CN116875732A - Composition, kit and application for detecting fungal infection pathogen - Google Patents
Composition, kit and application for detecting fungal infection pathogen Download PDFInfo
- Publication number
- CN116875732A CN116875732A CN202310951624.0A CN202310951624A CN116875732A CN 116875732 A CN116875732 A CN 116875732A CN 202310951624 A CN202310951624 A CN 202310951624A CN 116875732 A CN116875732 A CN 116875732A
- Authority
- CN
- China
- Prior art keywords
- composition
- probe
- detecting
- seq
- primer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000000203 mixture Substances 0.000 title claims abstract description 58
- 244000052769 pathogen Species 0.000 title claims abstract description 21
- 208000031888 Mycoses Diseases 0.000 title abstract description 7
- 206010017533 Fungal infection Diseases 0.000 title abstract description 5
- 230000001717 pathogenic effect Effects 0.000 title description 5
- 238000001514 detection method Methods 0.000 claims abstract description 38
- 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 abstract description 29
- 241000223218 Fusarium Species 0.000 claims abstract description 29
- 241000228212 Aspergillus Species 0.000 claims abstract description 25
- 230000004069 differentiation Effects 0.000 claims abstract description 10
- 238000007689 inspection Methods 0.000 claims abstract description 9
- 241001157813 Cercospora Species 0.000 claims abstract description 7
- 239000000523 sample Substances 0.000 claims description 72
- 238000011144 upstream manufacturing Methods 0.000 claims description 19
- 108020004707 nucleic acids Proteins 0.000 claims description 14
- 150000007523 nucleic acids Chemical class 0.000 claims description 14
- 102000039446 nucleic acids Human genes 0.000 claims description 14
- 244000053095 fungal pathogen Species 0.000 claims description 11
- 125000006853 reporter group Chemical group 0.000 claims description 9
- 239000003153 chemical reaction reagent Substances 0.000 claims description 8
- 238000003753 real-time PCR Methods 0.000 claims description 8
- 108090000790 Enzymes Proteins 0.000 claims description 6
- 102000004190 Enzymes Human genes 0.000 claims description 6
- 238000003908 quality control method Methods 0.000 claims description 6
- 241000894006 Bacteria Species 0.000 claims description 5
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 5
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 5
- 102100037111 Uracil-DNA glycosylase Human genes 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 210000003046 sporozoite Anatomy 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 3
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 abstract description 10
- 238000000034 method Methods 0.000 abstract description 9
- 230000035945 sensitivity Effects 0.000 abstract description 9
- 238000010222 PCR analysis Methods 0.000 abstract description 3
- 230000003321 amplification Effects 0.000 description 14
- 238000003199 nucleic acid amplification method Methods 0.000 description 14
- 230000000052 comparative effect Effects 0.000 description 9
- 208000015181 infectious disease Diseases 0.000 description 8
- 239000011324 bead Substances 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 241000233866 Fungi Species 0.000 description 4
- 206010061598 Immunodeficiency Diseases 0.000 description 4
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 4
- 201000005202 lung cancer Diseases 0.000 description 4
- 208000020816 lung neoplasm Diseases 0.000 description 4
- 239000006148 magnetic separator Substances 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 210000002345 respiratory system Anatomy 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 208000037026 Invasive Fungal Infections Diseases 0.000 description 3
- 206010036790 Productive cough Diseases 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 208000024794 sputum Diseases 0.000 description 3
- 210000003802 sputum Anatomy 0.000 description 3
- 241001225321 Aspergillus fumigatus Species 0.000 description 2
- 201000003883 Cystic fibrosis Diseases 0.000 description 2
- 208000004770 Fusariosis Diseases 0.000 description 2
- 206010051919 Fusarium infection Diseases 0.000 description 2
- 241001544359 Polyspora Species 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 229940091771 aspergillus fumigatus Drugs 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000007847 digital PCR Methods 0.000 description 2
- 239000000539 dimer Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000013399 early diagnosis Methods 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 230000001052 transient effect Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- UCTWMZQNUQWSLP-VIFPVBQESA-N (R)-adrenaline Chemical compound CNC[C@H](O)C1=CC=C(O)C(O)=C1 UCTWMZQNUQWSLP-VIFPVBQESA-N 0.000 description 1
- 229930182837 (R)-adrenaline Natural products 0.000 description 1
- VHVPQPYKVGDNFY-DFMJLFEVSA-N 2-[(2r)-butan-2-yl]-4-[4-[4-[4-[[(2r,4s)-2-(2,4-dichlorophenyl)-2-(1,2,4-triazol-1-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]piperazin-1-yl]phenyl]-1,2,4-triazol-3-one Chemical compound O=C1N([C@H](C)CC)N=CN1C1=CC=C(N2CCN(CC2)C=2C=CC(OC[C@@H]3O[C@](CN4N=CN=C4)(OC3)C=3C(=CC(Cl)=CC=3)Cl)=CC=2)C=C1 VHVPQPYKVGDNFY-DFMJLFEVSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- UDGUGZTYGWUUSG-UHFFFAOYSA-N 4-[4-[[2,5-dimethoxy-4-[(4-nitrophenyl)diazenyl]phenyl]diazenyl]-n-methylanilino]butanoic acid Chemical compound COC=1C=C(N=NC=2C=CC(=CC=2)N(C)CCCC(O)=O)C(OC)=CC=1N=NC1=CC=C([N+]([O-])=O)C=C1 UDGUGZTYGWUUSG-UHFFFAOYSA-N 0.000 description 1
- WNDDWSAHNYBXKY-UHFFFAOYSA-N ATTO 425-2 Chemical compound CC1CC(C)(C)N(CCCC(O)=O)C2=C1C=C1C=C(C(=O)OCC)C(=O)OC1=C2 WNDDWSAHNYBXKY-UHFFFAOYSA-N 0.000 description 1
- 241000589291 Acinetobacter Species 0.000 description 1
- 241000588626 Acinetobacter baumannii Species 0.000 description 1
- 241000222518 Agaricus Species 0.000 description 1
- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=C[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-KKGHZKTASA-N 0.000 description 1
- 201000002909 Aspergillosis Diseases 0.000 description 1
- 241000228197 Aspergillus flavus Species 0.000 description 1
- 208000036641 Aspergillus infections Diseases 0.000 description 1
- 241001465318 Aspergillus terreus Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 235000017166 Bambusa arundinacea Nutrition 0.000 description 1
- 235000017491 Bambusa tulda Nutrition 0.000 description 1
- 241001330002 Bambuseae Species 0.000 description 1
- 241000222122 Candida albicans Species 0.000 description 1
- 241000222178 Candida tropicalis Species 0.000 description 1
- 241001647372 Chlamydia pneumoniae Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000606768 Haemophilus influenzae Species 0.000 description 1
- 241000712431 Influenza A virus Species 0.000 description 1
- 241000713196 Influenza B virus Species 0.000 description 1
- 241000588747 Klebsiella pneumoniae Species 0.000 description 1
- 241000589242 Legionella pneumophila Species 0.000 description 1
- 241000132887 Lomentospora prolificans Species 0.000 description 1
- 241001518729 Monilinia Species 0.000 description 1
- 241000187479 Mycobacterium tuberculosis Species 0.000 description 1
- 241000202934 Mycoplasma pneumoniae Species 0.000 description 1
- 206010029803 Nosocomial infection Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 201000005702 Pertussis Diseases 0.000 description 1
- 235000015334 Phyllostachys viridis Nutrition 0.000 description 1
- 241000235645 Pichia kudriavzevii Species 0.000 description 1
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 241000725643 Respiratory syncytial virus Species 0.000 description 1
- 241000158504 Rhodococcus hoagii Species 0.000 description 1
- 241000187560 Saccharopolyspora Species 0.000 description 1
- 241000132889 Scedosporium Species 0.000 description 1
- 241000852049 Scedosporium apiospermum Species 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 241000193998 Streptococcus pneumoniae Species 0.000 description 1
- 241001523006 Talaromyces marneffei Species 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 description 1
- 229960003942 amphotericin b Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229960004099 azithromycin Drugs 0.000 description 1
- MQTOSJVFKKJCRP-BICOPXKESA-N azithromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)N(C)C[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 MQTOSJVFKKJCRP-BICOPXKESA-N 0.000 description 1
- 239000011425 bamboo Substances 0.000 description 1
- 238000010322 bone marrow transplantation Methods 0.000 description 1
- 229940095731 candida albicans Drugs 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229960005139 epinephrine Drugs 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229960004884 fluconazole Drugs 0.000 description 1
- RFHAOTPXVQNOHP-UHFFFAOYSA-N fluconazole Chemical compound C1=NC=NN1CC(C=1C(=CC(F)=CC=1)F)(O)CN1C=NC=N1 RFHAOTPXVQNOHP-UHFFFAOYSA-N 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 150000003278 haem Chemical class 0.000 description 1
- 229940047650 haemophilus influenzae Drugs 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 229960004130 itraconazole Drugs 0.000 description 1
- 229940045505 klebsiella pneumoniae Drugs 0.000 description 1
- 229940115932 legionella pneumophila Drugs 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 229960004393 lidocaine hydrochloride Drugs 0.000 description 1
- YECIFGHRMFEPJK-UHFFFAOYSA-N lidocaine hydrochloride monohydrate Chemical compound O.[Cl-].CC[NH+](CC)CC(=O)NC1=C(C)C=CC=C1C YECIFGHRMFEPJK-UHFFFAOYSA-N 0.000 description 1
- 238000007403 mPCR Methods 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229960002260 meropenem Drugs 0.000 description 1
- DMJNNHOOLUXYBV-PQTSNVLCSA-N meropenem Chemical compound C=1([C@H](C)[C@@H]2[C@H](C(N2C=1C(O)=O)=O)[C@H](O)C)S[C@@H]1CN[C@H](C(=O)N(C)C)C1 DMJNNHOOLUXYBV-PQTSNVLCSA-N 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 229940031000 streptococcus pneumoniae Drugs 0.000 description 1
- 229960005404 sulfamethoxazole Drugs 0.000 description 1
- JLKIGFTWXXRPMT-UHFFFAOYSA-N sulphamethoxazole Chemical compound O1C(C)=CC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 JLKIGFTWXXRPMT-UHFFFAOYSA-N 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- IEDVJHCEMCRBQM-UHFFFAOYSA-N trimethoprim Chemical compound COC1=C(OC)C(OC)=CC(CC=2C(=NC(N)=NC=2)N)=C1 IEDVJHCEMCRBQM-UHFFFAOYSA-N 0.000 description 1
- 229960001082 trimethoprim Drugs 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 239000002699 waste material Substances 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
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/77—Fusarium
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Botany (AREA)
- Mycology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the field of molecular biology detection, and particularly relates to detection of pathogens related to fungal infection, and more particularly relates to detection of fusarium, cercospora, aspergillus and marneffei. The composition for joint inspection provided by the invention mainly utilizes a multiplex fluorescence PCR analysis method to detect different pathogens by detecting targets on different pathogens, so that detection and differentiation of fusarium, sporidium, aspergillus and marneffei basket are realized in a single-tube reaction system. The composition has higher detection sensitivity reaching 500 copies/mL, good specificity and more accurate detection.
Description
Technical Field
The invention belongs to the field of molecular biology detection, and particularly relates to detection of pathogens related to fungal infection, and more particularly relates to fusarium, cercospora, aspergillus and marneffei.
Background
It has been reported that the prevalence of fungal infections in organ transplantation and malignant patients is as high as 20% to 40% and often fatal infections, invasive mycoses (invasive fungal disease, IFD), i.e. invasive mycoses (invasive fungal infection, IFI), account for 8% to 15% of hospital acquired infections, and that the mortality rate is high, which is a focus of domestic attention.
Fusarium (Fusarium) is a rare pathogenic fungus that can cause severe invasive Fusarium infection. As immunocompromised patients increase, the incidence of invasive fusarium infection increases over the last few years. Fusarium is the second most common pathogen, second only to aspergillus, among high risk groups such as leukemia patients, solid organ transplant recipients and allogeneic bone marrow transplantation. Clinically, the mortality rate of infection by invasive fusarium is as high as 79% -81%, so early diagnosis and treatment are important to prevent further progression to more invasive or disseminated infections. The Saedosporium (Scedosporium) mainly comprises two kinds of Saedosporium cuspidatum (Scedosporium apiospermum) and Saedosporium polyspora (Scedosporium prolificans), and the Saedosporium cuspidatum and the Saedosporium polyspora are widely existing in polluted environments such as soil, sewage, decay and the like, and can invade various organs of a human body to cause various disease forms. The most common site of infection is the lung. As the number of infected individuals increases, more and more people recognize that cercospora is an important pathogen, and Shahid humiin clinically shows that cercospora now accounts for 25% of all non-aspergillus infections in organ transplant recipients in immunocompromised hosts; rougeron studies showed that the second ranking among filamentous fungi colonized the airways of patients with Cystic Fibrosis (CF).
Aspergillus is a common conditionally pathogenic fungus, which infects the lungs mainly via the respiratory tract, can also invade the skin, mucous membranes, and severe sepsis. Common pathogens include aspergillus fumigatus, aspergillus flavus, aspergillus terreus, etc., wherein aspergillus fumigatus is invasive aspergillus, and is frequently found in severe or immunocompromised patients, and is deficient in typical clinical manifestations. And fusarium and cerdospora are not easily distinguished from aspergillus and fusarium in terms of aspergillus staining histology.
Cappi and Sureau isolated Marneffei basket (Talaromyces marneffei, TM) in bamboo rats in 1959, marneffei basket infection occurred mainly in southeast Asia, south China or North east Indian, and in particular in patients with a history of edible rhizome medical diseases, deep fungal diseases were caused in immunocompromised patients, where the common sites of infection were the respiratory tract and skin. Mortality can be as high as 100% if not properly treated. In recent years, as the number of lung cancer patients increases, more and more lung cancer patients become infected with Marneffei basket. The symptoms of the patient suffering from lung cancer infected by the Marneffei basket are not specific, and the clinical manifestations are different according to the infection sites. Chest imaging characteristics of the patient infected by the Markov basket are very similar to those of the lung cancer, and the patient is easy to be misdiagnosed and missed clinically.
Fusarium, sacalisporium, aspergillus, marneffei basket, and has the advantages of strong invasiveness, long treatment time, poor prognosis, and high death rate. Therefore, early diagnosis and early treatment are key to improving cure rate. At present, the conventional detection means in the laboratory such as color dyeing inspection, fungus culture and the like have long time consumption and complex operation, and can accurately judge the result only by professional personnel. The high-efficiency and accurate fungus detection product can provide reliable basis and reference for clinical reasonable medication, thereby improving the treatment effect and improving the prognosis outcome.
Thus, there is a need in the art for a product that can simply and rapidly detect such pathogens for targeted therapeutic action, and that is highly sensitive and highly specific.
Disclosure of Invention
In view of this, in a first aspect, the present invention provides a composition for joint inspection and differentiation of fungal pathogens comprising at least 3 of the following 4 groups: :
an upstream primer, a downstream primer and a probe for detecting fusarium shown in SEQ ID NO. 1-3;
an upstream primer, a downstream primer and a probe for detecting the sporozoites shown in SEQ ID NO. 4-6;
an upstream primer, a downstream primer and a probe for detecting aspergillus as shown in SEQ ID NO. 7-9; or (b)
An upstream primer, a downstream primer and a probe for detecting the Marneffei basket bacteria are shown as SEQ ID NO. 10-12.
Further, the composition comprises:
an upstream primer, a downstream primer and a probe for detecting fusarium shown in SEQ ID NO. 1-3;
an upstream primer, a downstream primer and a probe for detecting the sporozoites shown in SEQ ID NO. 4-6;
an upstream primer, a downstream primer and a probe for detecting aspergillus as shown in SEQ ID NO. 7-9; and
an upstream primer, a downstream primer and a probe for detecting the Marneffei basket bacteria are shown as SEQ ID NO. 10-12.
The composition for joint inspection provided by the invention mainly utilizes a multiplex fluorescence PCR analysis method to detect different pathogens by detecting targets on different pathogens, so that detection and differentiation of at least 3 of fusarium, sporozoon, aspergillus and marneffei basket are realized simultaneously in a single-tube reaction system, and a targeted strategy is provided for subsequent treatment. The composition has higher detection sensitivity reaching 500 copies/mL, good specificity and more accurate detection.
Further, the composition includes an upstream primer, a downstream primer and a probe for detecting an internal standard.
In some specific embodiments, the internal standard is a human internal standard gene. In a specific embodiment, the internal standard is Rnase P.
Further, the fluorophores of the probes of the compositions of the invention are different from each other and do not interfere with each other.
As used herein, "distinct and non-interfering with each other" means that the fluorophores used for each probe in the composition are different and do not affect each other's detection, i.e., can be performed using different channels. For example, ATTO 425, quasar705, FAM, HEX, ROX and CY5 can be used, which groups do not have close absorbance values and can select different channels so as not to interfere with each other.
In some specific embodiments, the fluorescent reporter group of the fusarium probe is FAM; the fluorescent reporter group of the cercospora probe is HEX (or VIC); the fluorescence reporter group of the aspergillus probe is ROX; the fluorescence reporter group of the Marneffei basket probe is CY5.
Further, in some embodiments, the compositions of the present invention may include one or more of the above-described primer and probe pairs simultaneously. In the present invention, "pair" refers to matched upstream and downstream primers and probes that detect a target.
The compositions of the invention can be combined in any combination to detect any combination of 4 targets. Those skilled in the art can combine the primers and probe pairs as necessary to detect which targets are the corresponding targets. These combinations are included in the present invention.
For example, any 3 pairs of the above 4 pairs of primers and probes may be included, any 2 pairs of the above 4 pairs of primers and probes may be included, and any 1 pair of the above 4 pairs of primers and probes may be included.
In some specific embodiments, the compositions of the invention are used in fluorescent PCR.
Further, the 3' end of the probe also has a non-fluorescent quencher.
Further, the 3' -end of the probe also has a quenching group, such as BHQ1 or BHQ2.
In a specific embodiment, the 3' end of the probe is BHQ1.
In a particular embodiment, the ingredients of the composition of the invention are present in separate packages.
In a particular embodiment, the ingredients of the composition of the invention are present in the same package.
Further, the components of the composition of the present invention are present in a mixed form.
In a second aspect, the invention provides the use of a composition of the invention as described above for the preparation of a kit for the joint detection and differentiation of fungal pathogens, wherein the pathogens are at least 3 of fusarium, cercospora, aspergillus, and marneffe.
In a third aspect, the present invention provides a kit for joint detection and differentiation of fungal pathogens, the kit comprising a composition of the invention as described above.
Further, the kit also comprises a negative quality control and a positive quality control.
In a specific embodiment, the negative quality control is DEPC H 2 O, physiological saline. The positive quality control material is at least one of fragment plasmid or fragment DNA of Fusarium, sacaliopsis, aspergillus, and Marneffei.
Further, the kit also comprises dNTP, PCR buffer solution and Mg 2+ At least one of them.
Still further, the kit further comprises: at least one of a nucleic acid releasing reagent, a nucleic acid extracting reagent, and a DNA polymerase.
Further, the kit further comprises a nucleic acid releasing reagent, a nucleic acid extracting reagent, dNTPs, dUTP, uracil glycosylase (UDG), a DNA polymerase, a PCR buffer solution and Mg 2+ At least one of them.
Further, the concentration of the DNA polymerase is 3U/reaction to 15U/reaction, for example, the DNA polymerase may be Taq enzyme.
In a specific embodiment, the kit of the invention comprises Taq enzyme, mg 2+ dNTP (U) s, primers, probes and PCR buffer.
Common PCR buffer consists of Tris-HCl and MgCl 2 Buffer systems such as KCl and Triton X-100. The total volume in a typical single PCR reaction tube is 20. Mu.l to 200. Mu.l.
In a specific embodiment, the kit of the invention is compatible with digital PCR amplification systems, i.e., can be used directly on a digital PCR instrument for amplification.
In a fourth aspect, there is provided a method of joint inspection and differentiation of fungal pathogens for non-diagnostic purposes, the method comprising the steps of:
1) Extracting or releasing nucleic acid of a sample to be tested;
2) Performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of the present invention as described above or the kit of the present invention as described above;
3) The results were obtained and analyzed.
In the present invention, the sample for detection may be respiratory tract secretion, sputum, or the like, but is not limited thereto.
Further, the reaction conditions of the fluorescent quantitative PCR are as follows:
UNG enzyme reaction, wherein the temperature is 50 ℃, the time is 1-6 min, and 1 cycle is performed; activating Taq enzyme at 94 deg.c for 1-6 min for 1 cycle; denaturation at 94 deg.c for 5-20 sec, annealing at 55-60 deg.c for 10-60 sec, 30-50 cycles, and collecting fluorescence.
In a specific embodiment, there is provided the use of a composition for the preparation and differentiation of a joint inspection of fungal pathogens, said joint inspection comprising the steps of:
1) Extracting or releasing nucleic acid of a sample to be tested;
2) Performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of the present invention as described above or the kit of the present invention as described above;
3) The results were obtained and analyzed.
Further, the reaction conditions of the fluorescent quantitative PCR are as follows:
UNG enzyme reaction, wherein the temperature is 50 ℃, the time is 1-6 min, and 1 cycle is performed; activating Taq enzyme at 94 deg.c for 1-6 min for 1 cycle; denaturation at 94 deg.c for 5-20 sec, annealing at 55-60 deg.c for 10-60 sec, 30-50 cycles, and collecting fluorescence.
In this context, the term "non-diagnostic purpose" refers to information not intended to obtain whether an individual is infected with the above-mentioned pathogen and suffering from respiratory diseases. For example, the method may be used to detect the presence of the aforementioned pathogens in a test culture (e.g., respiratory secretions, sputum) in need thereof.
Drawings
FIG. 1 is a graph of the results of detection of Fusarium, saccharopolyspora, aspergillus and Marneffe by the compositions of the present invention;
FIG. 2 is a graph showing the sensitivity results of the compositions of the present invention for detecting Fusarium;
FIG. 3 is a graph showing the sensitivity results of detecting Saicosporin a composition of the present invention;
FIG. 4 is a graph showing the sensitivity results of detecting Aspergillus with the composition of the present invention;
FIG. 5 is a graph showing the sensitivity results of the compositions of the present invention for detecting Langmuir marneffei;
FIG. 6 is a graph showing the results of a composition-specific test of the present invention;
FIG. 7 is a graph showing the anti-interference results of the compositions of the present invention for detecting Fusarium;
FIG. 8 is a graph showing the anti-interference results of the composition of the present invention for detecting Saicosporium;
FIG. 9 is a graph showing the anti-interference results of the detection of Aspergillus by the composition of the present invention;
FIG. 10 is a graph showing the anti-interference results of the compositions of the present invention for detecting Langmuir marneffei;
FIG. 11 is a graph showing the results of detection of a comparative example composition of the present invention (Fusarium comparative example primer set);
FIG. 12 is a graph showing the results of detection of a comparative example composition of the present invention (Monilinia marneffei comparative example primer set);
FIG. 13 is a graph showing the results of detection of a comparative example composition of the present invention (Fusarium comparative example primer tetrad);
FIG. 14 is a graph showing the results of detection of a comparative example composition of the present invention (Malfei basket comparative primer tetrad).
Detailed Description
The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.
Example 1, primers and probes used in the present invention
The primers and probes used in the present invention are shown in Table 1 below:
wherein, SEQ ID NO:3 the fluorescence report group of the probe is FAM; SEQ ID NO: the fluorescent reporter group of the 6 probe is HEX (or VIC); SEQ ID NO:9 is ROX; SEQ ID NO:12 is CY5.
Example 2 method for detecting pathogens
1. Reagent preparation
1.1 taking out the components in the box, placing at room temperature, after the temperature of the components is balanced to the room temperature, uniformly mixing and instantly centrifuging for standby.
1.2 according to the number of the sample to be detected, negative control and positive control, taking corresponding amount of components according to the proportion (44 mu L/human part of PCR reaction solution and 1 mu L/human part of enzyme mixed solution), fully and uniformly mixing to form PCR mixed solution, and carrying out instantaneous centrifugation for later use.
1.3 transferring the prepared reagent to a sample processing area for later use.
2. Sample processing and sample addition
The detection sample is sputum. The method for extracting virus nucleic acid by using the magnetic bead method comprises the following steps of:
2.1 add 1.5mL of the frozen tube to glass beads, add 300. Mu.L of the treated sample and 300. Mu.L of LTE to 1.5mL of frozen tube containing mixed glass beads and grind using a grinder. And taking out the frozen tube for instantaneous centrifugation after grinding is finished, and sucking the supernatant as a sample to be measured for standby.
2.2, measuring a plurality of 1.5mL centrifuge tubes according to the number of samples to be measured, and adding 300 mu L of samples to be measured into each tube;
2.3 adding 500. Mu.L of the extraction solution and 50. Mu.L of proteinase K-magnetic bead mixture; covering with a tube cover, shaking and mixing for 30s, and heating at 60deg.C for 10min.
2.4 standing at room temperature for 1min, centrifuging at low speed, placing a centrifuge tube on a magnetic separator, and slowly sucking and discarding waste liquid after 5min (taking care of not touching magnetic beads adsorbed on the inner side of the tube wall);
2.5 adding 200. Mu.L of the washing liquid 1 and 600. Mu.L of the washing liquid 2, shaking and uniformly mixing for 30s, and placing the centrifuge tube in a magnetic separator after low-speed instantaneous centrifugation. Magnetically sucking for 3min, and completely sucking out and discarding the liquid.
2.6 placing the centrifuge tube in a centrifuge for low speed transient centrifugation and placing the centrifuge tube in a magnetic separator again. And magnetically sucking for 3min to completely suck the liquid at the bottom of the pipe.
2.7 adding 30-100 mu L of eluent S (80 mu L is recommended) into the solution, vibrating and uniformly mixing the solution for 30S, eluting magnetic beads on the wall of the centrifugal tube to the bottom of the centrifugal tube, and standing the centrifugal tube at room temperature for 3min; the centrifuge tube was again placed on a magnetic separator for 3min by low speed transient centrifugation, and the eluted nucleic acid was transferred to a clean 1.5mL centrifuge tube.
2.8 sucking 5. Mu.L of each of the above treated sample, positive control and negative control, adding into corresponding 0.2mL PCR reaction tube, adding 45. Mu.L PCR mixture into each tube, and covering the tube cap.
The real-time fluorescent PCR reaction system was configured as follows in table 2:
TABLE 2
PCR amplification is carried out on PCR instruments such as an ABI 7500 fluorescent quantitative PCR instrument, a Life Technologies Quant StudioTM fluorescent PCR instrument, a SLAN-96P full-automatic medical PCR analysis system and the like according to a certain temperature and time setting program. The preferred embodiment of the present invention is shown in Table 3.
TABLE 3 Table 3
Analysis of results:
1) The target detection signal is FAM, HEX (or VIC), ROX and CY5;
2) Setting of Baserine: baseline is typically set to 3-15 cycles, which can be specifically adjusted according to the actual situation. The adjustment principle is as follows: the region where the fluorescent signal is more stable before exponential amplification is selected, the starting point (Start) avoids the signal fluctuation in the initial stage of fluorescent collection, and the End point (End) is reduced by 1-2 cycles compared with the sample Ct of which the exponential amplification occurs at the earliest. Setting of Threshold: setting a principle that a threshold line just exceeds the highest point of a normal negative control;
3) Interpretation of results
If the sample FAM, HEX (VIC), ROX and CY5 channels have obvious S-shaped amplification curves and Ct value is less than or equal to 40, judging positive; if the sample FAM, HEX (VIC), ROX, CY5 channel has No amplification curve (No Ct) or Ct value > 40, then the sample is judged as negative.
1. For FAM channel, detecting typical S-type amplification curve, and reporting sample with Ct less than or equal to 40 as fusarium positive; typical S-type amplification curves were not detected for FAM channels, or Ct > 40, reported as Fusarium negative.
2. For samples with a typical S-type amplification curve detected by the HEX channel and Ct less than or equal to 40, reporting the samples as Saidospora positive; typical S-type amplification curves were not detected for HEX channels, or Ct > 40, reported as Saadoxosporean negative.
3. For a sample with a typical S-shaped amplification curve detected by the ROX channel and Ct less than or equal to 40, reporting that the sample is positive to aspergillus; typical S-type amplification curves were not detected for ROX channels, or Ct > 40, reported as Aspergillus negative.
4. For a sample with a typical S-type amplification curve detected by the ROX channel and Ct less than or equal to 40, the sample is reported to be positive to the Markilis basket bacteria; typical S-type amplification curves were not detected for the ROX channel, or Ct > 40, reported as Marneffe basket negative. Specifically, the results are shown in Table 4.
TABLE 4 Table 4
Example 3 detection results of test samples of the inventive composition
The primers and probes shown in example 1 were used for PCR detection of Fusarium, sacaliopsis, aspergillus and Marneffei in a macrostone fluorescence quantitative PCR apparatus according to the method of example 2, and the detection results are shown in FIG. 1, and it can be seen from the graph that the composition of the present invention can perform good detection on various fungi.
Example 4 sensitivity of the composition of the invention
LOD (sensitivity) tests were performed on each target using the composition of example 1 of the present invention at concentrations of 500000, 50000, 5000, 500, 50 copies/ml, respectively, to simulate clinical specimens for multiplex PCR tests on a macrostone fluorescent quantitative PCR instrument. The results are shown in FIGS. 2-5, which show that each channel can still be accurately detected for samples as low as 500 copies/mL, and the detection rate is 100%, which shows that the sensitivity of the composition of the invention is 500 copies/mL.
EXAMPLE 5 specificity of the composition of the invention
The composition has no cross reaction with other pathogens similar to common pathogens of respiratory tract and infection symptoms (such as klebsiella pneumoniae, streptococcus pneumoniae, haemophilus influenzae, pseudomonas aeruginosa, legionella pneumophila, pertussis bacillus, staphylococcus aureus, mycoplasma pneumoniae, chlamydia pneumoniae, mycobacterium tuberculosis, nontuberculous mycobacteria, acinetobacter baumannii, acinetobacter agaricus, escherichia coli, rhodococcus equi, candida albicans, candida tropicalis, candida krusei, influenza a virus, influenza b virus and respiratory syncytial virus). The results are shown in FIG. 6.
EXAMPLE 6 interference resistance of the composition of the invention
The system has strong anti-interference capability in 10 mug/mL mucin, 20 mug/mL heme, 0.5mg/mL meropenem, 20 mug/mL trimethoprim, 0.44mg/mL sulfamethoxazole, 0.8mg/mL amphotericin B, 0.1g/mL itraconazole, 0.5mg/mL fluconazole, 0.1g/mL azithromycin, 1mg/mL epinephrine and 4mg/mL lidocaine hydrochloride respectively, as shown in figures 7-10.
Comparative example 1, remaining poorly performing primers and probes designed according to the invention
Because of the base-pairing rules, dimers are formed between the primer and/or probe, but with little probability, this can be eliminated at the beginning of the design. However, when multiple pathogens are jointly detected, a plurality of primers and probes are arranged, dimers are easy to occur between the primers and the primers, between the probes and the probes or between the primers and the probes, so that the conservation of design (which is crucial to the accuracy of detection) is ensured, and the mutual interference among different primer probes is considered, so that the primer probes need to be carefully designed.
Thus, the inventors have devised that the remaining primers and probes constitute a different detection system (sequences not shown) for detecting the above pathogens as well. Specific detection results are shown in fig. 11-14, and the comparison example primer probe of fusarium and the comparison example primer probe of marneffei basket are shown in the figures, so that the detection effect of a single detection system is good, however, the detection is affected when the single detection system is respectively placed in a four-joint detection system, ct and fluorescence values are poor, and the superiority of the composition is further illustrated.
Claims (10)
1. A composition for joint inspection and differentiation of fungal pathogens comprising at least 3 of the following 4 groups:
an upstream primer, a downstream primer and a probe for detecting fusarium shown in SEQ ID NO. 1-3;
an upstream primer, a downstream primer and a probe for detecting the sporozoites shown in SEQ ID NO. 4-6;
an upstream primer, a downstream primer and a probe for detecting aspergillus as shown in SEQ ID NO. 7-9; or (b)
An upstream primer, a downstream primer and a probe for detecting the Marneffei basket bacteria are shown as SEQ ID NO. 10-12.
2. The composition of claim 1, wherein the composition comprises:
an upstream primer, a downstream primer and a probe for detecting fusarium shown in SEQ ID NO. 1-3;
an upstream primer, a downstream primer and a probe for detecting the sporozoites shown in SEQ ID NO. 4-6;
an upstream primer, a downstream primer and a probe for detecting aspergillus as shown in SEQ ID NO. 7-9; and
an upstream primer, a downstream primer and a probe for detecting the Marneffei basket bacteria are shown as SEQ ID NO. 10-12.
3. The composition of claim 1, further comprising an upstream primer, a downstream primer, and a probe for detecting an internal standard.
4. A composition according to claim 3, wherein the fluorescent reporter group of fusarium is FAM; the fluorescent reporter group of the cercospora is HEX or IVC; the fluorescence reporter group of aspergillus is ROX; the fluorescence reporter group of the Marneffei basket probe is CY5.
5. The composition according to any one of claims 1 to 4, wherein the components of the composition are present in a mixed form.
6. Use of a composition according to any one of claims 1 to 5 in the preparation of a kit for joint inspection and differentiation of fungal pathogens, wherein the pathogens are at least 3 of fusarium, cerdospora, aspergillus, and marneffe basket.
7. A kit for joint detection and differentiation of fungal pathogens comprising a composition according to any one of claims 1 to 5.
8. The kit of claim 7, further comprising a negative quality control and a positive quality control.
9. The kit of claim 7 or 8, further comprising: nucleic acid releasing reagent, nucleic acid extracting reagent, DNA polymerase, dNTP, dUTP, UDG enzyme, PCR buffer solution and Mg 2+ At least one of them.
10. Use of a composition for preparing and differentiating between fungal pathogens, said joint test comprising the steps of:
1) Extracting or releasing nucleic acid of a sample to be tested;
2) Performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of any one of claims 1 to 5 or the kit of any one of claims 7 to 9;
3) The results were obtained and analyzed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310951624.0A CN116875732A (en) | 2023-07-31 | 2023-07-31 | Composition, kit and application for detecting fungal infection pathogen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310951624.0A CN116875732A (en) | 2023-07-31 | 2023-07-31 | Composition, kit and application for detecting fungal infection pathogen |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116875732A true CN116875732A (en) | 2023-10-13 |
Family
ID=88258629
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310951624.0A Pending CN116875732A (en) | 2023-07-31 | 2023-07-31 | Composition, kit and application for detecting fungal infection pathogen |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116875732A (en) |
-
2023
- 2023-07-31 CN CN202310951624.0A patent/CN116875732A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114752711B (en) | Composition, kit and method for detecting and parting monkeypox virus and application thereof | |
| WO2009110473A1 (en) | Method of distinguishing inflammatory pathogen causing acute respiratory infection | |
| CN106987626B (en) | Primer and probe for rapidly detecting various fungi and identifying strains and application thereof | |
| CN112410472B (en) | Primer probe combination for detecting mycoplasma pneumoniae, chlamydia pneumoniae and adenovirus and detection kit | |
| CN111690760A (en) | Method for accurately detecting cryptococcus neoformans | |
| WO2020224195A1 (en) | Primer-probe combination for aspergillus species detection and distinguishing, kit, detection method and use thereof | |
| CN111004862A (en) | Primer and probe for rapidly detecting and identifying cryptococcus and application thereof | |
| WO2010121578A1 (en) | Method for in vitro diagnostics of invasive aspergillosis | |
| CN117512204B (en) | Primer and probe combination for multiplex detection of aspergillus, cryptococcus and yersinia, kit and application | |
| CN110894534A (en) | Primer, probe, kit and detection method for detecting mycoplasma genitalium | |
| CN112680541B (en) | LNA-Taqman-multiplex fluorescence PCR technology and application thereof in rapid detection of candida | |
| CN111455094B (en) | Composition, kit, application and method for detecting deep infection fungi | |
| CN113897422A (en) | Multiplex PCR primer probe set and kit for detecting pathogenic mucor | |
| CN116287420A (en) | Composition for detecting different types of fungi, kit and application thereof | |
| Hizel et al. | Polymerase chain reaction in the diagnosis of invasive aspergillosis. | |
| CN116875732A (en) | Composition, kit and application for detecting fungal infection pathogen | |
| El-Morsy et al. | Allergic fungal rhinosinusitis: detection of fungal DNA in sinus aspirate using polymerase chain reaction | |
| CN115838814A (en) | Fungus and bacterium quadruple nucleic acid detection kit and detection method and application thereof | |
| CN116240312A (en) | Novel composition for combined detection of coronavirus, HIV and monkey pox virus | |
| CN116949207A (en) | Composition for fungus joint inspection, kit containing same and application of composition | |
| CN106929601A (en) | A kind of type kit for detecting nucleic acid of highly sensitive HPV 6,11 | |
| CN113151570B (en) | Loop-mediated isothermal amplification LAMP technology based on LNA modification and application of LAMP technology in rapid detection of candida | |
| CN116987811A (en) | Composition and kit for fungus joint inspection and application of composition and kit | |
| CN115725794B (en) | Compositions, methods and uses for novel coronavirus and monkey pox virus joint detection | |
| CN115807129B (en) | Novel coronavirus and monkey pox virus joint inspection and typing composition |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |