CN117305297A - Helicobacter pylori nucleic acid detection primer probe composition and integrated microfluidic chip kit - Google Patents
Helicobacter pylori nucleic acid detection primer probe composition and integrated microfluidic chip kit Download PDFInfo
- Publication number
- CN117305297A CN117305297A CN202210714405.6A CN202210714405A CN117305297A CN 117305297 A CN117305297 A CN 117305297A CN 202210714405 A CN202210714405 A CN 202210714405A CN 117305297 A CN117305297 A CN 117305297A
- Authority
- CN
- China
- Prior art keywords
- freeze
- primer probe
- helicobacter pylori
- probe composition
- ipc
- 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 74
- 239000000203 mixture Substances 0.000 title claims abstract description 53
- 241000590002 Helicobacter pylori Species 0.000 title claims abstract description 45
- 229940037467 helicobacter pylori Drugs 0.000 title claims abstract description 45
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 22
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 22
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 22
- 238000001514 detection method Methods 0.000 title abstract description 34
- 238000000746 purification Methods 0.000 claims abstract description 11
- 239000003153 chemical reaction reagent Substances 0.000 claims description 42
- 238000000034 method Methods 0.000 claims description 24
- 238000004108 freeze drying Methods 0.000 claims description 23
- 238000003908 quality control method Methods 0.000 claims description 21
- 108020004414 DNA Proteins 0.000 claims description 20
- 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 16
- 239000006166 lysate Substances 0.000 claims description 15
- 239000011324 bead Substances 0.000 claims description 14
- 239000003480 eluent Substances 0.000 claims description 14
- ODLHGICHYURWBS-LKONHMLTSA-N trappsol cyclo Chemical compound CC(O)COC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)COCC(O)C)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1COCC(C)O ODLHGICHYURWBS-LKONHMLTSA-N 0.000 claims description 11
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 10
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 10
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 10
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 10
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 10
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 10
- 229940098773 bovine serum albumin Drugs 0.000 claims description 10
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 10
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 10
- 229930195725 Mannitol Natural products 0.000 claims description 9
- 239000000594 mannitol Substances 0.000 claims description 9
- 235000010355 mannitol Nutrition 0.000 claims description 9
- 235000010979 hydroxypropyl methyl cellulose Nutrition 0.000 claims description 8
- 229920003088 hydroxypropyl methyl cellulose Polymers 0.000 claims description 8
- 239000013612 plasmid Substances 0.000 claims description 8
- 239000013530 defoamer Substances 0.000 claims description 7
- 239000002158 endotoxin Substances 0.000 claims description 7
- 239000001866 hydroxypropyl methyl cellulose Substances 0.000 claims description 7
- UFVKGYZPFZQRLF-UHFFFAOYSA-N hydroxypropyl methyl cellulose Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(O)C(CO)O3)O)C(CO)O2)O)C(CO)O1 UFVKGYZPFZQRLF-UHFFFAOYSA-N 0.000 claims description 7
- 229920006008 lipopolysaccharide Polymers 0.000 claims description 7
- 239000004373 Pullulan Substances 0.000 claims description 6
- 229920001218 Pullulan Polymers 0.000 claims description 6
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 239000013504 Triton X-100 Substances 0.000 claims description 6
- 229920004890 Triton X-100 Polymers 0.000 claims description 6
- 229960000789 guanidine hydrochloride Drugs 0.000 claims description 6
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 claims description 6
- 239000001103 potassium chloride Substances 0.000 claims description 6
- 235000019423 pullulan Nutrition 0.000 claims description 6
- 239000001632 sodium acetate Substances 0.000 claims description 6
- 235000017281 sodium acetate Nutrition 0.000 claims description 6
- 102000053602 DNA Human genes 0.000 claims description 5
- 239000001226 triphosphate Substances 0.000 claims description 5
- 235000011178 triphosphate Nutrition 0.000 claims description 5
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 claims description 5
- 239000002518 antifoaming agent Substances 0.000 claims description 3
- 238000003745 diagnosis Methods 0.000 claims description 2
- 201000010099 disease Diseases 0.000 claims description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 9
- 230000003321 amplification Effects 0.000 abstract description 8
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 8
- 238000000605 extraction Methods 0.000 abstract description 5
- 244000052769 pathogen Species 0.000 abstract description 3
- 230000001717 pathogenic effect Effects 0.000 abstract description 3
- 238000012123 point-of-care testing Methods 0.000 abstract description 2
- 238000005070 sampling Methods 0.000 abstract description 2
- 238000012360 testing method Methods 0.000 description 12
- 108090000623 proteins and genes Proteins 0.000 description 9
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 8
- 241000186660 Lactobacillus Species 0.000 description 4
- 229940039696 lactobacillus Drugs 0.000 description 4
- 235000011164 potassium chloride Nutrition 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000010791 quenching Methods 0.000 description 3
- 230000000171 quenching effect Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 208000007107 Stomach Ulcer Diseases 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 208000010643 digestive system disease Diseases 0.000 description 2
- 208000000718 duodenal ulcer Diseases 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 241000711573 Coronaviridae Species 0.000 description 1
- 241000592183 Eidolon Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 108010046334 Urease Proteins 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 210000001198 duodenum Anatomy 0.000 description 1
- 230000002550 fecal effect Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000001821 nucleic acid purification Methods 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 238000013492 plasmid preparation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 229940126409 proton pump inhibitor Drugs 0.000 description 1
- 239000000612 proton pump inhibitor Substances 0.000 description 1
- 238000012134 rapid urease test Methods 0.000 description 1
- 125000006853 reporter group Chemical group 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 210000002784 stomach Anatomy 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
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- 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
- C12Q1/6851—Quantitative amplification
-
- 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
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
-
- 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
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- 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)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of biological detection, and particularly relates to a helicobacter pylori nucleic acid detection primer probe composition and an integrated microfluidic chip kit. The primer probe composition provided by the invention has the advantages of good sensitivity and accuracy, high amplification efficiency, simplicity and convenience in operation, rapidness and time saving when used for detecting helicobacter pylori. The invention integrates nucleic acid extraction, purification, amplification and detection by utilizing an integrated chip microfluidic mode, solves the problem of rapid pathogen detection, realizes a POCT detection scheme of 'sample in-out', directly adds the sample into equipment after sampling, and purifies and amplifies the sample to obtain the result within 60 minutes.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a helicobacter pylori nucleic acid detection primer probe composition and an integrated microfluidic chip kit.
Background
Helicobacter pylori (h.pyrri) is one of the micro anaerobic bacterial pathogens. The bacterium is associated with gastric neoplasms associated with acidic digestive diseases of the stomach and duodenum. Pyri has been detected in individuals of all ages worldwide with prevalence ranging between 20% -80%. Marshall and Warren have accidentally found this bacterium in 1983, which has led to a significant change in our understanding of acid digestive diseases. Later in 2005, they obtained "nobel medical or physiological rewards" for the discovery of helicobacter pylori. About 80% and 95% of Gastric Ulcers (GU) and Duodenal Ulcers (DU), respectively, are reported to be caused by h.pyrri.
At present, there are at least seven diagnostic methods for helicobacter pylori: bacterial culture, rapid urease assay, urea breath assay, histology, PCR, serology and stool antigen assay. When microorganisms are few or the patient is taking acid inhibitors (proton pump inhibitors), the sensitivity of bacterial culture, rapid urease tests, urea breath tests, histological and fecal antigen tests is limited. The real-time fluorescent quantitative PCR technology is the most widely applied molecular detection method at present, and not only realizes quantitative detection, but also has the characteristics of high sensitivity, strong specificity, high degree of automation and the like. For example, patent CN101608210a discloses a probe and primer for detecting helicobacter pylori. .
Based on the problem, the applicant has developed for several years, and a novel on-site rapid nucleic acid detection device CarryOn P1000Q is disclosed in the prior application CN202110057507.0, and the device is a hand-held full-automatic closed nucleic acid qPCR analysis system, can complete on-site automatic nucleic acid detection of the novel coronavirus within 60 minutes, and can realize rapid detection of 'sample in and result out'. The application is based on the technology, and further develops a reagent type invention which can be better matched with the device, namely a microfluidic chip kit for detecting helicobacter pylori.
The existing detection kit and method have longer operation time and complicated operation steps, so that the helicobacter pylori detection kit with high sensitivity, strong specificity, wide coverage, simple operation, time saving and cost saving and the use method thereof are needed to be provided.
Disclosure of Invention
In order to solve the problems in the prior art, the application provides a helicobacter pylori nucleic acid detection primer probe composition and an integrated microfluidic chip kit. The primer probe for detecting helicobacter pylori has the advantages of better sensitivity and accuracy, high amplification efficiency, simple operation, rapidness and time saving.
In order to achieve the above object, the present invention provides the following technical solutions:
in one aspect, the invention provides a primer probe composition for detecting helicobacter pylori, which comprises a primer probe composition for detecting helicobacter pylori and a primer probe composition for detecting internal reference.
Specifically, the primer probe composition for detecting helicobacter pylori is an HP primer probe composition;
specifically, the primer probe composition for detecting the internal reference is an IPC primer probe composition.
Further specifically, the method comprises the steps of,
(1) The HP primer probe composition comprises:
F(SEQ ID NO:1)：5'-ACCTGCTGGAACATTACTGAC-3'；
R(SEQ ID NO:2)：5'-GCCCTCCAACAACTAGCAT-3'；
P(SEQ ID NO:3)：5'-CTGGTAGTCCACGCCCTAAACGA-3'；
(2) The IPC primer probe composition comprises:
F(SEQ ID NO:4)：5'-AGTTGCAGTGTAACCGTCATGTA-3'；
R(SEQ ID NO:5)：5'-TCGACGAGACTCTGCTGTTAA-3'；
P(SEQ ID NO:6)：5'-CAGTAATCTGCGTCGCACGTGTGCA-3'。
specifically, the HP primer probe composition is used for detecting the 16SrRNA gene.
Specifically, the 5 'end of the probe is marked with a fluorescence report group, and the 3' end of the probe is marked with a quenching group; the fluorescent reporter group is one or more of CY3, CY5, CY5.5, FAM, HEX, VIC, JOE or ROX, and the quenching group is a fluorescence quenching group.
More specifically, the probe for detecting HP is labeled with FAM, and the probe for detecting the internal reference IPC is labeled with CY5.
In another aspect, the invention provides application of the primer probe composition in preparation of helicobacter pylori detection products.
In particular, the products include, but are not limited to, stand-alone reagents, chips or kits.
In yet another aspect, the present invention provides a product for detecting helicobacter pylori, the product comprising the primer probe composition described above.
In particular, the products include, but are not limited to, stand-alone reagents, chips or kits.
Specifically, the product also comprises a purifying reagent, a freeze-dried IPC quality control product and a freeze-dried reagent.
The purification reagent comprises a lysate, an eluent and magnetic beads, wherein the lysate comprises guanidine hydrochloride, sodium acetate and Triton X-100, the eluent comprises Tris-HCl, the volume ratio of the lysate to the eluent is 1:2-2.5, and the magnetic beads are dried magnetic beads.
Further specifically, the sequence of the freeze-dried IPC quality control product is SEQ ID NO. 7.
Further specifically, the lyophilized IPC quality control is a DNA plasmid.
Further specifically, the freeze-drying system of the freeze-drying IPC quality control product comprises mannitol, trehalose, 2-hydroxypropyl-beta-cyclodextrin, bovine serum albumin, defoamer, tris-HCl pH8.0, naCl and Tween20.
Further specifically, the freeze-dried IPC quality control product is packaged in an integrated micro-fluidic chip in the form of freeze-dried balls.
Further specifically, the lyophilization reagents include lipopolysaccharide and hydroxypropyl methylcellulose.
Further specifically, the freeze-drying reagent also comprises mannitol, trehalose, pullulan, 2-hydroxypropyl-beta-cyclodextrin, deoxyribonucleic acid polymerase, bovine serum albumin, defoamer, deoxynucleoside triphosphate dNTPs, KCl, tris-HCl pH9.0 and MgCl 2 、Tween20。
In still another aspect, the invention also provides a microfluidic chip kit for detecting helicobacter pylori, which comprises the primer probe composition.
Specifically, the product also comprises a purifying reagent, a freeze-dried IPC quality control product and a freeze-dried reagent.
The purification reagent comprises a lysate, an eluent and magnetic beads, wherein the lysate comprises guanidine hydrochloride, sodium acetate and Triton X-100, the eluent comprises Tris-HCl, the volume ratio of the lysate to the eluent is 1:2-2.5, and the magnetic beads are dried magnetic beads.
Further specifically, the lysate comprises 6M guanidine hydrochloride, 0.4M sodium acetate at pH4.7 and 2% Triton X-100, and the eluate comprises 10mM Tris-HCl at pH 8.5.
Further specifically, the sequence of the freeze-dried IPC quality control product is SEQ ID NO. 7.
Further specifically, the lyophilized IPC quality control is a DNA plasmid.
Further specifically, the freeze-drying system of the freeze-drying IPC quality control product comprises mannitol, trehalose, 2-hydroxypropyl-beta-cyclodextrin, bovine serum albumin, defoamer, tris-HCl pH8.0, naCl and Tween20.
Further specifically, the freeze-dried IPC quality control product is packaged in an integrated micro-fluidic chip in the form of freeze-dried balls.
Further specifically, the lyophilization reagents include lipopolysaccharide and hydroxypropyl methylcellulose.
Further specifically, the freeze-drying reagent also comprises mannitol, trehalose, pullulan, 2-hydroxypropyl-beta-cyclodextrin, deoxyribonucleic acid polymerase, bovine serum albumin, defoamer, deoxynucleoside triphosphate dNTPs, KCl, tris-HCl pH9.0 and MgCl 2 、Tween20。
Specifically, the kit completes the extraction, purification, amplification and detection of the nucleic acid of the sample on an integrated chip.
In particular, the microfluidic chip kit can be used for Carryon P1000Q rapid nucleic acid detection equipment.
In yet another aspect, the invention provides the use of the primer probe composition, product or kit described above for the detection of helicobacter pylori.
In still another aspect, the present invention provides a method for detecting helicobacter pylori, the method being a non-disease diagnosis and treatment method, the method comprising detecting helicobacter pylori in a sample to be detected using the primer probe composition, product or kit described above.
Specifically, the method comprises the following steps:
(1) Extracting DNA of a sample and purifying by adopting the purifying reagent;
(2) Amplifying the sample DNA extracted and purified in step (1) using the primer probe composition described above;
(3) And analyzing the amplification result.
Further specifically, the steps are performed on a microfluidic integrated chip by using CarryOn P1000Q rapid nucleic acid detection equipment.
Compared with the prior art, the kit provided by the invention has the following beneficial effects:
(1) The primer probe composition provided by the invention has the advantages of good sensitivity and accuracy, high amplification efficiency, simplicity and convenience in operation, rapidness and time saving when used for detecting helicobacter pylori.
(2) The problem of pathogen rapid detection is solved by utilizing an integrated chip microfluidic mode, a POCT detection scheme of sample inlet-outlet is realized, equipment is directly added after sampling, and the results are obtained within 60 minutes after extraction, purification, amplification and detection of nucleic acid are integrated.
Drawings
FIG. 1 is a graph of sensitivity detection results.
FIG. 2 is a graph of the results of the reproducibility of the kit.
FIG. 3 is a graph showing the results of specific tests, wherein A is the result of human genome test, B is the result of E.coli genome test, and C is the result of lactobacillus genome test.
Fig. 4 is a schematic diagram of a microfluidic chip.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
The examples are not to be construed as a specific technique or condition, and are carried out according to the techniques or conditions described in the literature in the art (e.g., refer to J. Sam Brooks et al, J. Mi. Cloning Experimental guidelines, third edition, scientific Press, et al, huang Peitang et al) or according to the specifications of the product.
1. Reagent and instrument
(1) The information on the reagents used in this application is detailed in Table 1 below.
Table 1 reagents and manufacturer
(2) The instrument information used in this application is detailed in table 2 below.
Table 2 instrument
| Instrument for measuring and controlling the intensity of light | Manufacturer' s | Model number |
| One ten thousandth balance | METTLER-TOLEDO, Inc. | AL104 |
| qPCR instrument | Thermo Fisher Scientific | ABI 7500 |
| Inching eidolon | HANGZHOU MIU INSTRUMENTS Co.,Ltd. | MIX-28+ |
| Palm type centrifugal machine | Linbel instruments manufacturing Co.Ltd in Jiangsu sea | LX-100 |
| Freeze dryer | Sihuan Keyi (Tianjin) Technology Co.,Ltd. | LGJ-20 |
The PCR reaction apparatus used in the present application is described in patent 202110057507.0, and the chip apparatus for nucleic acid detection is described in 202110055537.8.
Example 1 primer probe composition for detecting helicobacter pylori
The primer probe composition comprises a primer probe composition for detecting helicobacter pylori and a primer probe composition for detecting internal reference.
The primer probe composition for detecting helicobacter pylori is an HP primer probe composition;
the primer probe composition for detecting the internal reference is an IPC primer probe composition.
(1) The HP primer probe composition comprises:
F(SEQ ID NO:1)：5'-ACCTGCTGGAACATTACTGAC-3'；
R(SEQ ID NO:2)：5'-GCCCTCCAACAACTAGCAT-3'；
P(SEQ ID NO:3)：5'-CTGGTAGTCCACGCCCTAAACGA-3'；
(2) The IPC primer probe composition comprises:
F(SEQ ID NO:4)：5'-AGTTGCAGTGTAACCGTCATGTA-3'；
R(SEQ ID NO:5)：5'-TCGACGAGACTCTGCTGTTAA-3'；
P(SEQ ID NO:6)：5'-CAGTAATCTGCGTCGCACGTGTGCA-3'。
the primer probe composition of helicobacter pylori is used for detecting helicobacter pylori 16SrRNA genes.
Example 2 microfluidic chip kit for detecting helicobacter pylori
(1) The primer probe composition described in example 1 was included, and each primer probe concentration was 0.2. Mu.M.
(2) The method also comprises a purifying reagent, a freeze-dried IPC quality control product and a freeze-drying reagent.
1) The purification reagent comprises 400 mu L of lysate and 850 mu L of eluent, 15 mu L of magnetic beads, the lysate comprises 6M guanidine hydrochloride, 0.4M sodium acetate with pH of 4.7 and 2% Triton X-100, the eluent comprises 10mM Tris-HCl with pH of 8.5, the purification reagent is added into a sample layer in a chip, and the magnetic bead drying method is disclosed in patent 202110222434.6.
2) The freeze-dried reaction reagent system is shown in the following table 3.
Table 3 freeze-dried reagent system
| Composition of the components | (1×) |
| Lipopolysaccharide Fucoid | 0.05％ |
| Hydroxypropyl methylcellulose HPMC | 0.006％ |
| Mannitol | 2.5％ |
| Trehalose | 2.5％ |
| Pullulan Pullulan | 0.2％ |
| 2-hydroxypropyl-beta-cyclodextrin HP-beta-CD | 0.5％ |
| Deoxyribose nucleic acid polymerase Taq glychol-free | 0.3U/μL |
| Bovine serum albumin BSA | 0.5mg/mL |
| Defoaming agent SE-15 | 0.009％ |
| Deoxynucleoside triphosphate dNTPs | 0.2mM |
| Potassium chloride KCl | 50mM |
| Tris-HCl, pH9.0 | 20mM |
| MgCl of magnesium chloride 2 | 3.5mM |
| Tween20 | 0.05％ |
The above-described reagents of table 3 were added to a silica gel mold and lyophilized according to the lyophilization procedure of the freeze dryer of table 5 below.
3) The freeze-drying IPC quality control product comprises an IPC plasmid preparation and freeze-drying reagent system.
The IPC sequence (SEQ ID NO: 7) was synthesized by the entire gene of the plant Biotechnology Co., ltd.) and ligated to pUC18 vector to obtain a plasmid containing the IPC sequence (SEQ ID NO: 7).
Table 4 freeze-dried IPC reagent system
| Composition of the components | |
| DNA plasmid | 100copies/μL |
| Mannitol | 10％ |
| Trehalose | 10％ |
| 2-hydroxypropyl-beta-cyclodextrin | 0.5％ |
| Bovine serum albumin BSA | 1mg/mL |
| Defoaming agent SE-15 | 0.036％ |
| Tris-HCl pH8.0 | 10mM |
| NaCl | 50mM |
| Tween20 | 0.05％ |
The above-mentioned reagents of Table 4 were lyophilized into pellets by dropping every 5. Mu.L into liquid nitrogen, and then lyophilized in a penicillin bottle according to the lyophilization procedure of the lyophilization machine of Table 5 below.
Table 5 lyophilization procedure
EXAMPLE 3 methods of Using helicobacter pylori kits
The detection of helicobacter pylori described in the present application was performed using CarryOn P1000Q, and the specific detection procedure is shown in Table 6 below.
TABLE 6
| Step (a) | Operation of | Time | Accumulated time | Environment (environment) |
| 1. Starting up the device | Manual work | 5 seconds | 00：05 | Quarantine site |
| 2. Preparing a sample | Manual work | For 1 minute | 01：05 | Quarantine site |
| 3. Taking a proper amount of sample and adding the sample into a chip | Manual work | 15 seconds | 01：20 | Quarantine site |
| 4. Clicking the "detect" button of the home page | Manual work | 5 seconds | 01：25 | Quarantine site |
| 5. Scanning chip two-dimensional code identification chip and inserting equipment | Manual work | 15 seconds | 01：40 | Quarantine site |
| 6. One-click operation device | Manual work | 5 seconds | 01：45 | Quarantine site |
| 7. Sample processing | Automatic machine | For 1 minute | 02：45 | On-chip |
| 8. Nucleic acid extraction purification | Automatic machine | 14 minutes | 16：45 | On-chip |
| 9. Reagent re-dissolution | Automatic machine | For 10 minutes | 26：45 | On-chip |
| 10. Real-time fluorescence PCR (45 cycles) | Automatic machine | 31 minutes | 57：45 | On-chip |
| 11. Interpretation, output and reporting of the result | Automatic machine | 0 seconds | 57：45 | On-chip |
| 12. Taking out the chip and discarding the waste to a biosafety waste bin | Manual work | 10 seconds | 57：55 | Quarantine site |
| 13. Resetting the device, sterilizing the interior, waiting for inspection or shutting down | Automatic machine | 20 seconds | 58：15 | Inside the device |
The real-time fluorescence quantitative PCR reaction procedure is: 55 ℃ for 10min;95 ℃ for 1min; (95 ℃ C. 10s,60 ℃ C. 20 s), 45cycles.
Experimental example 1 sensitivity test
The 16SrRNA gene sequence of helicobacter pylori is synthesized by the total genes of the proxy Bailey biotechnology Co Ltd, is connected to pUC18 vector, and is constructed and obtained to contain the plasmid of helicobacter pylori 16SrRNA gene (Genbank: NR_044761.1:401-900, SEQ ID NO: 8), and the concentration is adjusted to 1X 10 6 copies、1×10 5 copies、1×10 4 copies、1×10 3 copies、1×10 2 Samples of the extracted nucleic acids were tested according to the system and procedure of examples 1-3 above, with 200. Mu.L of each reaction, and the test results are shown in Table 7 below, and the test curves are shown in FIG. 1.
TABLE 7
As can be seen from Table 7, the primer composition and the kit thereof have good sensitivity to 1X 10 2 Samples of cobies were tested.
Experimental example 2 accuracy test
The 16SrRNA gene sequence of helicobacter pylori is synthesized by the total genes of the proxy Bailey biotechnology Co Ltd, is connected to pUC18 vector, and is constructed and obtained to contain the plasmid of helicobacter pylori 16SrRNA gene (Genbank: NR_044761.1:401-900, SEQ ID NO: 8), and the concentration is adjusted to 5 multiplied by 10 2 The nucleic acid samples extracted were tested according to the system and procedure of examples 1-3 described above, with 200. Mu.L of each reaction, and repeated six times, the test results are shown in Table 8 below, and the test results are shown in FIG. 2.
TABLE 8
As can be seen from Table 8, the primer composition and the kit thereof have better accuracy.
Experimental example 3 specificity detection
The nucleic acid extraction or nucleic acid purification device (see patent CN 202110057697.6) is used to extract human genome, coligenome, lactobacillus genome and other pathogen nucleic acid. Wherein the human genome and the E.coli genome are 1 μg, and the concentration of the lactobacillus genome is 1×10 4 Copies/mL was used for the experiment.
The above-extracted samples were examined according to the system and procedure of examples 1 to 3 described above, and the examination results are shown in Table 9 below.
TABLE 9
Wherein, ct value of detection results of human genome, escherichia coli genome and lactobacillus genome is internal reference amplification result. The helicobacter pylori primer probe composition described in the present application was not amplified to the product. Therefore, the detection result is negative.
As shown in FIG. 3, the primer composition and the kit thereof have better specificity as shown in Table 9.
Comparative example 1
The comparative example 1 was different from the experimental example 2 in that the lyophilized reagent used in the comparative example 1 did not contain lipopolysaccharide and hydroxypropyl methylcellulose, and the other steps were the same as those in the experimental example 3, and the extracted nucleic acid sample was subjected to repeated six times of detection, and the detection results are shown in the following table 10.
Table 10
As can be seen from Table 10, the accuracy of the lyophilized reagent was significantly reduced when it did not contain lipopolysaccharide or hydroxypropyl methylcellulose.
The foregoing is a description of embodiments of the invention, which are specific and detailed, but are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.
Sequence listing
<110> Beijing midwifery Instrument science and technology Co., ltd
<120> helicobacter pylori nucleic acid detection primer probe composition and integrated microfluidic chip kit
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 1
acctgctgga acattactga c 21
<210> 2
<211> 19
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 2
gccctccaac aactagcat 19
<210> 3
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 3
ctggtagtcc acgccctaaa cga 23
<210> 4
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 4
agttgcagtg taaccgtcat gta 23
<210> 5
<211> 21
<212> DNA
<213> agttgcagtg taaccgtcat gta
<400> 5
tcgacgagac tctgctgtta a 21
<210> 6
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 6
cagtaatctg cgtcgcacgt gtgca 25
<210> 7
<211> 100
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 7
agttgcagtg taaccgtcat gtaccagtaa tctgcgtcgc acgtgtgcac ctagtctaat 60
cacttatgac tcagataact taacagcaga gtctcgtcga 100
<210> 8
<211> 500
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 8
aggttttagg attgtaaact ccttttgtta gagaagataa tgacggtatc taacgaataa 60
gcaccggcta actccgtgcc agcagccgcg gtaatacgga gggtgcaagc gttactcgga 120
atcactgggc gtaaagagcg cgtaggcggg atagtcagtc aggtgtgaaa tcctatggct 180
taaccataga actgcatttg aaactactat tctagagtgt gggagaggta ggtggaattc 240
ttggtgtagg ggtaaaatcc gtagagatca agaggaatac tcattgcgaa ggcgacctgc 300
tggaacatta ctgacgctga ttgcgcgaaa gcgtggggag caaacaggat tagataccct 360
ggtagtccac gccctaaacg atggatgcta gttgttggag ggcttagtct ctccagtaat 420
gcagctaacg cattaagcat cccgcctggg gagtacggtc gcaagattaa aactcaaagg 480
aatagacggg gacccgcaca 500
Claims (10)
1. A primer probe composition for detecting helicobacter pylori, characterized in that: the primer probe composition comprises a primer probe composition for detecting helicobacter pylori and a primer probe composition for detecting internal references;
the primer probe composition for detecting helicobacter pylori is an HP primer probe composition;
the primer probe composition for detecting the internal reference is an IPC primer probe composition;
the HP primer probe composition comprises:
F：SEQ ID NO:1：5'-ACCTGCTGGAACATTACTGAC-3'；
R：SEQ ID NO:2：5'-GCCCTCCAACAACTAGCAT-3'；
P：SEQ ID NO:3：5'-CTGGTAGTCCACGCCCTAAACGA-3'；
the IPC primer probe composition comprises:
F：SEQ ID NO:4：5'-AGTTGCAGTGTAACCGTCATGTA-3'；
R：SEQ ID NO:5：5'-TCGACGAGACTCTGCTGTTAA-3'；
P：SEQ ID NO:6：5'-CAGTAATCTGCGTCGCACGTGTGCA-3'。
2. use of the primer probe according to claim 1 for the preparation of a product for detecting helicobacter pylori.
3. The use according to claim 2, characterized in that: the product comprises independent reagents, chips or kits.
4. A product for detecting helicobacter pylori, characterized in that: the product comprising the primer probe composition of claim 1.
5. The product of claim 4, wherein: the product also comprises a purifying reagent, a freeze-dried IPC quality control product and a freeze-dried reagent.
6. The product according to claim 5, wherein: the purification reagent comprises a lysate, an eluent and magnetic beads, wherein the lysate comprises guanidine hydrochloride, sodium acetate and Triton X-100, the eluent comprises Tris-HCl, the volume ratio of the lysate to the eluent is 1:2-2.5, and the magnetic beads are dried magnetic beads.
7. The product according to claim 5, wherein: the sequence of the freeze-dried IPC quality control product is SEQ ID NO 7;
the freeze-dried IPC quality control product is a DNA plasmid;
the freeze-drying IPC quality control material freeze-drying system comprises mannitol, trehalose, 2-hydroxypropyl-beta-cyclodextrin, bovine serum albumin, a defoaming agent, tris-HCl pH8.0, naCl and Tween20;
the freeze-dried IPC quality control product is packaged in an integrated micro-fluidic chip in a freeze-dried ball mode.
8. The product according to claim 5, wherein: the freeze-drying reagent comprises lipopolysaccharide and hydroxypropyl methylcellulose;
the freeze-drying reagent also comprises mannitol, trehalose, pullulan, 2-hydroxypropyl-beta-cyclodextrin, deoxyribonucleic acid polymerase, bovine serum albumin, defoamer, deoxynucleoside triphosphate dNTPs, KCl, tris-HCl pH9.0 and MgCl 2 、Tween20。
9. An integrated microfluidic chip kit for detecting helicobacter pylori nucleic acid, which is characterized in that:
the kit comprises the primer probe according to claim 1;
the kit also comprises a purification reagent, a freeze-dried IPC quality control product and a freeze-dried reagent;
the purification reagent comprises a lysate, an eluent and magnetic beads, wherein the lysate comprises guanidine hydrochloride, sodium acetate and Triton X-100, the eluent comprises Tris-HCl, the volume ratio of the lysate to the eluent is 1:2-2.5, and the magnetic beads are dried magnetic beads;
the sequence of the freeze-dried IPC quality control product is SEQ ID NO 7;
the freeze-dried IPC quality control product is a DNA plasmid;
the freeze-drying system of the freeze-drying IPC quality control product comprises mannitol, trehalose, 2-hydroxypropyl-beta-cyclodextrin, bovine serum albumin, defoamer, tris-HCl pH8.0, naCl and Tween20;
the freeze-drying reagent comprises lipopolysaccharide and hydroxypropyl methylcellulose;
the freeze-drying reagent also comprises mannitol, trehalose, pullulan, 2-hydroxypropyl-beta-cyclodextrin, deoxyribonucleic acid polymerase, bovine serum albumin, defoamer, deoxynucleoside triphosphate dNTPs, KCl, tris-HCl pH9.0 and MgCl 2 、Tween20。
10. A method for detecting helicobacter pylori, said method being a non-disease diagnosis and treatment method characterized in that: the method comprising detecting helicobacter pylori in a sample to be tested using the primer probe of claim 1, the product of any one of claims 3 to 9 or the kit of claim 10.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210714405.6A CN117305297A (en) | 2022-06-22 | 2022-06-22 | Helicobacter pylori nucleic acid detection primer probe composition and integrated microfluidic chip kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210714405.6A CN117305297A (en) | 2022-06-22 | 2022-06-22 | Helicobacter pylori nucleic acid detection primer probe composition and integrated microfluidic chip kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117305297A true CN117305297A (en) | 2023-12-29 |
Family
ID=89235888
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210714405.6A Pending CN117305297A (en) | 2022-06-22 | 2022-06-22 | Helicobacter pylori nucleic acid detection primer probe composition and integrated microfluidic chip kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117305297A (en) |
-
2022
- 2022-06-22 CN CN202210714405.6A patent/CN117305297A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111286559A (en) | Primer, probe and kit for detecting African swine fever virus | |
| JP2006129828A (en) | Probe set for detecting phlogogenic bacterium of infectious disease, carrier and method for examining gene | |
| CN111172301A (en) | PCR fluorescence detection kit for clostridium difficile toxin B and application thereof | |
| CN117305297A (en) | Helicobacter pylori nucleic acid detection primer probe composition and integrated microfluidic chip kit | |
| CN105256041B (en) | The nucleotide special to aeromonas hydrophila O44, O24, O25 and O28 and application | |
| CN116904655A (en) | African swine fever nucleic acid detection primer probe composition and integrated microfluidic chip kit | |
| CN116904450A (en) | Primer probe composition for enterohemorrhagic escherichia coli nucleic acid detection and integrated microfluidic chip kit | |
| CN113512598A (en) | Real-time fluorescent nucleic acid isothermal amplification detection kit for bordetella pertussis, and special primer and probe thereof | |
| CN117305479A (en) | Mycobacterium tuberculosis detection primer probe composition and integrated microfluidic chip kit | |
| CN116904617A (en) | Primer probe composition for typhus nucleic acid detection and integrated microfluidic chip kit | |
| CN117344055A (en) | Enterovirus 71 type nucleic acid detection primer probe composition and integrated microfluidic chip kit | |
| CN116904618A (en) | Vibrio cholerae detection primer probe composition and integrated microfluidic chip kit | |
| CN117004765A (en) | Primer probe composition for detecting Marburg virus and integrated microfluidic chip kit | |
| CN116904631A (en) | Malaria nucleic acid detection primer probe composition and integrated microfluidic chip kit | |
| CN116875709A (en) | Bacillus anthracis detection primer probe composition and integrated microfluidic chip kit | |
| CN117305478A (en) | Chlamydia trachomatis, gonococcus and ureaplasma urealyticum detection primer probe composition and integrated microfluidic chip kit | |
| CN116904449A (en) | Primer probe composition for detecting equine pestivirus nucleic acid and integrated microfluidic chip kit | |
| CN116904446A (en) | Neisseria meningitidis detection primer probe composition and integrated microfluidic chip kit | |
| CN116904619A (en) | Yersinia pestis detection primer probe composition and integrated microfluidic chip kit | |
| CN116904656A (en) | Chikungunya virus nucleic acid detection primer probe composition and integrated microfluidic chip kit | |
| CN117004766A (en) | Primer probe composition for detecting Ebola virus and integrated microfluidic chip kit | |
| CN116904652A (en) | Primer probe composition for rift valley fever virus nucleic acid detection and integrated microfluidic chip kit | |
| CN117305515A (en) | Primer probe composition for hepatitis B virus nucleic acid detection and integrated microfluidic chip kit | |
| CN116904660A (en) | Primer probe composition for detecting Crimedes-Congo hemorrhagic fever virus and integrated microfluidic chip kit | |
| CN116904447A (en) | Norovirus nucleic acid detection primer probe composition and integrated microfluidic chip kit |
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 |