEQ ID NO: 29 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 29 over its entire length, or a complement thereof;
(8) the CryptococGus neoformans probe comprises the nucleotide sequence of SEQ ID NO: 32 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 32 over its entire length, or a complement thereof;
(9) the Cryptococcus deneoformans probe comprises the nucleotide sequence of SEQ ID NO: 33 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 33 over its entire length, or a complement thereof;
(10) the Cryptococcus gattii probe comprises the nucleotide sequence of SEQ ID NO: 34 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 34 over its entire length, or a complement thereof;
(11) the Fusarium spp. probe comprises the nucleotide sequence of SEQ ID NO: 35 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 35 over its entire length, or a complement thereof;
(12) the Trichosporon spp. probe comprises the nucleotide sequence of SEQ ID NO: 36 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 36 over its entire length, or a
complement thereof;
(13) the Schizosaccharomyces pombe probe comprises the nucleotide sequence of SEQ ID NO: 37 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 37 over its entire length, or a complement thereof; and
(14) the Candida spp. probe comprises the nucleotide sequence of SEQ ID NO: 31 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 31 over its entire length, or a complement thereof.
In various embodiments, the combination of primer sets comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13 or at least 14 different primer sets. The combination of primer sets may be selected from the group consisting of:
(1) Candida albicans primer set, Candida tropicaiis primer set, Candida giabrata primer set, Candida parapsiiosis primer set, Candida krusei primer set, Candida guilliermondii primer set, Candida auris primer set, Cryptococcus neoformans primer set, Cryptococcus deneoformans primer set, Cryptococcus gattii primer set, Fusarium spp. primer set and Trichosporon spp. primer set;
(2) Candida albicans primer set, Candida tropicaiis primer set, Candida giabrata primer set, Candida parapsiiosis primer set, Candida krusei primer set, Candida guilliermondii primer set and Candida auris primer set;'
(3) Candida albicans primer set, Candida tropicaiis primer set, Candida giabrata primer set, Candida parapsiiosis primer set and Candida krusei primer set;
(4) Candida albicans primer set, Candida tropicaiis primer set and Candida giabrata primer set;
(5) Candida albicans primer set, Candida tropicaiis primer set, Candida giabrata primer set, Candida parapsiiosis primer set, Candida krusei primer set, Candida auris primer set, Cryptococcus neoformans primer set, Cryptococcus deneoformans primer set, Cryptococcus gattii primer set;
(6) Cryptococcus neoformans primer set, Cryptococcus deneoformans primer set, Cryptococcus gattii primer set, Fusarium spp. primer set and Trichosporon spp. primer set;
(7) Candida albicans primer set and Candida tropicaiis primer set; and
(8) Cryptococcus neoformans primer set, Cryptococcus deneoformans primer set and Cryptococcus gattii primer set.
The combination of probes may comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13 or at least 14 different probes. In various embodiments, the different probes are each specific for a different pathogenic fungi species.
The combination of probes may be selected from the group consisting of:
(1) Candida tropicaiis probe, Candida giabrata probe, Candida parapsiiosis probe, Candida krusei probe, Candida guilliermondii probe, Candida auris probe, Cryptococcus neoformans probe, Cryptococcus deneoformans probe, Cryptococcus gattii probe, Fusarium spp. probe and Trichosporon spp. probe;
(2) Candida albicans probe, Candida tropicaiis probe, Candida giabrata probe, Candida parapsiiosis
probe, Candida krusei probe, Candida guilliermondii probe and Candida auris probe;
(3) Candida albicans probe, Candida tropicalis probe, Candida glabrata probe, Candida parapsitosis probe and Candida krusei probe;
(4) Candida albicans probe, Candida tropicalis probe and Candida glabrata probe;
(5) Candida albicans probe, Candida tropicalis probe, Candida glabrata probe, Candida parapsitosis probe, Candida krusei probe, Candida auris probe, Cryptococcus neoformans probe, Cryptococcus deneoformans probe, Cryptococcus gattii probe;
(6) Cryptococcus neoformans probe, Cryptococcus deneoformans probe, Cryptococcus gattii probe, Fusarium spp. probe and Trichosporon spp. probe;
(7) Candida albicans probe and Candida tropicalis probe; and
(8) Cryptococcus neoformans probe, Cryptococcus deneoformans probe and Cryptococcus gattii probe.
The method of the invention may be a method forthe diagnosis of fungal sepsis in a subject. The subject may be a human subject. In various embodiments, the sample is a blood, serum or plasma sample. In these methods for the diagnosis of fungal sepsis in a subject, the subject may be diagnosed with fungal sepsis, if one or more of the target nucleic acids are detected in the sample. Depending on the diagnosis and the detected microorganism an appropriate therapy may be selected, typically an antifungal agent that is effective against the identified pathogen.
In another aspect, the present invention relates to a kit for carrying out the method of the invention. Such kits typically comprise two or more amplification primer sets and two or more detection probes, wherein (A) the two or more amplification primer sets are directed to different target nucleic acids of at least two different fungal pathogen species involved in fungal sepsis and are independently selected from the group consisting of:
(1) a Candida albicans primer set targeting a nucleotide sequence in the rDNA of Candida albicans;
(2) a Candida tropicalis primer set targeting a nucleotide sequence in the rDNA of Candida tropicalis;
(3) a Candida glabrata primer set targeting a nucleotide sequence in the rDNA of Candida glabrata;
(4) a Candida parapsitosis primer set targeting a nucleotide sequence in the rDNA of Candida parapsitosis;
(5) a Candida krusei primer set targeting a nucleotide sequence in the rDNA of Candida krusei;
(6) a Candida guilliermondii primer set targeting a nucleotide sequence in the rDNA of Candida guilliermondii;
(7) a Candida auris primer set targeting a nucleotide sequence in the rDNA of Candida auris;
(8) a Cryptococcus neoformans primer set targeting a nucleotide sequence in the rDNA of Cryptococcus neoformans;
(9) a Cryptococcus deneoformans primer set targeting a nucleotide sequence in the rDNA of Cryptococcus deneoformans;
(10) a Cryptococcus gattii primer set targeting a nucleotide sequence in the rDNA of Cryptococcus gattii;
(11) a Fusarium spp. primer set targeting a nucleotide sequence in the rDNA of Fusarium spp. ;
(12) a Trichosporon spp. primer set targeting a nucleotide sequence in the rDNA of Trichosporon spp. ;
(13) a Schizosaccharomyces pombe primer set targeting a nucleotide sequences in the genomic DNA of SchizosaccharomyGes pombe; and
(14) a Candida spp. primer set targeting a nucleotide sequence in the rDNA of Candida spp.; and (B) the two or more detection probes comprise at least one probe specific for each of the amplicons produced by amplification of the target nucleic acids by each of the two or more primer pairs. in various embodiments of the kits of the invention, the primer sets and probes are defined as described above in relation to the described methods.
The invention further features the use of the kit of the invention for the diagnosis of fungal sepsis in a subject. The subject and the sample analyzed may be as described above for the methods of the invention. Also encompassed are methods for the diagnosis of fungal sepsis in a subject, wherein said kits are used, preferably to allow practicing the methods of the invention.
Brief Description of the Drawings
Figure 1 shows the test results of a test panel of 14 primer set/probe combinations for 14 different pathogenic fungi. In these experiments, PCR was used for amplification of genomic DNA of the respective target.
Detailed Description
The present invention is based on the inventors’ surprising finding that pathogens that are involved in and responsible for fungal infection and fungal sepsis and human patients can be rapidly detected and identified using a multiplex diagnostic assay that uses a panel of primers and probes that allow simultaneous detection and identification of fungal pathogens in a given sample. It has been found that the developed methods and assays allow specific detection of numerous fungal pathogens in a sample without suffering from high background signals, cross-reactivity or any other interference that may hamper nucleic acid amplification and detection.
The methods of the invention are therefore for the detection of the presence and/or amount of two or more target nucleic acids in a sample, wherein the two or more target nucleic acids are of fungal pathogens involved in fungal sepsis. While the described methods may qualitatively detect the presence or absence of a given target, with the presence being confirmed if the detected signal exceeds a predetermined threshold value, they are similarly suitable to quantitatively determine the target in a sample and thus provide information on the concentration levels of the target in the sample. Accordingly, the methods can detect either only the presence or additionally also the amount of two or more target nucleic acids in sample.
“Two or more” and “at least two”, as used interchangeably herein, refer to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 ,
12, 13, 14 or more. It is understood that it used in relation to a specific target nucleic acid, it relates not to the absolute number of identical target molecules but rather to different types of target molecules. For example, “two or more target nucleic acids” of at least two different fungal pathogens may comprise target nucleic acids of C. albicans and C. glabrata. The same applies if this term is used in relation to amplification primer sets and probes.
The sample used in the methods of the invention may be any type of sample and includes tissue and body fluid samples, such as those obtained from a subject, as well as cultured samples. The sample may thus be a blood, serum or plasma sample or a cultured blood sample or any other cultured sample. The sample may be subjected to various processing steps before being used in the methods of the invention, including common isolation and/or purification steps, for examples steps to extract DNA from cells that may be present in the sample.
The methods of the invention generally comprise the steps of amplification of the target nucleic acid(s) and the subsequent detection of the amplicons. For the amplification, the two or more oligonucleotide amplification primer sets are contacted with the sample, (suspected of) containing the target nucleic acid(s) under conditions that allow the hybridization of the respective primers to their target. The hybridization conditions are selected such that they allow the specific hybridization of the primer to its complementary target sequence. The hybridization conditions are typically selected such that they are strict enough to avoid unspecific interactions or interactions with nucleic acids that are not sufficiently, preferably not fully, complementary to the target-specific region of the primer. This means that in various embodiments, hybridization to nucleic acids that are not fully complementary but comprise one or more mismatches does not occur or does at least not stably occur under the selected hybridization conditions. These hybridization conditions therefore allow to minimize unspecific interaction and thus serve to reduce background signals in the following amplification.
The primers generally comprise a target-specific region, i.e. a sequence stretch that is fully complementary to the intended target sequence. In various embodiments, the primers consist of such a target-complementary region, i.e. the full length of the primer is designed and intended to hybridize to the target. The primers are typically used in pairs. Such primer pair generally comprises a forward and a reverse primer that are designed such that they flank the sequence of the target to be amplified. “Complementary”, as used herein in relation to nucleic acid interaction, relates to conventional Watson- Crick base pairing.
The primer pairs are selected from the following primers pairs:
(1) a Candida albicans primer set targeting a nucleotide sequence in the rDNA of Candida albicans;
(2) a Candida tropicalis primer set targeting a nucleotide sequence in the rDNA of Candida tropicalis;
(3) a Candida glabrata primer set targeting a nucleotide sequence in the rDNA of Candida glabrata;
(4) a Candida parapsilosis primer set targeting a nucleotide sequence in the rDNA of Candida parapsilosis;
(5) a Candida krusei primer set targeting a nucleotide sequence in the rDNA of Candida krusei;
(6) a Candida guilliermondii primer set targeting a nucleotide sequence in the rDNA of Candida guilliermondii;
(7) a Candida auris primer set targeting a nucleotide sequence in the rDNA of Candida auris;
(8) a Cryptococcus neoformans primer set targeting a nucleotide sequence in the rDNA of Cryptococcus neoformans;
(9) a Cryptococcus deneoformans primer set targeting a nucleotide sequence in the rDNA of Cryptococcus deneoformans;
(10) a Cryptococcus gatii primer set targeting a nucleotide sequence in the rDNA of Cryptococcus gattii;
(11) a Fusarium spp. primer set targeting a nucleotide sequence in the rDNA of Fusarium spp. ;
(12) a Trichosporon spp. primer set targeting a nucleotide sequence in the rDNA of Trichosporon spp. ;
(13) a Schizosaccharomyces pombe primer set targeting a nucleotide sequences in the genomic DNA of Schizosaccharomyces pombe; and
(14) a Candida spp. primer set targeting a nucleotide sequence in the rDNA of Candida spp.
It is understood that, for example, a “Candida aibicans primer set” refers to an oligonucleotide amplification primer set, as defined herein, that targets a region in the nucleic acid of Candida aibicans. The selected target is generally selected such that it is specific for the indicated species to avoid crossreactivity with other species. In the afore-mentioned example this means that the Candida albicans primer set recognizes and binds to a Candida albicans target sequence but does not hybridize to nucleic acid regions in the DNA of other Candida species, at least no under the hybridization conditions chosen or at least with significantly lower affinity.
“rDNA”, as used herein, refers to ribosomal DNA. In contrast, “genomic DNA” refers to the general genomic DNA of an organism.
It is understood that those primer pairs for which no species information is given, such as Fusarium spp., Trichosporon spp. and Candida spp. are not specific for a certain species but detect to rDNA of several, but not necessarily all, members of this family of organisms, but have little or no cross-reactivity with species of other families. As explained above, such primer sets are used to detect numerous species of the respective family without discriminating such as to cover also those species for which no specific primer set is included. In various embodiments, this may either serve as a confirmation that a species of a given family is indeed present, i.e. in case one of the specific primer pairs for this species also gives a positive signal, or that a species of that family is present but is none of those species for which specific primer sets have been included. The specific primer sets have been selected based on the relevance and importance of the respective fungal species in fungal sepsis. The 11 specific fungal pathogens for which specific primer sets have been described herein do together cover the pathogens found in more than 90% of fungal sepsis cases.
In various embodiments of these methods according to the invention,
(1) the target region of rDNA of Candida albicans is of ITS2 region thereof;
(2) the target region of rDNA of Candida tropicalis is of ITS2 region thereof;
(3) the target region of rDNA of Candida glabrata is of ITS1 region thereof;
(4) the target region of rDNA of Candida parapsilosis is of ITS2 region thereof,
(5) the target region of rDNA of Candida krusei is of ITS2 region thereof;
(6) the target region of rDNA of Candida guilliermondii is of ITS2 region thereof;
(7) the target region of rDNA of Candida auris is of ITS2 region thereof;
(8) the target region of rDNA of Cryptococcus neoformans is of IGS1 region thereof;
(9) the target region of rDNA of Cryptococcus deneoformans is of IGS1 region thereof;
(10) the target region of rDNA of Cryptococcus gattii is of IGS1 region thereof;
(11) the target region of rDNA of Fusarium spp. is of 26S region thereof;
(12) the target region of rDNA of Tnchosporon spp. is of 18S region thereof;
(13) the target region of genomic DNA of Schizosaccharomyces pombe is of Cytochrome B gene region; and/or
(14) the target region of rDNA of Candida spp. is of 18S region thereof.
“ITS”, as used herein, refers to the internal transcribed spacer region of rDNA. “iGS” refers to ribosomal intergenic spacer. The 26S and 18S regions are those regions of the rDNA that encode the 26S subunit of the ribosome and the 18S subunit of the ribosome, respectively. The cytochrome B gene is a gene that encodes for the mitochondrial protein cytochrome B and is found in the genomic DNA. The above target regions have been selected to ensure sufficient specificity for the respective species or family and avoid cross-reactivity with the other listed primer pairs.
In various embodiments, at least one primer of the Candida albicans primer set, the Candida tropicalis primer set, the Candida parapsilosis primer set, the Candida krusei primer set, the Candida guilliermondii primer set, and the Candida auris primer set comprises or consists of the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 1 over its entire length, or a complement thereof. The primer of SEQ ID NO:1 has sufficient complementarity to the target nucleic acids of all these species to form a target hybrid under the reaction conditions. The desired specificity of the detection is then brought about by the second primer of the pair, as described below.
The determination of the identity of nucleotide sequences is achieved by a sequence comparison. This comparison or alignment is typically based on the BLAST algorithm well-established and known in the art (See, e.g., Aitschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J, (1990): "Basic local alignment search tool", J. Mol. Biol. 215:403-410, and Aitschul, Stephan F., Thomas L. Madden, Alejandro A, Schaffer, Jinghui Zhang, Hheng Zhang, Webb Miller, and David J. Lipman (1997): "Gapped BLAST and PSi-BLAST: a new generation of protein database search programs"; Nucleic Acids Res., 25, S.3389-3402) and is in principle carried out by aligning stretches of nucleotides in the nucleotide
sequences with each other. Another aigorithm available in the art is the FASTA algorithm. Sequence comparisons (alignments), in particular multiple sequence comparisons, can be generated using computer programs. Commonly used are for example the Clustal series (See, e.g. Chenna et al. (2003): “Multiple sequence alignment with the Clustal series of programs,” Nucleic Acid Research 31 , 3497- 3500), T-Coffee (See, e.g., Notredame et al. (2000): “T-Coffee: A novel method for multiple sequence alignments”, J. Mol. Biol. 302, 205-217) or programs based thereon orthe respective algorithms. Further possible are sequence comparisons (alignments) with the computer program Vector NTI® Suite 10.3 (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, CA, USA) with the pre-set standard parameters, the AlignX-module of which is based on ClustalW. If not explicitly defined otherwise, sequence identity is determined using the BLAST algorithm.
Such a comparison allows determining the identity of two sequences and is typically expressed in % identity, i.e. the portion of identical nucleotides in the same or corresponding positions. If not explicitly stated otherwise, the sequence identities defined herein relate to the percentage over the entire length of the respective sequence, i.e. typically the reference sequence. If the reference sequence is 20 nucleotides in length, a sequence identity of 90 % means that 18 nucleotides in a query sequence are identical while 2 may differ.
In various embodiments, in particular in those in which the first primer of the primer pair comprises or consists of the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 1 over its entire length, or a complement thereof,
(1) at least one primer of the Candida albicans primer set comprises or consists of the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 2 over its entire length, or a complement thereof;
(2) at least one primer of the Candida tropicalis primer set comprises or consists of the nucleotide sequence of SEQ ID NO: 3 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 3 over its entire length, or a complement thereof;
(3) at least one primer of the Candida parapsilosis primer set comprises or consists of the nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 4 over its entire length, or a complement thereof;
(4) at least one primer of the Candida krusei primer set comprises or consists of the nucleotide sequence of SEQ ID NO: 5 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 5 over its entire length, or a complement thereof;
(5) at least one primer of the Candida guilliermondii primer set comprises or consists of the nucleotide sequence of SEQ ID NO: 6 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 6 over its entire length, or a complement thereof; and/or
(6) at least one primer of the Candida auris primer set comprises or consists of the nucleotide sequence of SEQ ID NO: 7 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 7 over its entire length, or a complement thereof.
In these embodiments (1) to (6), if the first primer of the primer pair is a primer that comprises or consists the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 1 over its entire length, or a complement thereof, the primer of embodiment (1) to (6) is the respective second primer of the primer pair. It is understood that if, in such embodiments, the first primer is a complement of SEQ ID NO:1 , the second primer is also the complement of the respective indicated sequence.
In various embodiments, the Candida glabrata primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 8 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 8 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 9 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 9 over its entire length , or a complement thereof. As described above, if one of these primer sequences is the complement of the indicated sequence, the other is also a complement of the given sequence. This similarly applies to ail other primer pairs described below.
In various embodiments, the Cryptococcus neoformans primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 10 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 10 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 11 over its entire length, or a complement thereof.
In various embodiments, the Cryptococcus deneoformans primer set comprises a primer having a nucleotide sequence of SEQ ID NO: 12 or the nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 12 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 13 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 13 over its entire length, or a complement thereof.
In various embodiments, the Cryplococcus gatlii primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 14 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 15 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 15 over its entire length, or a complement thereof.
In various embodiments, the Fusarium spp. primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 16 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 16 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 17 over its entire length, or a complement thereof.
In various embodiments, the Trichosporon spp. primer set comprises a primer having the nucleotide
sequence of SEQ ID NO: 18 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 18 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 19 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 19 over its entire length, or a complement thereof.
In various embodiments, the Schizosaccharomyces pombe primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 20 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 20 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 21 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 21 over its entire length, or a complement thereof.
In various embodiments the Candida spp. primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 22 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 22 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 23 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 23 over its entire length, or a complement thereof.
In various embodiments, any two or more of the primer sets described herein above may be used in a single multiplexing method. In various embodiments, the methods of the invention thus use at least 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14 primer sets as described herein. These primer sets are preferably different in that they are directed against different species or families of organisms and allow specific detection and identification thereof. In various embodiments, primer sets that are directed fo the same target but are directed against different target regions of the same target may also be included. However, the methods of the invention are preferably designed such that at least tow different pathogenic fungal species can be detected, i.e. at least two primer sets directed to different fungal pathogen species are included.
It is understood that the more general primer sets disclosed herein, namely those directed to Candida spp... Trichosporon spp., and Fusarium spp., are specific for the respective family of fungi, but cannot distinguish between the different species belonging to that family. However, their inclusion may still be preferable, as a positive signal with a Candida spp. primer set, while all specific Candida primer sets are negative indicates that the sample comprises a Candida species other than those specifically tested.
The combination of primer sets used in the methods of the invention may be selected from the groups comprising or consisting of:
(1) Candida aibicans primer set, Candida tropicalis primer set, Candida glabrata primer set, Candida parapsilosis primer set, Candida krusei primer set, Candida guilliermondii primer set, Candida auris primer set, Cryptococcus neoformans primer set, Cryptococcus deneoformans primer set, Cryptococcus galtii primer set, Fusarium spp. primer set and Trichosporon spp. primer set;
(2) Candida aibicans primer set, Candida tropicalis primer set, Candida glabrata primer set, Candida
parapsilosis primer set, Candida krusei primer set, Candida guilliermondii primer set and Candida auris primer set;
(3) Candida aibicans primer set, Candida tropicalis primer set, Candida giabrata primer set, Candida parapsilosis primer set and Candida krusei primer set;
(4) Candida albicans primer set, Candida tropicalis primer set and Candida giabrata primer set;
(5) Candida albicans primer set, Candida tropicalis primer set, Candida giabrata primer set, Candida parapsilosis primer set, Candida krusei primer set, Candida auris primer set, Cryptococcus neoformans primer set, Cryptococcus deneoforrnans primer set, Cryptococcus gattii primer set;
(6) Cryptococcus neoformans primer set, Cryptococcus deneoforrnans primer set, Cryptococcus gattii primer set, Fusarium spp. primer set and Trichosporon spp. primer set;
(7) Candida albicans primer set and Candida tropicalis primer set; and
(8) Cryptococcus neoformans primer set, Cryptococcus deneoforrnans primer set and Cryptococcus gattii primer set.
Any two or more of these primer set combinations (1) to (8) may be combined or supplemented by any one or more of the other primer sets described herein. These primer set combinations (1) to (8) preferably comprise the primers with the specific nucleotide sequences disclosed herein. in various embodiments, the primer set for Candida albicans disclosed herein as comprising the nucleic acid sequences of SEQ ID Nos:1 and 2 may be combined with further second primers for the other Candida species, namely those of SEQ ID Nos. 3-7 as well as with the primer set for C. giabrata, i,e. the primers with the sequences set forth in SEQ ID Nos. 8 and 9. in various embodiments, the primers of SEQ ID Nos. 1 , 2 and any one, two or more of 3, 4, 5, 6, and 7 are included. These are in various embodiments further combined with those of SEQ ID Nos. 8 and 9. In various embodiments, the primers of SEQ ID Nos. 1 and any two of 3-7 are combined, optionally further combined with those of SEQ ID Nos. 8 and 9. Such primer set combinations are specificaliy suited for methods for diagnosis of fungal sepsis caused by Candida species. In order to further cover not specificaliy named Candida species the Candida spp. primer set, i.e. of SEQ ID Nos, 22 and 23, may be additionally included in all aforementioned embodiments.
In the next step of the method, the amplification reaction is carried out. In this reaction the hybridized primer:target complexes are recognized by a polymerase and the respective target sequence amplified. The preferred method of amplification in the methods of the invention is PCR. PCR is a well-known and established technique and commonly known and practiced by those skilled in the art. Said amplification step generates a multitude of amplicons of all target nucleic acids present in the sample for which also a suitable primer set is included by thermocycling the reaction mixture in the presence of a thermostable DNA polymerase to allow multiple cycles of primer annealing and elongation.
The primer sets have been designed such that they have under the conditions of the described methods no significant cross-reactivity and thus allow specific detection of one or more, preferably 2, 3, 4, 5, 6 ,7,
8, 9, 10, or 11 species for which primer sets are described herein. These primer sets may therefore be used in combination to allow multiplexing. In the multiplexing methods of the invention, in various embodiments, at least 2, preferably 3 or more of the species-specific primer sets are used. These may be further combined with 1 , 2 or 3 of the family-specific primer sets, i.e. the Candida, the Fusarium or the Trichosporon primer sets. Ail these primer sets disclosed herein below with their nucleic acid sequences have been found to allow simultaneous use without significant interference or crossreactivity.
The primers used in the methods of the invention may comprise further portions in addition to their target-complementary nucleotide sequence necessary for target recognition and binding. These portions may include further nucleotide sequences that do not base-pair with the target and serve different purposes, for example providing ligation sites or restriction enzyme sites or for interaction with enzymes/proteins, such as the polymerase, or non-nucleotide moieties, such as labeling or coupling moieties, including biotin and various chromophores or fluorophores. in various embodiments, one of the primers of a given primer pair is biotinylated on its 5’ end.
In a following step of the methods of the invention, the presence and/or amount of the target nucleic acid(s) in the sample is detected by contacting the amplified sample / reaction mixture containing the amplicons of any target nucleic acid that may have been present in the sample with a combination of oligonucleotide detection probes comprising at least one probe specific for each of the targets of the amplification primer sets used. These probes are generally specific for the target nucleic acid sequence as well as the amplicons produced by the amplification of the target nucleic acids in that they hybridize to these (amplified) target nucleic acid sequences. This hybridization is again carried out under conditions that are sufficiently strict to avoid unspecific hybridization or prevent hybridization to nucleic acids that carry one or more mismatches. The thus formed probertarget hybrids are then detected and optionally quantified to determine the presence and/or amount of the respective target nucleic acids in the sample. Various detection methods are known in the art and can be chosen by those skilled in the art based on preference. Common detection methods include radio-labeled, fluorophore- or chromophore-labeled probes that can then be detected by a read-out of the radioactivity, the fluorescence or color, for example by using available instruments, such as scintillation counters and spectrometers. Other options include the use of probes coupled to a solid support, such as a nanoparticle or bead, including magnetic beads, or so-called micro-discs. One system that may advantageously used for probertarget complex detection is the rtCode™ system of PlexBio™.
In various embodiments, the combination of oligonucleotide detection probes comprises two or more probes selected from:
(1) a Candida albicans probe having a nucleotide sequence specific to a target region in the rDNA of Candida albicans',
(2) a Candida tropicalis probe having a nucleotide sequence specific to a target region in the rDNA of Candida tropicalis;
(3) a Candida glabrata probe having a nucleotide sequence specific to a target region in the rDNA of Candida glabrata;
(4) a Candida parapsilosis probe having a nucleotide sequence specific to a target region in the rDNA of Candida parapsilosis;
(5) a Candida krusei probe having a nucleotide sequence specific to a target region in the rDNA of Candida krusei;
(6) a Candida guilliermondii probe having a nucleotide sequence specific to a target region in the rDNA of Candida guilliermondii;
(7) a Candida auris probe having a nucleotide sequence specific to a target region in the rDNA of Candida auris;
(8) a Cryptococcus neoformans probe having a nucleotide sequence specific to a target region in the rDNA of Cryptococcus neoformans;
(9) a Cryptococcus deneoformans probe having a nucleotide sequence specific to a target region in the rDNA of Cryptococcus deneoformans;
(10) a Cryptococcus gattii probe having a nucleotide sequence specific to a target region in the rDNA of Cryptococcus gattii;
(11) a Fusarium spp. probe having a nucleotide sequence specific to a target region in the rDNA of Fusarium spp. ;
(12) a Trichosporon spp. probe having a nucleotide sequence specific to a target region in the rDNA of Trichosporon spp. ;
(13) a Schizosaccharomyces pombe probe having a nucleotide sequence specific to a target region in the genomic DNA of Schizosaccharomyces pombe; and
(14) a Candida spp. probe having a nucleotide sequence specific to a target region in the rDNA of Candida spp.
It is understood that the probes are selected in dependence of the primer sets used for amplification such that for each potential target a probe is used. Accordingly, the method uses suitable primer pairprobe combinations. The target regions for the probes are generally selected such that they are within the amplified sequence stretch, i.e. lie in the region that is amplified by the use of the primers in an amplification reaction. It is preferably that the to be detected sequence does not overlap with the primer binding sites, but rather lies between the sequence stretches bound by the forward and reverse primers. As a consequence, thereof, the target region of the probes used is the same as that to which the primers are directed. Therefore, in various embodiments in accordance with the preferred primer target regions,
(1) the target region of rDNA of Candida albicans is in the ITS2 region thereof;
(2) the target region of rDNA of Candida tropicalis is in the ITS2 region thereof;
(3) the target region of rDNA of Candida glabrata is in the ITS 1 region thereof;
(4) the target region of rDNA of Candida parapsilosis is in the ITS2 region thereof,
(5) the target region of rDNA of Candida krusei is in the ITS2 region thereof;
(6) the target region of rDNA of Candida guilliermondii is in the ITS2 region thereof;
(7) the target region of rDNA of Candida auris is in the ITS2 region thereof;
(8) the target region of rDNA of Cryptococcus neoformans is in the IGS1 region thereof;
(9) the target region of rDNA of Cryptococcus deneoformans is in the IGS1 region thereof;
(10) the target region of rDNA of Cryptococcus gattii is in the iGS1 region thereof;
(11) the target region of rDNA of Fusarium spp. is in the 26S region thereof;
(12) the target region of rDNA of Trichosporon spp. is in the 18S region thereof
(13) the target region of genomic DNA of Schizosaccharomyces pombe is in the Cytochrome B gene region; and
(14) the target region of rDNA of Candida spp. is in the 18S region thereof.
The probes used in the methods of the invention may comprise further portions in addition to their target- complementary nucleotide sequence necessary for target recognition and binding. These portions may include further nucleotide sequences that do not base-pair with the target and serve different purposes, for example providing ligation sites or restriction enzyme sites or for interaction with enzymes/proteins, such as the polymerase, or non-nucleotide moieties, such as labeling or coupling moieties, including biotin and various chromophores or fiuorophores, In various embodiments, such additional portions may provide for a linker function that allows coupling with a non-nucleotide moiety. For this purpose, these nucleotides or nucleotide sequences, for example dT sequences of 2 to 20 nucleotides in length, may be further modified with a functional group or a spacer including a functional group, such as an amino group. This may then allow coupling to various delectable labels, such as pi-code microdiscs (ex PlexBio, TW).
Probe sequences may be designed in accordance with the above-described principles. It is further important that the probe sequences are again sufficiently specific to avoid cross-reactivity between different targets. Therefore, within the amplified sequence stretches sufficiently different target regions for probe binding need to be identified that allow preferential binding of a specific probe forthe respective target over binding of any other probe directed to a different target. Herein disclosed are probe sequences that have been designed accordingly and can be used in combination without the risk of cross-reactivity and thus false positives and undesired interference with each other.
In various embodiments,
(1) the Candida albicans probe comprises the nucleotide sequence of SEQ ID NO: 24 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 24 over its entire length, or a complement thereof;
(2) the Candida tropicalis probe comprises the nucleotide sequence of SEQ ID NO: 25 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 25 over its entire length, or a complement thereof;
(3) the Candida giabrata probe comprises the nucleotide sequence of SEQ ID NO: 30 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 30 over its entire length, or a complement thereof;
(4) the Candida parapsilosis probe comprises the nucleotide sequence of SEQ ID NO: 26 or a
nucleotide sequence having at ieast 90% sequence identity to SEQ ID NO: 26 over its entire length, or a complement thereof;
(5) the Candida krusei probe comprises the nucleotide sequence of SEQ ID NO: 27 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 27 over its entire length, or a complement thereof;
(6) the Candida guilliermondii probe comprises the nucleotide sequence of SEQ ID NO: 28 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 28 over its entire length, or a complement thereof;
(7) the Candida auris probe comprises the nucleotide sequence of SEQ ID NO: 29 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 29 over its entire length, or a complement thereof;
(8) the Cryptococcus neoformans probe comprises the nucleotide sequence of SEQ ID NO: 32 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 32 over its entire length, or a complement thereof;
(9) the Cryptococcus deneoformans probe comprises the nucleotide sequence of SEQ ID NO: 33 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 33 over its entire length, or a complement thereof;
(10) the Cryptococcus gattii probe comprises the nucleotide sequence of SEQ ID NO: 34 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 34 over its entire length, or a complement thereof;
(11) the Fusarium spp. probe comprises the nucleotide sequence of SEQ ID NO: 35 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 35 over its entire length, or a complement thereof;
(12) the Trichosporon spp. probe comprises the nucleotide sequence of SEQ ID NO: 36 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 36 over its entire length, or a complement thereof;
(13) the Schizosaccharomyces pombe probe comprises the nucleotide sequence of SEQ ID NO: 37 or a nucleotide sequence having at least 90'% sequence identity to SEQ ID NO: 37 over its entire length, or a complement thereof; and
(14) the Candida spp. probe comprises the nucleotide sequence of SEQ ID NO: 31 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 31 over its entire length, or a complement thereof.
For the determination of the sequence identity, the same as described above in relation to the primers applies.
The combination of probes may comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13 or at least 14 different probes. In various embodiments, the different probes are each specific for a different pathogenic fungi species. As described above, the different probes are selected such that at least one probe for each potentially
amplified target, i.e. those targets for which primers have been used/included, is present to ensure that each target that has successfully been amplified is subsequently detected. The respective preferred combinations of probes are thus equivalent to the preferred combinations of primer sets disclosed herein. Accordingly, the combination of probes may be selected from the group comprising or consisting of:
(1) Candida tropicalis probe, Candida giabrata probe, Candida parapsilosis probe, Candida krusei probe, Candida guilliermondii probe, Candida auris probe, Cryptococcus neoformans probe, Cryptococcus deneoformans probe, Cryptococcus gattii probe, Fusarium spp. probe and Trichosporon spp. probe;
(2) Candida albicans probe, Candida tropicaiis probe, Candida giabrata probe, Candida parapsilosis probe, Candida krusei probe, Candida guilliermondii probe and Candida auris probe;
(3) Candida albicans probe, Candida tropicalis probe, Candida giabrata probe, Candida parapsilosis probe and Candida krusei probe;
(4) Candida albicans probe, Candida tropicalis probe and Candida giabrata probe;
(5) Candida albicans probe, Candida tropicalis probe, Candida giabrata probe, Candida parapsilosis probe, Candida krusei probe, Candida auris probe, Cryptococcus neoformans probe, Cryptococcus deneoformans probe, Cryptooocous gattii probe;
(6) Cryptococcus neoformans probe, Cryptococcus deneoformans probe, Cryptococcus gattii probe, Fusarium spp. probe and Trichosporon spp. probe;
(7) Candida albicans probe and Candida tropicalis probe; and
(8) Cryptococcus neoformans probe, Cryptococcus deneoformans probe and Cryptococcus gattii probe.
The method of the invention may be a method for the diagnosis of fungal sepsis in a subject. Specifically, the invention also covers a method for the diagnosis of fungal sepsis in a subject that comprises a first step of obtaining a sample from said subject and then subjecting said sample to the methods of the invention forthe detection of the presence and/or amount of two or more target nucleic acids in a sample, wherein the two or more target nucleic acids are of fungal pathogens involved in fungal sepsis. The respective analysis steps are carried out in vitro, i.e. using the sample obtained from the subject.
The subject may be a human subject. In various embodiments, the sample is a blood, serum or plasma sample. Alternatively, other sample types may be used, including tissue or other body fluid samples, although blood, plasma and serum samples are the most common.
In these methods for the diagnosis of fungal sepsis in a subject, the subject may be diagnosed with fungal sepsis, if one or more of the target nucleic acids are detected in the sample. In other words, if in the methods for the detection of two or more target nucleic acids in a sample described herein one or more of said targets is successfully identified, this may in a further step then lead to diagnosing the subject with fungal infection and fungal sepsis. It is understood that for the final diagnosis of fungal sepsis other factors may also be considered, including medical conditions, such as fever and the like.
Depending on the diagnosis and the detected microorganism an appropriate therapy may be selected, typically an antifungal agent that is effective against the identified pathogen. For all fungal pathogens specifically described herein, appropriate antifungal treatment options exist and suitable medicaments are available in the art. The skilled person can thus readily select an appropriate antifungal agent depending on the results of the inventive methods.
The invention further features kits for carrying out the methods of the invention. Such kits typically comprise a number of agents and reagents useful and necessary to perform the methods of the invention. In various embodiments, the kits thus comprise at least the primer sets and corresponding probes disclosed herein, i.e. two or more amplification primer sets and two or more detection probes. The primers and probes are preferably those that have been described in relation to the methods herein. in addition to the primers and probes, the kit may comprise a number of further agents that may selected from, without limitation, a polymerase, buffers, mononucleotides, and all other agents necessary for a PCR reaction and subsequent isolation/purification of the amplicons.
The invention further features the use of these kits for the diagnosis of fungal sepsis in a subject, i.e. for use in the methods of diagnosis described herein above. This means that also encompassed are methods for the diagnosis of fungal sepsis in a subject, wherein said kits are used, preferably to allow practicing the methods of the invention. The subject and the sample analyzed may be as described above for the methods of the invention.
All embodiments disclosed herein in relation to the methods of the invention similarly apply to the kits and uses disclosed and vice versa.
Primers and probes described above are listed below:
Primers:
Probes:
Examples
Example 1: Fungal sepsis panel and testing thereof
A panel of primer/probe combinations was tested using DNAs of different fungal species. For each PGR reaction genomic DNA (2 pg) of one fungus was used as a template. The full multiplex primer mixture was used for each conditions. Said primer mixture comprised primers for Candida albicans; Candida
tropicalis, Candida giabrata, Candida parapsiiosis, Candida krusei, Candida guilliermondii, Candida auris, Cryptococcus neoformans, Cryptococcus deneoformans, Cryptococcus gattii, Fusarium spp., Trichosporon spp., Schizosaccharomyces pombe. and Candida spp. having the nucleic acid sequences set forth in SEQ ID Nos. 1 -23. PCR conditions were 94°C for 1 min; 40x cycles of 94°C for 30 sec, 60°C for 1 min; and 72°C for 10 min.
In order to make the amplicon detectable in a subsequent step of the method, one of the primers for each target is conjugated with biotin at the 5’ end as shown in the list below (5Biosg):
The probes (SEQ ID NO 24-37) of individual targets were modified with adding a short oligo (dT) fragment at the 5’ end. The short oligo (dT) fragment serves as a linker between the pi-Code microdiscs (ex PlexBio, TW) and the probe sequence. In addition, the 5’ end of the oligo (dT) fragment was modified with an amino group with a (C6) spacer arm as shown below (5AmMC6).
Probes:
For probe hybridization, multiplex nCode™ probes (ex PlexBio™) were prepared in advance. The probe mixture comprised probes for each of the targets of the primer sets having the nucleotide sequences set forth in SEQ ID Nos. 24-37. The mixture was added into each well of a 96-well plate for later use.
20|j I of each PCR product was denatured at 95°C for 5 min and chilled on ice for 2 min. The denatured PCR product was then added to an individual well with the probe mixture and probe hybridization was done in an automated IntelliPlex 1000 device (PlexBio™). Detection of the formed probe:target hybrids was done by putting the plate in an automated BioPlex 100 detection device (PlexBio™) and the data obtained collected and subsequently analyzed.
The results of this analysis are shown in Figure 1. Each sample was detected specifically by the corresponding probe(s), for example, Candida albicans gDNA was detected by the specific probe Probe_C.aibi_JTS2 and the pan-Candida probe Probe_Candida_spp_18S. The strain of Cryptococcus neoformans is a hybrid strain, it contains chromosomes of both C. neoformans and C.deneoformans, therefore, both probes (Probe_Cr.neof_.IGS1 and Probe_Cr.deneofo_.IGS1) were positive. There is no cross-reaction among different fungal species. Every well had all the different piCode-probes, only the specific probe(s) showed strong positive signal, other probes just showed background signals with MFI ~ 2000. The probe of Candida albicans has no cross-reaction with Candida dubliniesis, although these two strains are very similar and some commercially available test assays claim that they cannot discriminate these two species. SAFE Control probe is a probe with random artificial sequence conjugated with Biotin at the 3’ end, which is supposed to be positive after the test. Blank Control probe
is with the same artificial sequence which is supposed to be negative since no amplicons are supposed to hybridize on it.
The primers used in the panel were found not to interfere with each other. The same applies to the probes used. Each sample only needed one round of PCR to obtain the amplicon.
Claims
1 . Method for the detection of the presence and/or amount of two or more target nucleic acids in a sample, wherein the two or more target nucleic acids are of fungal pathogens involved in fungal sepsis, the method comprising: i) contacting the sample with a combination of two or more oligonucleotide amplification primer sets under conditions that allow hybridization of the oligonucleotide primers to the target nucleic acids of the fungal pathogens, wherein the two or more primer sets are directed to different target nucleic acids of at least two different fungal pathogen species involved in fungal sepsis and are independently selected from the group consisting of:
(1) a Candida albicans primer set targeting a nucleotide sequence in the rDNA of Candida albicans;
(2) a Candida tropicalis primer set targeting a nucleotide sequence in the rDNA of Candida tropicalis;
(3) a Candida glabrata primer set targeting a nucleotide sequence in the rDNA of Candida glabrata;
(4) a Candida parapsilosis primer set targeting a nucleotide sequence in the rDNA of Candida parapsilosis;
(5) a Candida krusei primer set targeting a nucleotide sequence in the rDNA of Candida krusei;
(6) a Candida guilliermondii primer set targeting a nucleotide sequence in the rDNA of Candida guilliermondii;
(7) a Candida auris primer set targeting a nucleotide sequence in the rDNA of Candida auris;
(8) a Cryptococcus neoformans primer set targeting a nucleotide sequence in the rDNA of Cryptococcus neoformans;
(9) a Cryptococcus deneoformans primer set targeting a nucleotide sequence in the rDNA of Cryptococcus deneoformans;
(10) a Cryptococcus gatii primer set targeting a nucleotide sequence in the rDNA of Cryptococcus gattii;
(11) a Fusarium spp. primer set targeting a nucleotide sequence in the rDNA of Fusarium spp.;
(12) a Trichosporon spp. primer set targeting a nucleotide sequence in the rDNA of Trichosporon spp. ;
(13) a Schizosaccharomyces pombe primer set targeting a nucleotide sequences in the genomic DNA of Schizosaccharomyces pombe; and
(14) a Candida spp. primer set targeting a nucleotide sequence in the rDNA of Candida spp.; and ii) amplifying the target nucleic acid(s) hybridized to any one or more of the oligonucleotide primers by PCR; and iii) detecting the presence and/or amount of the target nucleic acid(s) in the sample by contacting the PCR amplified sample of step ii) with combination of oligonucleotide detection probes comprising at least one probe specific for each of the amplicons produced by the amplification of the target nucleic
acids in step ii) under conditions that allow the hybridization of the probes to their specific target nucleic acid sequences and determining the presence and/or amount of probe :target hybrids.
2. The method according to Ciaim 1 , wherein:
(1) the target region of rDNA of Candida albicans is of ITS2 region thereof;
(2) the target region of rDNA of Candida tropicalis is of ITS2 region thereof;
(3) the target region of rDNA of Candida glabrata is of ITS1 region thereof;
(4) the target region of rDNA of Candida parapsilosis is of ITS2 region thereof,
(5) the target region of rDNA of Candida krusei is of ITS2 region thereof;
(6) the target region of rDNA of Candida guiiliermondii is of ITS2 region thereof;
(7) the target region of rDNA of Candida auris is of ITS2 region thereof;
(8) the target region of rDNA of Cryptococcus neoformans is of IGS1 region thereof;
(9) the target region of rDNA of Cryptococcus deneoformans is of IGS1 region thereof;
(10) the target region of rDNA of Cryptococcus gattii is of IGS1 region thereof;
(11) the target region of rDNA of Fusarium spp. is of 26S region thereof;
(12) the target region of rDNA of Trichosporon spp. is of 18S region thereof;
(13) the target region of genomic DNA of Schizosaccharomyces pombe is of Cytochrome B gene region; and
(14) the target region of rDNA of Candida spp. is of 18S region thereof.
3. The method according to Claim 1 or 2, wherein: at least one primer of the Candida albicans primer set, the Candida tropicalis primer set, the Candida parapsilosis primer set, the Candida krusei primer set, the Candida guiiliermondii primer set, and the Candida auris primer set comprises the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 1 over its entire length, or a complement thereof.
4. The method according to any one of Claims 1-3, wherein:
(1) at least one primer of the Candida albicans primer set comprises the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 2 over its entire length, or a complement thereof;
(2) at least one primer of the Candida tropicalis primer set comprises the nucleotide sequence of SEQ ID NO: 3 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 3 over its entire length, or a complement thereof;
(3) at least one primer of the Candida parapsilosis primer set comprises the nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 4 over its entire length, or a complement thereof;
(4) at least one primer of the Candida krusei primer set comprises the nucleotide sequence of SEQ ID NO: 5 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 5 over its entire length, or a complement thereof;
(5) at least one primer of the Candida guiiliermondii primer set comprises the nucleotide sequence
of SEQ ID NO: 6 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 6 over its entire length, or a complement thereof; and
(6) at least one primer of the Candida auris primer set comprises the nucleotide sequence of SEQ ID NO: 7 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 7 over its entire length, or a complement thereof.
5. The method according to any one of Claims 1-4, wherein
(1) the Candida glabrata primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 8 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 8 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 9 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 9 over its entire length, or a complement thereof;
(2) the Cryptococcus neoformans primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 10 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 10 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 11 over its entire length, or a complement thereof;
(3) the Cryptococcus deneoformans primer set comprises a primer having a nucleotide sequence of SEQ ID NO: 12 or the nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 12 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 13 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 13 over its entire length, or a complement thereof;
(4) the Cryptococcus gattii primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 14 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 15 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 15 over its entire length, or a complement thereof;
(5) the Fusarium spp. primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 16 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 16 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 17 over its entire length, or a complement thereof;
(6) the Trichosporon spp. primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 18 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 18 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 19 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 19 over its entire length, or a complement thereof;
(7) the Schizosaccharomyces pombe primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 20 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 20 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID
NO: 21 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 21 over its entire length, or a complement thereof; and
(8) the Candida spp. primer set comprises a primer having the nucleotide sequence of SEQ ID NO: 22 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 22 over its entire length, or a complement thereof, and a primer having the nucleotide sequence of SEQ ID NO: 23 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 23 over its entire length, or a complement thereof.
6. The method according to any one of Claims 1-5, wherein the combination of oligonucleotide detection probes comprises two or more probes selected from:
(1) a Candida albicans probe having a nucleotide sequence specific to a target region in the rDNA of Candida albicans;
(2) a Candida tropicalis probe having a nucleotide sequence specific to a target region in the rDNA of Candida Iropicalis;
(3) a Candida glabrata probe having a nucleotide sequence specific to a target region in the rDNA of Candida glabrata;
(4) a Candida parapsilosis probe having a nucleotide sequence specific to a target region in the rDNA of Candida parapsilosis;
(5) a Candida krusei probe having a nucleotide sequence specific to a target region in the rDNA of Candida krusei;
(6) a Candida guilliermondii probe having a nucleotide sequence specific to a target region in the rDNA of Candida guilliermondii;
(7) a Candida auris probe having a nucleotide sequence specific to a target region in the rDNA of Candida auris;
(8) a Cryptococcus neoformans probe having a nucleotide sequence specific to a target region in the rDNA of Cryptococcus neoformans;
(9) a Cryptococcus deneoformans probe having a nucleotide sequence specific to a target region in the rDNA of Cryptococcus deneoformans;
(10) a Cryptococcus gaitii probe having a nucleotide sequence specific to a target region in the rDNA of Cryptococcus gaitii;
(11) a Fusarium spp. probe having a nucleotide sequence specific to a target region in the rDNA of . probe having a nucleotide sequence specific to a target region in the rDNA
yces pombe probe having a nucleotide sequence specific to a target region in the genomic DNA of Schizosaccharomyces pombe; and
(14) a Candida spp. probe having a nucleotide sequence specific to a target region in the rDNA of Candida spp.
7. The method according to Claim 6, wherein:
(1) the target region of rDNA of Candida albicans is in the iTS2 region thereof;
(2) the target region of rDNA of Candida tropicalis is in the ITS2 region thereof;
(3) the target region of rDNA of Candida glabrata is in the ITS 1 region thereof;
(4) the target region of rDNA of Candida parapsiiosis is in the ITS2 region thereof,
(5) the target region of rDNA of Candida krusei is in the iTS2 region thereof;
(6) the target region of rDNA of Candida guiiliermondii is in the iTS2 region thereof;
(7) the target region of rDNA of Candida auris is in the iTS2 region thereof;
(8) the target region of rDNA of Cryptococcus neoformans is in the iGS1 region thereof;
(9) the target region of rDNA of Cryptococcus deneoformans is in the iGS1 region thereof;
(10) the target region of rDNA of Cryptococcus gattii is in the IGS1 region thereof;
(11) the target region of rDNA of Fusarium spp. is in the 26S region thereof;
(12) the target region of rDNA of Trichosporon spp. is in the 18S region thereof
(13) the target region of genomic DNA of Schizosaccharomyces pombe is in the Cytochrome B gene region; and
(14) the target region of rDNA of Candida spp. is in the 18S region thereof.
8. The method according to any one of Claim 6 or 7, wherein:
(1) the Candida albicans probe comprises the nucleotide sequence of SEQ ID NO: 24 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 24 over its entire length, or a complement thereof;
(2) the Candida tropicalis probe comprises the nucleotide sequence of SEQ ID NO: 25 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 25 over its entire length, or a complement thereof;
(3) the Candida glabrata probe comprises the nucleotide sequence of SEQ ID NO: 30 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 30 over its entire length, or a complement thereof;
(4) the Candida parapsiiosis probe comprises the nucleotide sequence of SEQ ID NO: 26 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 26 over its entire length, or a complement thereof;
(5) the Candida krusei probe comprises the nucleotide sequence of SEQ ID NO: 27 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 27 over its entire length, or a complement thereof;
(6) the Candida guiiliermondii probe comprises the nucleotide sequence of SEQ ID NO: 28 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 28 over its entire length, or a complement thereof;
(7) the Candida auris probe comprises the nucleotide sequence of SEQ ID NO: 29 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 29 over its entire length, or a complement thereof;
(8) the Cryptococcus neoformans probe comprises the nucleotide sequence of SEQ ID NO: 32 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 32 over its entire length, or
a complement thereof;
(9) the Cryptococcus deneoformans probe comprises the nucleotide sequence of SEQ ID NO: 33 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 33 over its entire length, or a complement thereof;
(10) the Cryptococcus gattii probe comprises the nucleotide sequence of SEQ ID NO: 34 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 34 over its entire length, or a complement thereof;
(11) the Fusarium spp. probe comprises the nucleotide sequence of SEQ ID NO: 35 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 35 over its entire length, or a complement thereof;
(12) the Trichosporon spp. probe comprises the nucleotide sequence of SEQ ID NO: 36 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 36 over its entire length, or a complement thereof;
(13) the Schizosaccharomyces pombe probe comprises the nucleotide sequence of SEQ ID NO: 37 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 37 over its entire length, or a complement thereof; and
(14) the Candida spp. probe comprises the nucleotide sequence of SEQ ID NO: 31 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 31 over its entire length, or a complement thereof.
9. The method according to any one of Claims 1 -8, wherein the combination of primer sets comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13 or at least 14 different primer sets.
10. The method according to any one of Claims 1 to 9, wherein the combination of primer sets is selected from the group consisting of:
(1) Candida albicans primer set, Candida tropicalis primer set, Candida glabrata primer set, Candida parapsilosis primer set, Candida krusei primer set, Candida guilliermondii primer set, Candida auris primer set, Cryptococcus neoformans primer set, Cryptococcus deneoformans primer set, Cryptococcus gattii primer set, Fusarium spp. primer set and Trichosporon spp. primer set;
(2) Candida albicans primer set, Candida tropicalis primer set, Candida glabrata primer set, Candida parapsilosis primer set, Candida krusei primer set, Candida guilliermondii primer set and Candida auris primer set;'
(3) Candida albicans primer set, Candida tropicalis primer set, Candida glabrata primer set, Candida parapsilosis primer set and Candida krusei primer set;
(4) Candida albicans primer set, Candida tropicalis primer set and Candida glabrata primer set;
(5) Candida albicans primer set, Candida tropicalis primer set, Candida glabrata primer set, Candida parapsilosis primer set, Candida krusei primer set, Candida auris primer set, Cryptococcus neoformans primer set, Cryptococcus deneoformans primer set, Cryptococcus gattii primer set;
(6) Cryptococcus neoformans primer set, Cryptococcus deneoformans primer set, Cryptococcus
gattii primer set, Fusarium spp. primer set and Trichosporon spp. primer set;
(7) Candida albicans primer set and Candida tropicalis primer set; and
(8) Cryptococcus neoformans primer set, Cryptococcus deneoformans primer set and Cryptococcus gattii primer set;
11. The method according to any one of Claims 1 to 10, wherein combination of probes comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least S, at least 9, at least 10, at least 11 , at least
12, at least 13 or at least 14 different probes, wherein preferably the different probes are each specific for a different pathogenic fungi species.
12. The method according to any one of Claims 1 to 11 , wherein the combination of probes is selected from the group consisting of:
(1) Candida tropicalis probe, Candida giabrata probe, Candida parapsilosis probe, Candida krusei probe, Candida guilliermondii probe, Candida auris probe, Cryptococcus neoformans probe, Cryptococcus deneoformans probe, Cryptococcus gattii probe, Fusarium spp. probe and Trichosporon spp. probe;
(2) Candida albicans probe, Candida tropicalis probe, Candida giabrata probe, Candida parapsilosis probe, Candida krusei probe, Candida guilliermondii probe and Candida auris probe;
(3) Candida albicans probe, Candida tropicalis probe, Candida giabrata probe, Candida parapsilosis probe and Candida krusei probe;
(4) Candida albicans probe, Candida tropicalis probe and Candida giabrata probe;
(5) Candida albicans probe, Candida tropicalis probe, Candida giabrata probe, Candida parapsilosis probe, Candida krusei probe, Candida auris probe, Cryptococcus neoformans probe, Cryptococcus deneoformans probe, Cryptococcus gattii probe;
(6) Cryptococcus neoformans probe, Cryptococcus deneoformans probe, Cryptococcus gattii probe, Fusarium spp. probe and Trichosporon spp. probe;
(7) Candida albicans probe and Candida tropicalis probe; and
(8) Cryptococcus neoformans probe, Cryptococcus deneoformans probe and Cryptococcus gattii probe.
13. Kit for carrying out the method of any one of claims 1 to 12, wherein the kit comprises two or more amplification primer sets and two or more detection probes, wherein
(A) the two or more amplification primer sets are directed to different target nucleic acids of at least two different fungal pathogen species involved in fungal sepsis and are independently selected from the group consisting of:
(1) a Candida albicans primer set targeting a nucleotide sequence in the rDNA of Candida albicans;
(2) a Candida tropicalis primer set targeting a nucleotide sequence in the rDNA of Candida tropicalis;
(3) a Candida giabrata primer set targeting a nucleotide sequence in the rDNA of Candida
glabrata:
(4) a Candida parapsilosis primer set targeting a nucleotide sequence in the rDNA of Candida parapsilosis:
(5) a Candida krusei primer set targeting a nucleotide sequence in the rDNA of Candida krusei:
(6) a Candida guilliermondii primer set targeting a nucleotide sequence in the rDNA of Candida guilliermondii:
(7) a Candida auris primer set targeting a nucleotide sequence in the rDNA of Candida auris:
(8) a CryptocoGcus neoformans primer set targeting a nucleotide sequence in the rDNA of CryptoGOCGus neoformans:
(9) a Cryptococcus deneoformans primer set targeting a nucleotide sequence in the rDNA of CryptocoGcus deneoformans:
(10) a Cryptococcus gattii primer set targeting a nucleotide sequence in the rDNA of CryptocGccus gattii:
(11) a Fusarium spp. primer set targeting a nucleotide sequence in the rDNA of Fusarium spp.:
(12) a Trichosporon spp. primer set targeting a nucleotide sequence in the rDNA of Trichosporon spp. ;
(13) a Schizosaccharomyces pombe primer set targeting a nucleotide sequences in the genomic DNA of Schizosaccharomyces pombe: and
(14) a Candida spp. primer set targeting a nucleotide sequence in the rDNA of Candida spp.: and
(B) the two or more detection probes comprise at least one probe specific for each of the amplicons produced by amplification of the target nucleic acids by each of the two or more primer pairs.
14. The method according to any one of claims 1 to 12, wherein the method is a method for the diagnosis of fungal sepsis in a subject.
15. The method according to claim 14, wherein the subject is a human.
16. The method according to claim 14 or 15, wherein if one or more of the target nucleic acids are detected in the sample, the subject is diagnosed with fungal sepsis.
17. Use of the kit of claim 13 for the diagnosis of fungal sepsis in a subject.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SG10202250839C | 2022-08-26 | ||
| SG10202250839C | 2022-08-26 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2024043830A1 true WO2024043830A1 (en) | 2024-02-29 |
Family
ID=90014181
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/SG2023/050556 WO2024043830A1 (en) | 2022-08-26 | 2023-08-15 | Methods for the detection and identification of fungal species |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2024043830A1 (en) |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5827695A (en) * | 1997-08-04 | 1998-10-27 | Novartis Finance Corporation | Detection of wheat fungal pathogens using the polymerase chain reaction |
| US20050164243A1 (en) * | 1999-05-28 | 2005-07-28 | Terry Smith | Nucleic acid probes and methods for detecting clinically important fungal pathogens |
| JP2006191922A (en) * | 2004-12-15 | 2006-07-27 | Shimadzu Corp | System for estimating strain of microorganism and method for the same |
| US20080248970A1 (en) * | 2005-09-27 | 2008-10-09 | Morrison Christine J | Compositions and Methods for the Detection of Candida Species |
| WO2014133153A1 (en) * | 2013-03-01 | 2014-09-04 | 株式会社ヤクルト本社 | Method for collective detection of pathogenic fungi |
| JP2018038345A (en) * | 2016-09-09 | 2018-03-15 | 丸善製薬株式会社 | New strain of sporidiobolus pararoseus, food and drink, method for producing food and drink, skin cosmetics, and method for producing skin cosmetics |
| US20200270708A1 (en) * | 2019-02-25 | 2020-08-27 | Bakotic Pathology Associates, LLC | Methods of screening for causative agents of onychodystrophy |
| CN112143821A (en) * | 2020-09-30 | 2020-12-29 | 北京师范大学 | Reagent for detecting candida albicans, cryptococcus neoformans and klebsiella pneumoniae by FRET and application thereof |
-
2023
- 2023-08-15 WO PCT/SG2023/050556 patent/WO2024043830A1/en unknown
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5827695A (en) * | 1997-08-04 | 1998-10-27 | Novartis Finance Corporation | Detection of wheat fungal pathogens using the polymerase chain reaction |
| US20050164243A1 (en) * | 1999-05-28 | 2005-07-28 | Terry Smith | Nucleic acid probes and methods for detecting clinically important fungal pathogens |
| JP2006191922A (en) * | 2004-12-15 | 2006-07-27 | Shimadzu Corp | System for estimating strain of microorganism and method for the same |
| US20080248970A1 (en) * | 2005-09-27 | 2008-10-09 | Morrison Christine J | Compositions and Methods for the Detection of Candida Species |
| WO2014133153A1 (en) * | 2013-03-01 | 2014-09-04 | 株式会社ヤクルト本社 | Method for collective detection of pathogenic fungi |
| JP2018038345A (en) * | 2016-09-09 | 2018-03-15 | 丸善製薬株式会社 | New strain of sporidiobolus pararoseus, food and drink, method for producing food and drink, skin cosmetics, and method for producing skin cosmetics |
| US20200270708A1 (en) * | 2019-02-25 | 2020-08-27 | Bakotic Pathology Associates, LLC | Methods of screening for causative agents of onychodystrophy |
| CN112143821A (en) * | 2020-09-30 | 2020-12-29 | 北京师范大学 | Reagent for detecting candida albicans, cryptococcus neoformans and klebsiella pneumoniae by FRET and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US5426026A (en) | PCR identification of four medically important candida species using one primer pair and four species-specific probes | |
| EP3049537B1 (en) | Detection of methicillin-resistant staphylococcus aureus in biological samples | |
| US7816080B2 (en) | Identifying organisms by detecting intronic nucleic acid or encoded proteins | |
| Kanbe et al. | Identification of the pathogenic Aspergillus species by nested PCR using a mixture of specific primers to DNA topoisomerase II gene | |
| US7871779B2 (en) | Molecular identification of Aspergillus species | |
| Xafranski et al. | A quick and low-cost PCR-based assay for Candida spp. identification in positive blood culture bottles | |
| US6605439B2 (en) | Extraction, amplification and sequential hybridization of fungal cell DNA and process for detection of fungal cells in clinical material | |
| JP6298040B2 (en) | Batch detection method for pathogenic fungi | |
| US5708159A (en) | Probe for diagnosing infectious diseases which hybridizes with DNA from candida albicans | |
| EP3610044B1 (en) | Detection and delineation of microorganisms using the ilv3 gene | |
| WO2024043830A1 (en) | Methods for the detection and identification of fungal species | |
| Kim et al. | Evaluation of the Punch-it™ NA-Sample kit for detecting microbial DNA in blood culture bottles using PCR-reverse blot hybridization assay | |
| CN111424112A (en) | Single-tube nested PCR (polymerase chain reaction) primer pair for detecting coffee leaf rust bacteria and detection method | |
| CN116377118A (en) | Multiplex PCR detection method for three pathogenic bacteria of Solanaceae and application thereof | |
| CA2670793A1 (en) | Nucleic acids probes for detection of yeast and fungal species | |
| KR101520931B1 (en) | Composition for diagnosing sepsis and method thereof | |
| Chen et al. | Development of Penicillium italicum-specific primers for rapid detection among fungal isolates in citrus | |
| US20100311040A1 (en) | Dna Fragments, Primers, Kits, and Methods for Amplification the Detection and Identification of Clinically Relevant Candida Species | |
| US9745636B2 (en) | SWI5 gene as a diagnostic target for the identification of fungal and yeast species | |
| EP2231866B1 (en) | Methods and oligonucleotides for detection of mastitis causing bacteria | |
| US6518020B1 (en) | Haemobartonella PCR methods and materials | |
| JP5621992B2 (en) | Methods for detecting and identifying Candida species | |
| JP2003506066A (en) | PCR-based detection and quantification of Tapesiaallundae and Tapesiaacuformis | |
| EP1370679B1 (en) | Methods of making and uses of compositions that modulate intronic region-encoded protein function | |
| US20090191561A1 (en) | Methods and oligonucleotides for detection of mastitis causing bacteria |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 23857831 Country of ref document: EP Kind code of ref document: A1 |