CN117660700A - Primer probe combination and kit for detecting three RNA viruses infecting respiratory tract of dogs - Google Patents
Primer probe combination and kit for detecting three RNA viruses infecting respiratory tract of dogs Download PDFInfo
- Publication number
- CN117660700A CN117660700A CN202410089134.9A CN202410089134A CN117660700A CN 117660700 A CN117660700 A CN 117660700A CN 202410089134 A CN202410089134 A CN 202410089134A CN 117660700 A CN117660700 A CN 117660700A
- Authority
- CN
- China
- Prior art keywords
- canine
- primer probe
- seq
- amplification
- probe combination
- 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
- 239000000523 sample Substances 0.000 title claims abstract description 124
- 241000282472 Canis lupus familiaris Species 0.000 title abstract description 35
- 241001493065 dsRNA viruses Species 0.000 title abstract description 14
- 210000002345 respiratory system Anatomy 0.000 title abstract description 14
- 230000003321 amplification Effects 0.000 claims abstract description 96
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 96
- 241001353878 Canine parainfluenza virus Species 0.000 claims abstract description 44
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 42
- 241000712083 Canine morbillivirus Species 0.000 claims abstract description 40
- 241001594994 Canine respiratory coronavirus Species 0.000 claims abstract description 40
- 241000282465 Canis Species 0.000 claims abstract description 36
- 241000700605 Viruses Species 0.000 claims description 46
- 238000000034 method Methods 0.000 claims description 35
- 102000004190 Enzymes Human genes 0.000 claims description 25
- 108090000790 Enzymes Proteins 0.000 claims description 25
- 239000002773 nucleotide Substances 0.000 claims description 25
- 125000003729 nucleotide group Chemical group 0.000 claims description 25
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 24
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 20
- 108700039887 Essential Genes Proteins 0.000 claims description 18
- 238000010791 quenching Methods 0.000 claims description 18
- 230000000171 quenching effect Effects 0.000 claims description 18
- 102100037111 Uracil-DNA glycosylase Human genes 0.000 claims description 15
- 101710160987 Uracil-DNA glycosylase Proteins 0.000 claims description 15
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 11
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 11
- 230000000694 effects Effects 0.000 claims description 11
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 10
- 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 claims description 9
- 102100034343 Integrase Human genes 0.000 claims description 9
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims description 9
- 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 claims description 7
- 239000000872 buffer Substances 0.000 claims description 7
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 claims description 6
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 claims description 6
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 claims description 6
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 claims description 6
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 claims description 6
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 5
- 108700008625 Reporter Genes Proteins 0.000 claims description 4
- WCKQPPQRFNHPRJ-UHFFFAOYSA-N 4-[[4-(dimethylamino)phenyl]diazenyl]benzoic acid Chemical compound C1=CC(N(C)C)=CC=C1N=NC1=CC=C(C(O)=O)C=C1 WCKQPPQRFNHPRJ-UHFFFAOYSA-N 0.000 claims description 3
- MPLHNVLQVRSVEE-UHFFFAOYSA-N texas red Chemical compound [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 33
- 230000028327 secretion Effects 0.000 abstract description 14
- 230000000241 respiratory effect Effects 0.000 abstract description 12
- 208000024891 symptom Diseases 0.000 abstract description 9
- 238000002955 isolation Methods 0.000 abstract description 8
- 230000036541 health Effects 0.000 abstract description 5
- 239000008280 blood Substances 0.000 abstract description 4
- 210000004369 blood Anatomy 0.000 abstract description 4
- 230000007613 environmental effect Effects 0.000 abstract description 4
- 208000035473 Communicable disease Diseases 0.000 abstract description 2
- 239000012634 fragment Substances 0.000 description 26
- 150000007523 nucleic acids Chemical group 0.000 description 19
- 238000006243 chemical reaction Methods 0.000 description 18
- 239000003068 molecular probe Substances 0.000 description 17
- 108020004707 nucleic acids Proteins 0.000 description 17
- 102000039446 nucleic acids Human genes 0.000 description 17
- 239000013612 plasmid Substances 0.000 description 17
- 238000003752 polymerase chain reaction Methods 0.000 description 13
- 238000003908 quality control method Methods 0.000 description 13
- 238000012360 testing method Methods 0.000 description 11
- 108020004414 DNA Proteins 0.000 description 9
- 208000015181 infectious disease Diseases 0.000 description 9
- 244000052769 pathogen Species 0.000 description 9
- 125000006853 reporter group Chemical group 0.000 description 8
- 238000011144 upstream manufacturing Methods 0.000 description 8
- 230000001717 pathogenic effect Effects 0.000 description 7
- 238000003753 real-time PCR Methods 0.000 description 7
- 239000000443 aerosol Substances 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 241000711573 Coronaviridae Species 0.000 description 5
- 238000000137 annealing Methods 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 238000004321 preservation Methods 0.000 description 5
- 238000010839 reverse transcription Methods 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- 238000010257 thawing Methods 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- 241000711506 Canine coronavirus Species 0.000 description 4
- 102000053602 DNA Human genes 0.000 description 4
- 238000011529 RT qPCR Methods 0.000 description 4
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 4
- 230000005540 biological transmission Effects 0.000 description 4
- 239000007850 fluorescent dye Substances 0.000 description 4
- 230000001900 immune effect Effects 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 3
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000011901 isothermal amplification Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 229960005486 vaccine Drugs 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 208000003322 Coinfection Diseases 0.000 description 2
- 206010012735 Diarrhoea Diseases 0.000 description 2
- 241001559185 Mammalian rubulavirus 5 Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 241000711504 Paramyxoviridae Species 0.000 description 2
- 208000002606 Paramyxoviridae Infections Diseases 0.000 description 2
- 108020004682 Single-Stranded DNA Proteins 0.000 description 2
- 206010058874 Viraemia Diseases 0.000 description 2
- 208000036142 Viral infection Diseases 0.000 description 2
- 206010047700 Vomiting Diseases 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 210000003292 kidney cell Anatomy 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 210000003563 lymphoid tissue Anatomy 0.000 description 2
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000013112 stability test Methods 0.000 description 2
- 229940035893 uracil Drugs 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- 235000001892 vitamin D2 Nutrition 0.000 description 2
- 230000008673 vomiting Effects 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- NOIIUHRQUVNIDD-UHFFFAOYSA-N 3-[[oxo(pyridin-4-yl)methyl]hydrazo]-N-(phenylmethyl)propanamide Chemical compound C=1C=CC=CC=1CNC(=O)CCNNC(=O)C1=CC=NC=C1 NOIIUHRQUVNIDD-UHFFFAOYSA-N 0.000 description 1
- 241000588779 Bordetella bronchiseptica Species 0.000 description 1
- 241000680578 Canid alphaherpesvirus 1 Species 0.000 description 1
- 241000282421 Canidae Species 0.000 description 1
- 241000701114 Canine adenovirus 2 Species 0.000 description 1
- 241001466804 Carnivora Species 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 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 description 1
- 101001065501 Escherichia phage MS2 Lysis protein Proteins 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 241000712079 Measles morbillivirus Species 0.000 description 1
- 241000266847 Mephitidae Species 0.000 description 1
- 208000007101 Muscle Cramp Diseases 0.000 description 1
- 241000282339 Mustela Species 0.000 description 1
- 241000204031 Mycoplasma Species 0.000 description 1
- 108090001074 Nucleocapsid Proteins Proteins 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 241000282335 Procyon Species 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 206010039101 Rhinorrhoea Diseases 0.000 description 1
- 208000005392 Spasm Diseases 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 0.000 description 1
- 102000006943 Uracil-DNA Glycosidase Human genes 0.000 description 1
- 108010072685 Uracil-DNA Glycosidase Proteins 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 239000000370 acceptor Substances 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000011217 control strategy Methods 0.000 description 1
- 230000037029 cross reaction Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007847 digital PCR Methods 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 238000007849 hot-start PCR Methods 0.000 description 1
- 230000004727 humoral immunity Effects 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000003317 immunochromatography Methods 0.000 description 1
- 239000003547 immunosorbent Substances 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 208000010753 nasal discharge Diseases 0.000 description 1
- 210000002850 nasal mucosa Anatomy 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 210000001328 optic nerve Anatomy 0.000 description 1
- 210000002741 palatine tonsil Anatomy 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000002787 reinforcement Effects 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 210000003437 trachea Anatomy 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- 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
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the field of biotechnology, in particular to a primer probe combination and a kit for detecting three RNA viruses infecting the respiratory tract of dogs. The primer probe combination, the amplification reagent or the kit provided by the invention can detect canine distemper virus, canine parainfluenza virus and canine respiratory coronavirus, and a detection sample covers an oral secretion sample, an eye secretion sample, a nasal secretion sample, a blood sample, a tissue sample and an environmental sample. The invention can rapidly and accurately detect dogs with respiratory symptoms observed in the environments of families, dog houses, pet hospitals, communities, police offices, customs and the like, realizes early detection, early isolation and early treatment, effectively ensures the health of dogs and avoids the expansion of epidemic situation of canine infectious diseases.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a primer probe combination and a kit for detecting three RNA viruses infecting the respiratory tract of dogs.
Background
With the increase of the living pressure and the living standard of modern people, more and more people start to keep pet dogs and treat the dogs as own relatives and friends. The increase of the number of the pet dogs is accompanied with the reinforcement of the transmissibility of the canine-related pathogenic pathogens, and when the dogs are abnormal in appearance, the dogs can be timely and effectively diagnosed, and corresponding treatment measures are adopted, so that the requirements of the owners of dogs keeping the dogs are increasingly met.
Canine distemper virus (canine distemper virus, CDV), canine parainfluenza virus (canine parainfluenzavirus, CPIV) and canine respiratory coronavirus (canine respiratory coronavirus, CRCoV) are three common canine respiratory RNA viruses, which have diverse transmission routes such as direct contact with bodily fluids, secretions, excretions, etc. of infected dogs, and transmission through air droplets, food water sources, etc. The symptoms after infection of the three viruses are similar, can cause symptoms of cough, sneeze, nasal discharge and fever of dogs, can cause complications of pneumonia of dogs and the like in severe cases, and can cause nerve symptoms such as muscle spasm and the like, diarrhea and vomiting symptoms, wherein the diarrhea and vomiting symptoms can be caused by other pathogens or parasites. Two or three of the three RNA viruses may simultaneously cause mixed infection in dogs. Mixed infections in newborn, geriatric or immunocompromised dogs can cause more severe physical symptoms and even death.
The main detection methods of the three RNA viruses are virus separation, immunological detection and molecular detection.
Virus isolation: successful isolation, culture and identification of viruses from disease agents is the "gold standard" for current diagnosis of viral infections. The virus separation needs to remove the pollution of other germs and impurities in the sample, then the steps of preparing virus suspension culture cells to inoculate the virus sample to canine kidney cells (MDCK) and the like are carried out, and the requirements on laboratories and detection personnel are high while strict and sterile environments are needed. Virus isolation typically takes a long time (about 1-2 weeks) to obtain results and low concentrations of virus samples or cross-reactions with other virus or cellular components cannot be detected, leading to the appearance of false positive results.
Immunological detection: immunological detection is a technique for detecting viruses and their specific antibodies by utilizing the specific reaction of an antigen and an antibody. Antibodies are part of the immune response of dogs to infection. Antibodies against a certain virus are usually detectable the first few weeks after infection, the presence of which indicates that the dog is infected with the virus, whether severe, mild or asymptomatic at the time. This method is not suitable for timely detection, but rather for retrospectively determining the size or extent of infection of an epidemic in a study canine population.
And (3) molecular detection: including Polymerase Chain Reaction (PCR) techniques, gene chip techniques, real-time fluorescent quantitative PCR techniques, digital PCR techniques, recombinant Polymerase Amplification (RPA) techniques, and the like, by detecting the presence of virus in a sample by detecting virus-specific nucleic acid fragments or gene fragments, specific viral nucleic acids can be found at the beginning of infection, thereby enabling early diagnosis and treatment, and reducing the severity and risk of spread of disease.
Disadvantages of virus isolation: 1. complicated experimental operations including steps of culturing cells, inoculating virus samples to canine kidney cells (MDCK), observing by a microscope and the like, and a strictly sterile environment have high requirements on laboratories and detection personnel; 2. virus isolation typically requires a long time (about 1-2 weeks) to achieve results, delaying diagnosis and treatment; 3. the sensitivity is low, and a low-concentration virus sample cannot be detected; 4. the specificity of virus isolation is limited and may cross-react with other viral or cellular components, leading to the appearance of false positive results.
Immunological tests include enzyme-linked immunosorbent and immunochromatography, etc., and have the following disadvantages: 1. the development period of the monoclonal specific antibody is long, and large-scale and stable production is difficult to realize; 2. it is not possible to effectively distinguish whether the antibodies are generated due to viral infection or after vaccination; 3. the sensitivity is low, and the virus cannot be effectively detected in the early stage of infection of dogs with the virus; 4. multiple assays for viruses cannot be performed.
Molecular detection includes real-time quantitative PCR techniques (qPCR), polymerase chain reaction techniques (PCR), isothermal amplification (RPA, LAMP) techniques, and the like, wherein the disadvantages of the polymerase chain reaction techniques are: 1. after the reaction is finished, the amplified nucleic acid product needs to be uncapped for agarose nucleic acid electrophoresis detection, so that aerosol pollution of the amplified product is very easy to cause false positive to the subsequent amplification reaction; 2. agarose electrophoresis increases the operation steps and detection time, resulting in time waste. Disadvantages of isothermal amplification are: 1. sensitivity is still not as good as PCR detection methodologies; 2. the cost of purchasing isothermal amplification polymerase is high; 3. the stability of the enzyme is inferior to that of the DNA polymerase used for PCR; 4. multiple assays of pathogens cannot be performed in the same tube.
At present, no implementation scheme for simultaneously carrying out triple detection on canine distemper virus, canine parainfluenza virus and canine respiratory coronavirus by using real-time fluorescence quantitative PCR is available.
Disclosure of Invention
In view of this, the present invention provides primer probe combinations and kits for detecting three canine respiratory tract-infecting RNA viruses.
The invention provides a primer probe combination and a kit for detecting three RNA viruses infecting the respiratory tract of dogs. The primer probe combination, the amplification reagent or the kit provided by the invention can detect canine distemper virus, canine parainfluenza virus and canine respiratory coronavirus, and a detection sample covers an oral secretion sample, an eye secretion sample, a nasal secretion sample, a blood sample, a tissue sample and an environmental sample. The invention can rapidly and accurately detect dogs with respiratory symptoms observed in the environments of families, dog houses, pet hospitals, communities, police offices, customs and the like, realizes early detection, early isolation and early treatment, effectively ensures the health of dogs and avoids the expansion of epidemic situation of canine infectious diseases.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a primer probe combination, which comprises the following components:
the primer has a nucleotide sequence shown as SEQ ID No. 1-2, 4-5, 7-8 and/or 10-11; and
(II) the probe has a nucleotide sequence shown as SEQ ID No.3, 6, 9 and/or 12; or (b)
(III) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences with the nucleotide sequence shown in (I) or (II), and having the same or similar functions as the nucleotide sequence shown in (I) or (II); or (b)
(IV) a nucleotide sequence having at least 80% sequence homology with the nucleotide sequence of any one of (I) to (III).
In some embodiments of the invention, the primer probe combination comprises:
primers for detecting nucleotide sequences shown in SEQ ID No. 1-2 and probes for detecting nucleotide sequences shown in SEQ ID No.3 of canine distemper virus;
primers for detecting nucleotide sequences shown in SEQ ID Nos. 4 to 5 and probes for detecting nucleotide sequences shown in SEQ ID No.6 of canine parainfluenza virus;
primers for detecting nucleotide sequences shown in SEQ ID Nos. 7 to 8 and probes for detecting nucleotide sequences shown in SEQ ID No.9 of canine respiratory coronavirus;
primers for detecting the nucleotide sequences shown in SEQ ID Nos. 10 to 11 and probes for detecting the nucleotide sequences shown in SEQ ID No.12 of the canine housekeeping gene.
In some embodiments of the invention, the probe is labeled with a fluorescent reporter at the 5 'end and a quencher at the 3' end;
the fluorescent reporter gene comprises 6' -FAM, VIC, ROX, CY5, TET, JOR, HEX, CY3, NED, TXR, TAMRA, texas red, SX670, CY5.5 and/or CY7;
the quenching groups include BHQ1, BHQ2, TAMRA, BHQ0, BHQ3, dabcyl, MGB, SQ1, SQ2, and/or SQX.
In some embodiments of the invention, the fluorescent reporter gene comprises 6' -FAM, VIC, ROX and/or CY5; the quenching group comprises BHQ1 and/or BHQ2.
In some embodiments of the invention, the 5' end of the probe of the nucleotide sequence shown in SEQ ID No.3 is labeled with a fluorescent reporter group of 6-FAM; the 3' end of the quenching group comprises BHQ1.
In some embodiments of the invention, the 5' end of the probe of the nucleotide sequence shown in SEQ ID No.6 is labeled with a fluorescent reporter group as VIC; the 3' end of the quenching group comprises BHQ1.
In some embodiments of the invention, the 5' end of the probe of the nucleotide sequence shown in SEQ ID No.3 is labeled with a fluorescent reporter group as ROX; the 3' end of the quenching group comprises BHQ2.
In some embodiments of the invention, the 5' end of the probe of the nucleotide sequence shown in SEQ ID No.12 is labeled with a fluorescent reporter group CY5; the 3' end of the quenching group comprises BHQ2.
The invention also provides an amplification reagent comprising the primer probe combination.
In some embodiments of the invention, the amplification reagents further comprise an amplification buffer, a DNA polymerase, a reverse transcriptase, and/or a UNG enzyme;
the amplification buffer comprises KCl, mgCl2, (NH 4) 2 SO 4 One or more of dNTP, tris-HCl or DMSO.
In some embodiments of the invention, the final concentration of the primers in the primer probe combination is 100 to 1000nM; the final concentration of the probe in the primer probe combination is 50-500 nM;
the concentration of KCl is 20-100 mmol; the concentration of MgCl2 is 0.5-5 mmol; said (NH 4) 2 SO 4 The concentration of (2) is 0-10 mmol; the dNTPs include dATP, dCTP, dGTP and dTT; the concentrations of dATP, dCTP, dGTP and dTT were 0.2mmol; the concentration of the Tris-HCl is 5-20 mmol; the concentration of DMSO is 0-10%;
the enzyme activity of the DNA polymerase is 5U; the enzyme activity of the reverse transcriptase is 200U; the enzyme activity of the UNG enzyme is 5U.
In some embodiments of the invention, the final concentration of primer in the primer probe combination is 300nM; the final concentration of the probes in the primer probe combination is 100nM;
the concentration of KCl is 50mmol; the concentration of MgCl2 is 1.5mmol; said (NH 4) 2 SO 4 Is 2mmol; the concentration of Tris-HCl is 10mmol; the DMSO concentration was 5%.
Based on the above study, the invention also provides the application of any of the following in preparing a kit for detecting canine distemper virus, canine parainfluenza virus and canine respiratory coronavirus:
(A) The primer probe combination; and/or
(B) And the amplification reagent.
The invention also provides a kit comprising the primer probe combination or the amplification reagent.
In some embodiments of the invention, the kit further comprises a positive quality control and/or a negative quality control;
the positive quality control product comprises a canine distemper virus target fragment, a canine parainfluenza virus target fragment and a canine respiratory tract coronavirus target fragment;
the canine distemper virus target fragment, the canine parainfluenza virus target fragment and the canine respiratory coronavirus target fragment are sequentially provided with:
1) Nucleotide sequences as shown in SEQ ID Nos. 13, 14 and 15; or (b)
2) A nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences with the nucleotide sequence shown in 1), and functionally identical or similar to the nucleotide sequence shown in 1); or (b)
3) A nucleotide sequence having at least 80% sequence homology with the nucleotide sequence of 1) or 2).
The invention also provides a method for simultaneously detecting viruses, which is based on qRT-PCR detection of samples according to any of the following items:
(A) The primer probe combination; and/or
(B) The amplification reagent; and/or
(C) The kit;
the viruses include canine distemper virus, canine parainfluenza virus and canine respiratory coronavirus.
In some embodiments of the invention, the method for simultaneously detecting viruses comprises the steps of:
step 1: taking a sample to be tested;
step 2: detecting by adopting the primer probe combination, the amplification reagent or the kit to obtain a Ct value;
step 3: judging whether a virus exists in the sample according to the Ct value;
the judgment criteria include:
when the Ct value of the 6' -FAM channel is less than or equal to 36, the sample is judged to be positive to the canine distemper virus, and when the Ct value is more than 36, the sample is judged to be negative to the canine distemper virus;
when the Ct value of the VIC channel is less than or equal to 36, the canine parainfluenza virus is judged to be positive, and when the Ct value is more than 36, the canine parainfluenza virus is judged to be negative;
when the Ct value of the ROX channel is less than or equal to 36, the canine respiratory coronavirus is judged to be positive, and when the Ct value is more than 36, the canine respiratory coronavirus is judged to be negative;
the viruses include canine distemper virus, canine parainfluenza virus and canine respiratory coronavirus.
In some embodiments of the invention, the sample to be tested comprises one or more of an oral secretion sample, an ocular secretion sample, a nasal secretion sample, a blood sample, a tissue sample, or an environmental sample.
In some embodiments of the invention, the detected reaction procedure comprises:
【1】 Reverse transcription is carried out at 45-55 ℃ for 1-10min; 【2】 Pre-denaturing at 92-98 ℃ for 10s-10min, [ 3 ] denaturing at 92-98 ℃ for 2-30s; annealing at 56-62 deg.c for 2-50s and circulation times of 38-45 times.
In some embodiments of the invention, the detected reaction procedure comprises:
【1】 Reverse transcription (cycle 1) at 50℃for 5min; 【2】 Pre-denaturing at 95 ℃ for 3min (1 cycle), [ 3 ] denaturing at 95 ℃ for 10s; annealing at 58℃was extended for 30s (45 cycles), with 58℃being set at the same time to collect fluorescence.
The invention also provides a method for amplifying viruses, which is based on any of the following:
(A) The primer probe combination; and/or
(B) The amplification reagent; and/or
(C) The kit;
the viruses include canine distemper virus, canine parainfluenza virus and canine respiratory coronavirus.
In some embodiments of the invention, the method of amplifying a virus comprises the steps of:
step 1: taking a sample to be tested;
step 2: amplifying by using the primer probe combination, the amplification reagent or the kit;
the viruses include canine distemper virus, canine parainfluenza virus and canine respiratory coronavirus.
In some embodiments of the invention, the sample to be tested comprises one or more of an oral secretion sample, an ocular secretion sample, a nasal secretion sample, a blood sample, a tissue sample, or an environmental sample.
In some embodiments of the invention, the amplification reaction procedure comprises:
【1】 Reverse transcription is carried out at 45-55 ℃ for 1-10min; 【2】 Pre-denaturing at 92-98 ℃ for 10s-10min, [ 3 ] denaturing at 92-98 ℃ for 2-30s; annealing at 56-62 deg.c for 2-50s and circulation times of 38-45 times.
In some embodiments of the invention, the amplification reaction procedure comprises:
【1】 Reverse transcription (cycle 1) at 50℃for 5min; 【2】 Pre-denaturing at 95 ℃ for 3min (1 cycle), [ 3 ] denaturing at 95 ℃ for 10s; annealing at 58℃was extended for 30s (45 cycles), with 58℃being set at the same time to collect fluorescence.
The invention provides a primer probe combination and a kit for detecting three RNA viruses infecting the respiratory tract of dogs. The invention can carry out on-machine amplification by adding a proper amount of sample nucleic acid into an amplification reagent, the reagent still has stable amplification performance after uncovering, freeze thawing and illumination for many times, the amplification sensitivity is 1 copy/mu L, and the total time of extraction and amplification is 1.5 hours; the reagent is provided with the detection of the canine housekeeping gene target and the UNG enzyme, and the detection result of the canine housekeeping gene target can monitor the processes of sample sampling, transportation, extraction and amplification, so that false negative is avoided; the addition of UNG enzyme can avoid the pollution of aerosol during sample addition and avoid false positive.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows a reverse transcription-real-time fluorescent quantitative PCR (RT-qPCR) schematic diagram;
FIG. 2 shows a graph of amplification results of the kit of the present invention with a positive quality control as a template;
FIG. 3 is a graph showing the amplification results of canine distemper virus in example 2 under templates with different plasmid concentrations;
FIG. 4 is a graph showing amplification results of canine parainfluenza virus in example 2 under templates of different plasmid concentrations;
FIG. 5 is a graph showing the amplification results of canine respiratory coronavirus in example 2 under templates of different plasmid concentrations;
FIG. 6 is a graph showing the amplification results of canine housekeeping gene in example 2 under templates of different plasmid concentrations;
FIG. 7 is a graph showing the amplification result of canine distemper virus in non-specific test in example 3;
FIG. 8 is a graph showing amplification results of canine parainfluenza virus in non-specific test in example 3;
FIG. 9 is a graph showing amplification results of canine respiratory coronavirus in example 3 under a nonspecific test;
FIG. 10 is a graph showing the amplification results of canine housekeeping gene under non-specific test in example 3;
FIG. 11 is a graph showing the amplification results of canine distemper virus in example 4 under the test of uncapping and freeze-thaw stability;
FIG. 12 is a graph showing the amplification results of canine parainfluenza virus in example 4 under the test of uncapping and freeze-thaw stability;
FIG. 13 is a graph showing the amplification results of canine respiratory coronavirus in example 4 under the decap and freeze-thaw stability tests;
FIG. 14 is a graph showing the amplification results of canine housekeeping genes in example 4 under the decapping and freeze-thaw stability test;
FIG. 15 is a graph showing the amplification comparison result of canine distemper virus in comparative example using the primer probe combination of the present invention and the primer probe combination of the control group;
FIG. 16 is a graph showing the amplification results of canine parainfluenza virus in comparative examples using the primer probe combinations of the present invention and the control primer probe combinations;
FIG. 17 is a graph showing the results of amplification comparison of canine respiratory coronavirus in comparative example using the primer probe combinations of the present invention and the control primer probe combinations;
FIG. 18 is a graph showing the results of amplification comparison of canine housekeeping genes in comparative examples using the primer probe combinations of the present invention and the primer probe combinations of the control group.
Detailed Description
The invention discloses a primer probe combination and a kit for detecting three RNA viruses infecting the respiratory tract of dogs, and the technical parameters can be properly improved by the person skilled in the art by referring to the content of the invention. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
Term interpretation:
canine distemper virus (canine distemper virus, CDV): canine distemper virus CDV is a large (100-250 nm) ssRNA pantropic virus, belongs to measles virus genus of Paramyxoviridae, and has genome of non-segmented and non-overlapping negative strand RNA, and consists of 15616 nucleotides. The virus can survive for several months at-10 ℃ and can survive for a long time at-70 ℃ or under freeze-drying condition; the dried virus is stable at room temperature and is easily inactivated above 32 ℃. The virus is unstable at pH 3.0, fashionable and stable above pH 4.5, and pH 7.0 is beneficial to the preservation of the virus. Is relatively unstable in the host, is transmitted mainly by aerosol droplet secretions of infected animals, and is also highly transmissible to dogs and other carnivores (such as ferrets, raccoons, skunks and foxes). The virus initially replicates in the lymphoid tissue of the respiratory tract, and cell-associated viremia causes all lymphoid tissue infections, followed by respiratory, gastrointestinal and genitourinary epithelium, and central and optic nerve infections. Severity is regulated by the specific humoral immunity of the host during viremia, and some infected dogs may shed viral particles within months. There are vaccines.
Canine parainfluenza virus (canine parainfluenza virus, CPIV): CPIV, also known as Simian virus 5,or parainfluenza virus 5,canine parainfluenza 2,or simply canine parainfluenza,2016 years, was ultimately renamed to mammalian orthomumps virus type 5 (Mammalian orthorubulavirus). CPIV is a respiratory virus of enveloped single-stranded negative-sense RNA belonging to Paramyxoviridae and the genus Lu Bula. CPIV follows a polar transcription mechanism, and mRNA abundance decreases from 3 'end to 5' end in sequence, so that the expression quantity of N protein is highest, and the expression quantity of L protein is lowest. CPIV is excreted from the respiratory tract of infected animals up to 2 weeks after infection, usually by air transmission. Viruses spread rapidly in doghouses or acceptors where a large number of dogs are kept. CPIV can be isolated from up to 50% of dogs with respiratory disease in the kennel environment prior to vaccine introduction.
Canine respiratory coronavirus (canine respiratory coronavirus, CRcov): canine coronaviruses are positive-stranded, non-segmented RNA viruses, in two forms: enterocanine coronavirus (canine coronavirus type I, CCoV) and respiratory canine coronavirus (canine coronavirus type II, CRCoV). CRCoV is transmitted by direct dog-to-dog contact or by air and enters the lungs, causing acute respiratory symptoms. CRCoV is most commonly found in the nasal mucosa, nasal tonsils and trachea, which are also detected in the lung and bronchial lymph nodes. Nucleocapsid genes are highly conserved in each coronavirus group, thereby ensuring detection of variant CRCoV strains. The canine respiratory coronavirus is not transmitted to other species. There is currently no vaccine against this respiratory virus.
False positive: meaning that in a PCR reaction, nonspecific amplification or contaminating amplification is caused by some reason (such as contamination of the environment with aerosols generated from the DNA product of the pre-amplification), thereby producing an amplification product similar to the target sequence or amplification caused by the nucleic acid of the sample, thereby exhibiting a target positive result, which is called false positive.
False negative: the result shows negative, but in reality the subject may be infected with a pathogen of some kind. This may be due to improper experimental operation, reagent quality problems, improper sample handling, or low pathogen concentrations.
UNG enzyme (uracil-N-glycylase): the alias uracil N-glycosylase can selectively hydrolyze and break uracil glycosidic bond in dU-containing double-stranded or single-stranded DNA, and the DNA chain with missing base is further hydrolyzed and broken under alkaline medium and high temperature, so that the DNA chain is eliminated. The optimal activation temperature of UNG enzyme is 50deg.C, 95deg.C. Most commonly, the most important contaminant in PCR reactions is the PCR product, which is a DNA strand containing dU, because the anti-pollution hot start PCR kit replaces dTTP with dUTP. The heat preservation step of 50 ℃ is added before the PCR is started, and the UNG enzyme can degrade uracil bases in the existing U-DNA pollutants in the reaction system, and DNA chain breaks under the condition of subsequent denaturation, so that amplification caused by polluted DNA is eliminated, and the specificity and accuracy of an amplification result are ensured. Meanwhile, UNG enzyme is inactivated, and the newly amplified product U-DNA is not degraded.
CT value: refers to the threshold Cycle number (Ct), i.e., the number of cycles that each reaction tube experiences when the fluorescent signal within that tube reaches a set threshold. The threshold is typically 10 times the standard deviation of the baseline, at which time PCR amplification enters the exponential amplification phase. The baseline is typically a fluorescent signal of 3-15 cycles. The Ct value has a linear relation with the logarithm of the initial copy number of the template, and the higher the initial template concentration is, the smaller the Ct value is; the lower the initial template amount concentration, the greater the Ct value.
The technical problems to be solved by the invention are as follows:
1. the common pathogenic RNA virus triple detection of the respiratory tract of dogs is rapidly and accurately carried out: the pathogens in the pet body are effectively identified, misdiagnosis and missed diagnosis are avoided, a pet doctor and a pet owner are helped to find diseases in time and take corresponding treatment measures, and further development and transmission of the diseases are avoided;
2. and (3) internal standard control: the kit comprises a primer and a probe of the canine housekeeping gene, can effectively monitor the sampling, transportation, nucleic acid extraction and amplification processes of the canine samples, and avoids false negative results;
3. avoiding aerosol pollution: the kit contains UNG enzyme, and is effective in preventing false positive results caused by aerosol pollution.
4. The stability is excellent: under the condition of repeated freezing and thawing and uncovering, the detection activity and accuracy can be kept.
5. The operation is simple and convenient: the kit is simple and convenient to operate, and convenient for a pet doctor to use and operate;
6. improving the health level of pets: the detection of the pathogenic nucleic acid of the pet can help the pet owner to discover and treat diseases in the pet in time, improve the health level of the pet and prolong the service life of the pet;
7. monitoring epidemic situation dynamics: by checking and monitoring the viruses, epidemic situation dynamics can be mastered in time, corresponding prevention and control strategies and measures are formulated, and the health and safety of dogs are ensured.
The background technology adopted by the invention is reverse transcription-real-time fluorescence quantitative PCR (RT-qPCR), and the principle is shown in figure 1.
The molecular probe is a single-stranded DNA molecule with fluorescent markers, and the 5 'end and the 3' end of the molecular probe are respectively marked with a fluorescent group and a quenching group. When the probe is complete, the fluorescent signal emitted by the fluorescent group is absorbed by the quenching group; during PCR amplification, the 5'-3' exonuclease activity of the DNA polymerase enzyme cleaves and degrades the probe to separate the reporting fluorescent group from the quenching fluorescent group, so that a fluorescent monitoring system of the real-time fluorescent PCR instrument can receive a fluorescent signal. That is, every time a nucleic acid strand is amplified, a fluorescent molecule is formed, and the accumulation of fluorescent signals and the formation of PCR products are completely synchronized.
In the amplification process, RNA single strand in a sample is firstly reverse transcribed into cDNA (complementary DNA) double strand by reverse transcriptase in a reagent at 50 ℃, then variable-temperature amplification is carried out under the action of DNA polymerase, the instrument can carry out fluorescence detection at the end of each variable-temperature cycle, and the fluorescence reaches the theoretical maximum value at the end of the cycle. The method has high sensitivity and strong specificity, can relatively quantify, and can approximately estimate the virus copy number in the sample according to the CT value.
Nucleic acid combinations for detecting canine distemper virus, canine parainfluenza virus, and canine respiratory coronavirus include:
an upstream primer designed for canine distemper virus, the sequence of the upstream primer is 5'-tctgccggcaaagtaagctc-3'
(SEQ ID No. 1); abbreviated CDV-F;
downstream primer designed for canine distemper virus and having sequence of 5'-gtcctccgttgtcttggatgc-3'
(SEQ ID No. 2); abbreviated CDV-R;
molecular probes designed for canine distemper virus, and the sequences of the molecular probes are as follows
5'-cacttgccgccgagcttggcatcacca-3' (SEQ ID No. 3); for short, CDV-P, the 5 'end of the fluorescent probe is marked as a reporter group 6-FAM, and the 3' end is marked as a quenching group BHQ1;
an upstream primer designed for canine parainfluenza virus, the sequence of the upstream primer being 5'-gccgtggagaaatcaatgc-3' (SEQ ID No. 4); CPIV-F for short;
a downstream primer designed for canine parainfluenza virus, the downstream primer having the sequence 5'-tccctcgacttcggagtcaa-3' (SEQ ID No. 5); CPIV-R for short;
a molecular probe designed for canine parainfluenza virus, the sequence of the molecular probe is 5'-atgctgcacttgcagaagatctacctgacacac-3' (SEQ ID No. 6); CPIV-P for short, wherein the 5 'end of the fluorescent probe is marked as a reporter group VIC, and the 3' end is marked as a quenching group BHQ1;
an upstream primer designed for canine respiratory coronavirus, the sequence of the upstream primer being 5'-ttgaaggctcaggaaggtct-3' (SEQ ID No. 7); abbreviated CRCoV-F;
a downstream primer designed for canine respiratory coronavirus, the downstream primer having the sequence 5'-ctattgccagaattggctctac-3' (SEQ ID No. 8); abbreviated CRCoV-R;
a molecular probe designed for canine respiratory coronavirus, the sequence of the molecular probe is 5'-tccagatctacttcacgcgcatccagtagagc-3' (SEQ ID No. 9); for short CRCoV-P, the 5 'end of the fluorescent probe is marked as a reporter group ROX, and the 3' end is marked as a quenching group BHQ2;
an upstream primer designed for the canine housekeeping gene, the sequence of the upstream primer being 5'-aaaggaactcggcaaacaca-3' (SEQ ID No. 10); abbreviated CIC-F;
a downstream primer designed for the canine housekeeping gene, the sequence of the downstream primer being 5'-ggcagtgcctccaatactaga-3' (SEQ ID No. 11); abbreviated CIC-R;
molecular probes designed for canine housekeeping genes, wherein the sequences of the molecular probes are as follows
5'-accccgcctgtttaccaaaaacatcacctcc-3' (SEQ ID No. 12); for short CIC-P, the 5 'end of the fluorescent probe is marked as a reporter group CY5, and the 3' end is marked as a quenching group BHQ2.
The kit for detecting the canine distemper virus, the canine parainfluenza virus and the canine respiratory coronavirus comprises an amplification reagent, a positive quality control product, a negative quality control product, a sample collection and preservation tube-flocking swab composition and a kit instruction.
The amplification reagent comprises a primer probe combination of the nucleotide sequence, an amplification buffer solution, DNA polymerase, reverse transcriptase and UNG enzyme. The primer probe combination comprises the 4 pairs of primer groups and 4 molecular probes, the final concentration of the primer groups can be 100-1000nM (preferably 300 nM), the final concentration of the molecular probes can be 50-500nM (preferably 100 nM), and the amplification buffer comprises 20-100mmol KCl (preferably 50 mmol), 0.5-5mmol MgCl2 (preferably 1.5 mmol) and 0-10mmol (NH 4) 2 SO 4 (preferably 2 mmol), dNTPs (dATP, dCTP, dGTP, dTTP each 0.2 mmol), 5-20mmol Tris-HCl (preferably 10 mmol) and 0% -10% DMSO (preferably 5%); DNA polymerase, reverse transcriptase and UNG enzyme were all purchased from Zhuhai Sharp Biotech Co., ltd per reactionThe enzyme activities were 5U, 200U and 5U, respectively.
The positive quality control product is an artificially synthesized plasmid which contains a canine distemper virus target fragment, a canine parainfluenza virus target fragment, a canine respiratory tract coronavirus target fragment and a canine housekeeping gene target fragment at the same time, and the concentration is 10 copies/mu L;
the negative quality control product is TE buffer solution with pH of 7;
the sample collection and preservation tube and flocked swab composition are manufactured by Shenzhen Bertoni biological products Co.
The preparation steps of the kit are as follows:
combining and quantifying the above primers to a specific concentration (100-1000 nM, preferably 300 nM) and combining and quantifying the above molecular probes to a specific concentration (50-500 nM, preferably 100 nM) in a PCR-grade clean shop; combining and quantifying the materials contained in the amplification reagent into the concentration range; and respectively preparing a negative quality control product and a positive quality control product, and then assembling a sample collection and preservation tube-flocking swab composition, a kit instruction and a kit label.
1. The nucleic acid composition used in the invention is an oligonucleotide composition of primer/molecular probe, wherein the fluorescent groups used in the molecular probe are as follows:
| molecular probe name | Fluorescent group | Quenching group |
| CDV-P | 6’-FAM | BHQ1 |
| CPIV-P | VIC | BHQ1 |
| CRCoV-P | ROX | BHQ2 |
| CIC-P | CY5 | BHQ2 |
Other alternatives may be exchange of fluorophores, as may corresponding quenching groups.
In addition to the above fluorophores, TET, JOR, HEX, CY, NED, TXR, TAMRA, texas red, SX670, CY5.5, CY7 can be substituted for the above fluorophores;
in addition to the above-described quenching groups, TAMRA, BHQ0, BHQ3, dabcyl, MGB, SQ1, SQ2, SQX may be substituted for the above-described quenching groups.
3. The DNA polymerase, reverse transcriptase and UNG enzyme used in the present invention are all available from Zhuhai Sharp Biotech Inc. and other alternatives are available from other enzyme reagent manufacturers
4. The invention sets up the amplification reaction procedure on the fluorescent quantitative PCR instrument as follows: 【1】 5min (cycle one time) at 50 ℃; 【2】 95℃for 3min (cycle 1); 【3】 95℃10s,58℃30s (45 cycles), wherein 58℃is set at the same time to collect fluorescence.
Alternatively, [ 1 ] 50 ℃ (reverse transcription) can be set to 45-55 ℃ for 1-10min; 【2】 95 ℃ (pre-denaturation) can be set to 92-98 ℃, the time can be set to 10s-10min, 95 ℃ (denaturation) can be set to 92-98 ℃, and the time can be set to 2-30s;58 ℃ (annealing extension) can be set to 56-62 ℃, the time can be set to 2-50s, and the number of cycles can be set to 38-45 times.
The artificially synthesized plasmid sequences of the canine distemper virus target fragment, the canine parainfluenza virus target fragment, the canine respiratory tract coronavirus target fragment and the canine housekeeping gene of the nucleic acid group are as follows:
the synthetic plasmid sequence of canine distemper virus (SEQ ID No. 13):
ttcatggtggcgctcatcttggacatcaaacgatccccagggaacaagcctagaattgctgaaatgatttgtgatatagataactacattgtggaagctgggttagctagtttcatcctaactatcaagtttggcattgaaactatgtatccggctcttgggttgcatgagttttccggagaattaacaactattgaatccctcatgatgctatatcaacagatgggtgaaacagcaccgtacatggttatcttggaaaactctgttcaaaacaaatttagtgcagggtcctacccattgctctggagttatgctatgggggttggtgttgaacttgaaaactccatgggagggttaaatttcggtcgatcttactttgacccagcttacttcagactcgggcaagaaatggttaggagatctgccggcaaagtaagctctgcacttgccgccgagcttggcatcaccaaggaggaagctcagctagtgtcagaaatagcatccaagacaacagaggaccggacaattcgagctactggtcctaagcaatcccaaatcacttttctgcactcggaaagatccgaagtcgccaatcaacaacccccaaccatcaacaagaggtccgaaaaccagggaggagacaaataccccattcacttcagtgacgaaaggcttccagggtataccccagatgtcaacagttctgaatggagtgagtcacgctatgacacccaaattatccaagatgatggaaatgacgatgatcggaaatcgatggaagcaatcgccaagatgaggat
canine parainfluenza virus synthetic plasmid sequence (SEQ ID No. 14):
Gtgcttaaagcatatgagcgattcacgctcactcaagaactgcaagatcagagtgaggaaggtacaatcccacctacaacactaaaaccggtaatcagggtatttatactaacctctaataacccagagctaagatcccggcttcttctattctgcctacggattgttctcagtaatggtgcaagggattcccatcgctttggagcattactcacaatgttttcgctaccatcagccacaatgctcaatcatgtcaaattagctgaccagtcaccagaagctgatatcgaaagggtagagatcgatggctttgaggagggatcattccgcttaatccccaatgcacgttcaggtatgagccgtggagagatcaatgcctatgctgcacttgcagaagatctacctgacacactaaaccatgcaacacctttcgttgattccgaagtcgagggaactgcatgggatgagattgagactttcttagatatgtgttacagtgtcctaatgcaggcatggatagtgacttgcaagtgcatgactgcgccagaccaacctgctgcttctattgagaaacgcctgcaaaaatatcgtcagcaaggcaggatcaacccgagatatctcctgcaaccggaggctcgacgaataatccagaatgtaatccggaagggaatggtggtcagacatttcctcacctttgaactgcagcttgcccgagcacaaagccttgtatcaaataggtattatgctatggtaggggatgttggaaagtatatagagaattgtggaatgggaggcttctt
the canine respiratory coronavirus artificial plasmid sequence (SEQ ID No. 15):
Ctcagtttcaaaaaggaagggattttgaatttgcagagggacaaggtgtgcctattgcaccaggagtcccagctactgaagctaaggggtactggtacagacacaacagacgttcttttaaaacagccgatggcaaccagcgtcaactgctgccacgatggtatttttactatcttggaacaggaccgcatgccaaagaccagtatggcaccgatattgacggtttcttctgggtcgctagtaaccaggctgatgtcaataccccggctgacattcccgatcgggacccaagtagcgatgaggctattccgactaggtttccgcctggcacggtactccctcagggttactatattgaaggctcaggaaggtctgctcctaattccagatctacttcacgcgcatccagtagagcctctagtgcaggatcgcgtagtagagccaattctggcaacagaacccctacctctggtgtaacacctgatatggctgatcaaattgctagtcttgttctggcaaaacttggcaaggatgccactaagccacagcaagtaactaagcagactgccaaagaaatcagacagaaaattttgaataagccccgccagaagaggagccccaataaacaatgcactgttcagcagtgttttgggaagagaggccccaatcagaattttggtggtggagaaatgttaaaacttggaactagtgacccacagttccccattcttgcagaactcgcacccacagctggtgcgtttttctttggatcaagattagagttggccaaagtgcagaat
canine housekeeping gene artificial plasmid sequence (SEQ ID No. 16):
aaaagcagccaccaattgagaaagcgttccagctcaacaaacaatataacttaatcccaaccatactacatcaactcctaattataccccctgggtcattctatttaagtatagaagcaataatgctagtatgagtaacaagaaccattttctccccgcataagcttatatcaggaacggatagaccactgatagttaacaatctgataatatcaacccaaaaatgaaatacttatccaccccattgttaacccaacacaggtatgcattcaaggaaagattaaaaggagtaaaaggaactcggcaaacacaaaccccgcctgtttaccaaaaacatcacctccagcatttctagtattggaggcactgcctgcccggtgacacttgtttaacggccgcggtatcctgaccgtgcaaaggtagcataatcatttgttctctaaatagggacttgtatgaatggccacacgagggtttaactgtctcttactcccaatcagtgaaattgaccttcccgtgaagaggcgggaataccacaataagacgagaagaccctatggagctttaattaactaacccaaacttatggatactagatacctacaaggcataacataacaccattattatgagttagcaatttaggttggggtgacctcggaatataaaaaaactcccgagtgattaaaatttagacccacaagtcaaaatacaacatcacttattgatccaataatttttgatcaacggaacaagttaccctagggataacagcgcaatcctattcaagagtccatatc
the primer probe combination for detecting three RNA viruses infecting the respiratory tract of dogs and the raw materials and reagents used in the kit can be purchased from the market.
The invention is further illustrated by the following examples:
example 1
The final concentrations of the primer probe nucleic acid sets of the nucleic acid combinations of the invention are shown in Table 1.
TABLE 1 final concentration of primer probe nucleic acid composition of example 1
| Primer/molecular probe name | Final concentration (nM) was used |
| CDV-F | 300 |
| CDV-R | 300 |
| CDV-P | 100 |
| CPIV-F | 300 |
| CPIV-R | 300 |
| CPIV-P | 100 |
| CRCoV-F | 300 |
| CRCoV-R | 300 |
| CRCoV-P | 100 |
| CIC-F | 300 |
| CIC-R | 300 |
| CIC-P | 100 |
The total volume of the amplification reagents (comprising the primer probe combination, the amplification buffer, the DNA polymerase 5U, the reverse transcriptase 200U, UNG enzyme 5U) using the kit is 15uL, wherein the amplification buffer comprises KCl 50mmol, mgCl21.5mmol, (NH 4) 2 SO 4 2mmol, dNTP (dATP, dCTP, dGTP, dTTP each 0.2 mmol), tris-HCl10mmol and DMSO 5%, adding 10 μL of positive quality control template to prepare 25 μL of amplification system, covering the tube cover, and micro-centrifuging to add the amplification reaction tube into the amplification hole of the fluorescent quantitative PCR instrument. The amplification reaction procedure was set on the fluorescent quantitative PCR instrument as follows: 【1】 5min (cycle one time) at 50 ℃; 【2】 95℃for 3min (cycle 1); 【3】 95℃10s,58℃30s (45 cycles), wherein 58℃is set at the same time to collect fluorescence.
The data results are shown in figure 2.
The amplification curve is shown in FIG. 2, and the obtained Ct value is less than or equal to 32 by taking the positive quality control product as a template, so that the reagent can normally detect the positive quality control product. The positive Ct judgment of each target gene of the kit is shown in Table 2.
TABLE 2
Example 2
The effect of the amplification reagents on each concentration of target was examined according to the amplification reagents, amplification systems, and amplification reaction procedures described in example 1.
The synthetic plasmids (mixed plasmids with equal proportions) containing the Canine Distemper Virus (CDV) target fragment, the canine parainfluenza virus (CPIV) target fragment, the canine respiratory coronavirus (CRCoV) target fragment and the canine housekeeping gene (CIC) target fragment are respectively diluted to 1000 copies/mu L, 100 copies/mu L, 10 copies/mu L and 1 copy/mu L, and the diluted mixed plasmids are respectively and uniformly mixed with an amplification reagent for amplification so as to test the detection effect of the amplification reagent on targets with different concentrations. The data graphs of the detection results are shown in the following FIGS. 3 to 6.
The results show that the amplification reagents of the invention can perform good amplification detection at target nucleic acid concentrations of greater than or equal to 1 copy/. Mu.L, and at target nucleic acid concentrations of 1000 copies/. Mu.L, 100 copies/. Mu.L, 10 copies/. Mu.L, and 1 copy/. Mu.L, respectively, with corresponding Ct value intervals of 24+ -0.5, 27.5+ -0.5, 31+ -0.5, and 34.5+ -0.5.
EXAMPLE 3 non-specific amplification test
The same tube test was performed by taking the amplification reagents from the kit described in example 1, and adding the single detection target nucleic acid sample and the genome of other canine respiratory pathogens, including canine herpesvirus, canine adenovirus type 2, bordetella bronchiseptica, and canine mycoplasma, respectively, which were not the detection targets of the present reagents, and the human genome collected from the human mouth swab. The amplification system and the amplification reaction procedure described in example 1 were followed to observe whether or not there was a nonspecific amplification phenomenon, and the data of the detection results are shown in FIGS. 7 to 10.
The result shows that the primer probe combination has strong specificity, the detected target can be effectively amplified under the interference of other canine respiratory pathogen genomes and human genome nucleic acids, the Ct value is less than or equal to 36, and other canine respiratory pathogen genomes and human genome nucleic acid samples are not amplified, and the Ct value is not available.
Example 4 decap and freeze thaw test
After 10 times of uncapping and 10 times of repeated freezing and thawing of the amplification reagent, 10 mu L of artificially synthesized plasmid (mixed plasmid with equal proportion) simultaneously containing canine distemper virus target fragment, canine parainfluenza virus target fragment, canine respiratory coronavirus target fragment and canine housekeeping gene (CIC) target fragment is added, and the concentration is 1 copy/mu L and 15 times of repetition are carried out. The stability of the reagent after the uncapping and freeze-thawing treatment was examined according to the procedure of the amplification reaction described in example 1, and the examination results are shown in FIGS. 11 to 14.
The results show that under the conditions of repeated uncovering and freeze thawing, the amplification reagent can perform good amplification detection, and Ct values are less than or equal to 35.5, which indicates that the detection performance of the amplification reagent is not reduced and the stability is excellent.
Comparative example
According to the amplification reagents, amplification system and amplification reaction procedure described in example 1, the primer probe combinations of the present invention were amplified using 100 copies/. Mu.L of artificially synthesized plasmids (mixed plasmids of equal proportions) containing Canine Distemper Virus (CDV) target fragment, canine parainfluenza virus (CPIV) target fragment, canine respiratory coronavirus (CRCoV) target fragment and canine housekeeping gene (CIC) target fragment as templates, as shown in FIGS. 15 to 18, with another set of control primer probe combinations (primer probe combinations shown in Table 3) in the development process of the kit.
TABLE 3 Table 3
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. Primer probe combination, its characterized in that includes:
the primer has a nucleotide sequence shown as SEQ ID No. 1-2, 4-5, 7-8 and/or 10-11; and
(II) the probe has a nucleotide sequence shown as SEQ ID No.3, 6, 9 and/or 12; or (b)
(III) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences with the nucleotide sequence shown in (I) or (II), and having the same or similar functions as the nucleotide sequence shown in (I) or (II); or (b)
(IV) a nucleotide sequence having at least 80% sequence homology with the nucleotide sequence of any one of (I) to (III).
2. The primer probe combination of claim 1, comprising:
primers for detecting nucleotide sequences shown in SEQ ID No. 1-2 and probes for detecting nucleotide sequences shown in SEQ ID No.3 of canine distemper virus;
primers for detecting nucleotide sequences shown in SEQ ID Nos. 4 to 5 and probes for detecting nucleotide sequences shown in SEQ ID No.6 of canine parainfluenza virus;
primers for detecting nucleotide sequences shown in SEQ ID Nos. 7 to 8 and probes for detecting nucleotide sequences shown in SEQ ID No.9 of canine respiratory coronavirus;
primers for detecting the nucleotide sequences shown in SEQ ID Nos. 10 to 11 and probes for detecting the nucleotide sequences shown in SEQ ID No.12 of the canine housekeeping gene.
3. The primer probe combination of claim 1 or 2, wherein the probe is labeled with a fluorescent reporter gene at the 5 'end and a quencher group at the 3' end;
the fluorescent reporter gene comprises 6' -FAM, VIC, ROX, CY5, TET, JOR, HEX, CY3, NED, TXR, TAMRA, texas red, SX670, CY5.5 and/or CY7;
the quenching groups include BHQ1, BHQ2, TAMRA, BHQ0, BHQ3, dabcyl, MGB, SQ1, SQ2, and/or SQX.
4. An amplification reagent comprising the primer probe combination according to any one of claims 1 to 3.
5. The amplification reagent of claim 4, wherein the amplification reagent further comprises an amplification buffer, a DNA polymerase, a reverse transcriptase, and/or an UNG enzyme;
the amplification buffer comprises KCl, mgCl2, (NH 4) 2 SO 4 One or more of dNTP, tris-HCl or DMSO.
6. The amplification reagent of claim 4 or 5, wherein the final concentration of the primer in the primer probe combination is 100 to 1000nM; the final concentration of the probe in the primer probe combination is 50-500 nM;
the concentration of KCl is 20-100 mmol; the concentration of MgCl2 is 0.5-5 mmol; said (NH 4) 2 SO 4 The concentration of (2) is 0-10 mmol; the dNTPs include dATP, dCTP, dGTP and dTT; the concentrations of dATP, dCTP, dGTP and dTT were 0.2mmol; the concentration of the Tris-HCl is 5-20 mmol; the concentration of DMSO is 0-10%;
the enzyme activity of the DNA polymerase is 5U; the enzyme activity of the reverse transcriptase is 200U; the enzyme activity of the UNG enzyme is 5U.
7. The amplification reagent of any one of claims 4 to 6, wherein the final concentration of the primers in the primer probe combination is 300nM; the final concentration of the probes in the primer probe combination is 100nM;
the concentration of KCl is 50mmol; the concentration of MgCl2 is 1.5mmol; said (NH 4) 2 SO 4 Is 2mmol; the concentration of Tris-HCl is 10mmol; the DMSO concentration was 5%.
8. Use of any of the following in the preparation of a kit for detecting canine distemper virus, canine parainfluenza virus and canine respiratory coronavirus:
(A) A primer probe combination according to any one of claims 1 to 3; and/or
(B) An amplification reagent according to any one of claims 4 to 7.
9. Kit comprising a primer probe combination according to any one of claims 1 to 3 or an amplification reagent according to any one of claims 4 to 7.
10. A method for amplifying a virus, characterized in that a sample is amplified based on any of the following:
(A) A primer probe combination according to any one of claims 1 to 3; and/or
(B) An amplification reagent according to any one of claims 4 to 7; and/or
(C) The kit of claim 9;
the viruses include canine distemper virus, canine parainfluenza virus and canine respiratory coronavirus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410089134.9A CN117660700A (en) | 2024-01-22 | 2024-01-22 | Primer probe combination and kit for detecting three RNA viruses infecting respiratory tract of dogs |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410089134.9A CN117660700A (en) | 2024-01-22 | 2024-01-22 | Primer probe combination and kit for detecting three RNA viruses infecting respiratory tract of dogs |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117660700A true CN117660700A (en) | 2024-03-08 |
Family
ID=90075313
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410089134.9A Pending CN117660700A (en) | 2024-01-22 | 2024-01-22 | Primer probe combination and kit for detecting three RNA viruses infecting respiratory tract of dogs |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117660700A (en) |
-
2024
- 2024-01-22 CN CN202410089134.9A patent/CN117660700A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111020064B (en) | Novel coronavirus ORF1ab gene nucleic acid detection kit | |
| EP4015654A1 (en) | Composition, kit and method for detecting and typing coronaviruses | |
| CN110592290B (en) | Kit and method for detecting feline calicivirus | |
| CN111004870B (en) | Novel coronavirus N gene nucleic acid detection kit | |
| CN111440897A (en) | Probe and primer composition for rapidly detecting seven coronaviruses and other respiratory pathogens | |
| CN111321251A (en) | Composition, kit, method and application for detecting and typing pathogens causing respiratory tract infection | |
| CN111411172A (en) | Probe and primer composition for simultaneously detecting novel human coronavirus, influenza A virus and influenza B virus | |
| CN111808989A (en) | Coronavirus/influenza virus/rhinovirus nucleic acid combined detection kit and use method thereof | |
| KR102338861B1 (en) | PNA Probe and Primer for Detecting SARS-CoV-2 Causing Covid-19 Using RT-LAMP and Method for Detecting SARS-CoV-2 Infection Using Thereof | |
| CN113718045B (en) | DNA fragment, primer, probe and kit for detecting 4 kinds of Bordetella pertussis and specifically detecting Bordetella pertussis and application | |
| WO2023087868A1 (en) | Compositions, kits, methods for detecting and identifying pathogens that cause respiratory tract infections and use thereof | |
| CN113930547B (en) | RT-RAA fluorescence detection primer pair, kit and detection method for porcine epidemic diarrhea virus N gene | |
| CN116171333A (en) | Compositions and methods for detecting severe acute respiratory syndrome coronavirus 2 (SARS-COV-2), influenza A and influenza B | |
| KR20230030639A (en) | Methods for Detecting SARS-CoV-2, Influenza and RSV | |
| CN113652505A (en) | Method and kit for detecting novel coronavirus and VOC-202012/01 mutant strain thereof | |
| JP2023536962A (en) | Compositions and methods for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-2), influenza A and influenza B | |
| CN111471800A (en) | Kit for detecting novel coronavirus and amplification primer composition thereof | |
| KR102435209B1 (en) | Composition for simultaneously distinguishing and detecting influenza type A and type B viruses and type 2 severe acute respiratory syndrome coronavirus and detection method using the same | |
| CN116121458A (en) | Composition for detecting influenza A virus and influenza B virus and application thereof | |
| CN117660700A (en) | Primer probe combination and kit for detecting three RNA viruses infecting respiratory tract of dogs | |
| CN114395643A (en) | Double-channel digital PCR detection kit and method for African swine fever virus | |
| CN112111603A (en) | Composition, kit, application and method for detecting and typing respiratory tract related viruses | |
| CN111172320A (en) | Detection primer, kit and method for respiratory syncytial virus F gene | |
| CN101415844A (en) | Assay for SARS coronavirus by amplification and detection of nucleocapsid RNA sequence | |
| KR102395969B1 (en) | Primer set for detecting coronavirus using the loop-mediated isothermal amplification reaction and uses thereof |
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 |