CN116904619A - Yersinia pestis detection primer probe composition and integrated microfluidic chip kit - Google Patents
Yersinia pestis detection primer probe composition and integrated microfluidic chip kit Download PDFInfo
- Publication number
- CN116904619A CN116904619A CN202210543236.4A CN202210543236A CN116904619A CN 116904619 A CN116904619 A CN 116904619A CN 202210543236 A CN202210543236 A CN 202210543236A CN 116904619 A CN116904619 A CN 116904619A
- Authority
- CN
- China
- Prior art keywords
- primer probe
- probe composition
- ipc
- yersinia pestis
- product
- 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 75
- 239000000203 mixture Substances 0.000 title claims abstract description 56
- 241000607479 Yersinia pestis Species 0.000 title claims abstract description 54
- 238000001514 detection method Methods 0.000 title claims abstract description 45
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 15
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 15
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 15
- 230000003321 amplification Effects 0.000 claims abstract description 12
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 12
- 238000000746 purification Methods 0.000 claims abstract description 11
- 238000000605 extraction Methods 0.000 claims abstract description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 29
- 238000000034 method Methods 0.000 claims description 24
- 238000003908 quality control method Methods 0.000 claims description 24
- 108020004414 DNA Proteins 0.000 claims description 21
- 238000004108 freeze drying Methods 0.000 claims description 19
- 239000006166 lysate Substances 0.000 claims description 15
- 239000011324 bead Substances 0.000 claims description 14
- 239000003480 eluent Substances 0.000 claims description 14
- 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 11
- 239000013612 plasmid Substances 0.000 claims description 11
- 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 10
- 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 9
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 9
- 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 9
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 9
- 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 9
- 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 9
- 229940098773 bovine serum albumin Drugs 0.000 claims description 9
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 9
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 9
- 229930195725 Mannitol Natural products 0.000 claims description 8
- 239000000594 mannitol Substances 0.000 claims description 8
- 235000010355 mannitol Nutrition 0.000 claims description 8
- 102000053602 DNA Human genes 0.000 claims description 7
- 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
- 239000001632 sodium acetate Substances 0.000 claims description 6
- 235000017281 sodium acetate Nutrition 0.000 claims description 6
- 239000002518 antifoaming agent Substances 0.000 claims description 5
- 239000004373 Pullulan Substances 0.000 claims description 4
- 229920001218 Pullulan Polymers 0.000 claims description 4
- -1 carboxypropyl methylcellulose Chemical compound 0.000 claims description 4
- 239000013530 defoamer Substances 0.000 claims description 4
- 239000002274 desiccant Substances 0.000 claims description 4
- 239000002158 endotoxin Substances 0.000 claims description 4
- 229920006008 lipopolysaccharide Polymers 0.000 claims description 4
- 229920000609 methyl cellulose Polymers 0.000 claims description 4
- 239000001923 methylcellulose Substances 0.000 claims description 4
- 235000019423 pullulan Nutrition 0.000 claims description 4
- 239000001226 triphosphate Substances 0.000 claims description 4
- 235000011178 triphosphate Nutrition 0.000 claims description 4
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 claims description 4
- 201000010099 disease Diseases 0.000 claims description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000002405 diagnostic procedure Methods 0.000 claims 1
- 238000002560 therapeutic procedure Methods 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 9
- 238000005516 engineering process Methods 0.000 abstract description 3
- 244000052769 pathogen Species 0.000 abstract description 3
- 230000001717 pathogenic effect Effects 0.000 abstract description 2
- 238000012123 point-of-care testing Methods 0.000 abstract description 2
- 238000005070 sampling Methods 0.000 abstract description 2
- 206010035148 Plague Diseases 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 4
- 241000191967 Staphylococcus aureus Species 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 101100408676 Caenorhabditis elegans pmt-1 gene Proteins 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 3
- 101150033549 caf1 gene Proteins 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 108090000623 proteins and genes Proteins 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
- 241001465754 Metazoa Species 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 208000035742 Air-borne transmission Diseases 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241000711573 Coronaviridae Species 0.000 description 1
- 238000009007 Diagnostic Kit Methods 0.000 description 1
- 101100273027 Dictyostelium discoideum cafA gene Proteins 0.000 description 1
- 206010062713 Haemorrhagic diathesis Diseases 0.000 description 1
- 206010020741 Hyperpyrexia Diseases 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
- 206010035664 Pneumonia Diseases 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 101100394265 Schizosaccharomyces pombe (strain 972 / ATCC 24843) hba1 gene Proteins 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 241000607734 Yersinia <bacteria> Species 0.000 description 1
- 101100273031 Yersinia pestis caf1 gene Proteins 0.000 description 1
- 230000005557 airborne transmission Effects 0.000 description 1
- 238000004458 analytical method Methods 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
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 101150052693 cnot7 gene Proteins 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 208000031169 hemorrhagic disease Diseases 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000007937 lozenge 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
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 101150013341 pla gene Proteins 0.000 description 1
- 238000013492 plasmid preparation Methods 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 125000006853 reporter group Chemical group 0.000 description 1
- 238000012216 screening Methods 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
- 230000008961 swelling Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000001018 virulence Effects 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
-
- 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 application belongs to the technical field of biological detection, and particularly relates to a yersinia pestis detection primer probe composition and an integrated microfluidic chip kit. The primer probe composition provided by the application is used for detecting Yersinia pestis, has good sensitivity and accuracy, high amplification efficiency, and is simple and convenient to operate, rapid and time-saving. The application solves the problem of pathogen rapid detection by integrating nucleic acid extraction, purification, amplification and detection by utilizing a microfluidic chip technology, 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 application belongs to the technical field of biological detection, and particularly relates to a yersinia pestis detection primer probe composition and an integrated microfluidic chip kit.
Background
Plague (plague) is a virulent infectious disease of the class a caused by Yersinia pestis (Yersinia pestis), most of which is transmitted by fleas from animal hosts in wild rodents to other animals and humans. Continuous airborne transmission of aerosolized bacteria can also lead to primary plague. Clinically, it is mainly manifested as hyperpyrexia, swelling and pain of lymph nodes, bleeding tendency, lung inflammation and the like. The people are generally susceptible to Yersinia pestis, the infectious property of the Yersinia pestis is strong, and if the people are not treated, the death rate is as high as 30% -60%. Yersinia pestis is a gram-negative, bacillus, capsular, both aerobic and anaerobic, and is very resistant to the outside world, suitably at a temperature of 27-28 ℃. Plague is prevalent in asia, africa and the natural epidemic sources of america. Plague is usually associated with poverty and poor hygiene, and is often encountered in countries with crude medical infrastructure, and is particularly important in the preparation of plague biologicals because it is easily transmitted from person to person, resulting in high mortality rates and having a significant potential impact on public health.
Real-time PCR assays of yersinia pestis against its virulence plasmids pPCP1 (plagene) and pMT1 (caf 1, ymt genes) in the genome were compared to antigen immunochromatographic tests and organism cultures. Other monitoring means are found to be complex to operate, long in time consumption, low in sensitivity and not applicable to early diagnosis of diseases. The real-time fluorescent quantitative PCR technology is the most widely applied molecular detection method for screening pathogenic bacteria at present, and not only realizes the quantification of pathogenic bacteria nucleic acid, but also has the characteristics of high sensitivity, strong specificity, high detection speed, high degree of automation and the like. For example, patent CN101824468A discloses a primer set for yersinia pestis detection, a rapid diagnostic kit and a detection method, and the amplification reaction can be completed in less than 1 hour, so that the detection cost is low, the time consumption is short, the yield is high, the specificity is high, and the verification rate is high.
For the nucleic acid detection of pathogens, the existing detection system or method in the market generally needs sample treatment before detection, based on the problem, the inventor and the prior application CN202110057507.0 disclose a novel on-site rapid nucleic acid amplification detector CarryOn P1000Q, the device is a 'hand-held full-automatic closed nucleic acid qPCR analysis system', the on-site automatic nucleic acid detection of the novel coronavirus can be completed within 60 minutes, and the rapid detection of 'sample in and result out' can be realized. The application is further developed on the basis of the technology, and can be better matched with the reagent application of the equipment, namely the micro-fluidic chip kit of yersinia pestis.
The existing detection kit and method have longer operation time and complicated operation steps, so that the Yersinia pestis 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 primer probe composition for yersinia pestis detection and an integrated microfluidic chip kit. The primer probe composition for detecting yersinia pestis has the advantages of good sensitivity and accuracy, high amplification efficiency, simplicity and convenience in operation, rapidness and time saving.
In order to achieve the above object, the present application provides the following technical solutions:
in one aspect, the application provides a primer probe composition for detecting yersinia pestis, which comprises a primer probe composition for detecting yersinia pestis and a primer probe composition for detecting internal reference.
Specifically, the primer probe composition for detecting yersinia pestis is a Ypst 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 Ypst primer probe composition comprises:
F(SEQ ID NO:1)：5'-ATTTAACTGCAAGCACCACT-3'；
R(SEQ ID NO:2)：5'-TATAGCCGCCAAGAGTAAGCGTA-3'；
P(SEQ ID NO:3)：5'-ACTCTTGTTGAACCAGCCCGCATC-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 primer probe composition of Yersinia pestis is used for detecting caf1 gene of pMT1 plasmid of Yersinia pestis.
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.
Further specifically, the probe for detecting Yersinia pestis is labeled with FAM, and the probe for detecting internal reference IPC is labeled with CY5.
On the other hand, the application provides application of the primer probe composition in preparation of yersinia pestis detection products.
In particular, the products include, but are not limited to, stand-alone reagents, chips or kits.
In yet another aspect, the application provides a product for detecting yersinia pestis, said 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-drying reagent and an IPC quality control product.
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 freeze-drying agent comprises lipopolysaccharide, carboxypropyl methylcellulose, mannitol, trehalose, pullulan, 2-hydroxypropyl-beta-cyclodextrin, deoxyribonucleic acid polymerase, bovine serum albumin, defoamer, deoxynucleoside triphosphate, KCI, tris-HCI (pH 9.0), mgCl 2 、Tween 20。
Further specifically, the IPC quality control product exists in a micro-fluidic chip in the form of freeze-dried balls;
the artificial synthetic sequence of the IPC quality control product is SEQ ID NO 7;
the IPC quality control product is DNA plasmid;
the freeze-drying system of the IPC quality control product comprises mannitol, trehalose, bovine serum albumin, 2-hydroxypropyl-beta-cyclodextrin, an antifoaming agent, tris-HCl (pH 8.0), naCl and Tween20.
In still another aspect, the application also provides a microfluidic chip kit for detecting Yersinia pestis, wherein the kit comprises the primer probe composition.
Specifically, the kit also comprises a purification reagent, a freeze-drying reagent and an IPC quality control product.
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 freeze-drying agent comprises lipopolysaccharide, carboxypropyl methylcellulose, mannitol, trehalose, pullulan, 2-hydroxypropyl-beta-cyclodextrin, deoxyribonucleic acid polymerase, bovine serum albumin, defoamer, deoxynucleoside triphosphate, KCI, tris-HCI (pH 9.0), mgCl 2 、Tween 20。
The IPC quality control product exists in the micro-fluidic chip in the form of freeze-dried balls;
the artificial synthetic sequence of the IPC quality control product is SEQ ID NO 7;
the IPC quality control product is DNA plasmid;
the freeze-drying system of the IPC quality control product comprises mannitol, trehalose, bovine serum albumin, 2-hydroxypropyl-beta-cyclodextrin, an antifoaming agent, tris-HCl (pH 8.0), naCl and Tween20.
The kit completes the extraction, purification, amplification and detection of the nucleic acid of the sample on a microfluidic chip.
In particular, the microfluidic chip kit can be used for a Carryon P1000Q rapid nucleic acid amplification detector.
In yet another aspect, the application provides the use of the primer probe composition, product or kit described above for detecting yersinia pestis.
In yet another aspect, the application provides a method for detecting Yersinia pestis, the method is a non-disease diagnosis and treatment method, the method comprises detecting Yersinia pestis in a sample to be detected by using the primer probe composition, the product or the kit.
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 carried out on a microfluidic integrated chip by using a CarryOn P1000Q rapid nucleic acid amplification detector.
Compared with the prior art, the kit provided by the application has the following beneficial effects:
(1) The primer probe composition provided by the application is used for detecting Yersinia pestis, has good sensitivity and accuracy, high amplification efficiency, and is simple and convenient to operate, rapid and time-saving.
(2) The problem of pathogen rapid detection is solved by using a microfluidic method on a chip, a POCT detection scheme of 'sample in-out' is realized, and the sample is directly added into equipment after sampling, purified and amplified, and the result is obtained within 60 minutes.
Drawings
FIG. 1 is a graph of sensitivity detection results.
Fig. 2 is a diagram of accuracy detection results.
FIG. 3 is a graph showing the results of specificity detection, wherein A is the result of Pseudomonas aeruginosa detection, B is the result of Staphylococcus aureus detection, and C is the result of Escherichia coli detection.
Fig. 4 is a schematic diagram of a microfluidic chip.
Detailed Description
The present application will be described in further detail with reference to the following examples, which are not intended to limit the present application, but are merely illustrative of the present application. 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 the present application is shown in Table 1 below.
Table 1 reagents and manufacturer
(2) The instrument information used in the present 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 |
| Vortex mixing instrument | HANGZHOU MIU INSTRUMENTS Co.,Ltd. | MIX-28+ |
| PalmCentrifugal machine | Linbel instruments manufacturing Co.Ltd in Jiangsu sea | LX-100 |
| Freeze dryer | Sihuan Keyi (Tianjin) Technology Co.,Ltd. | LGJ-20 |
The PCR reaction device used in the application is shown in patent 202110057507.0, and the chip device for detecting nucleic acid is shown in 202110055537.8.
Example 1 primer probe composition for detection of Yersinia pestis
The primer probe composition comprises a primer probe composition for detecting yersinia pestis and a primer probe composition for detecting internal reference.
The primer probe composition for detecting yersinia pestis is Ypst primer probe composition.
The primer probe composition for detecting the internal reference is an IPC primer probe composition.
(1) The Ypst primer probe composition comprises:
F(SEQ ID NO:1)：5'-ATTTAACTGCAAGCACCACT-3'；
R(SEQ ID NO:2)：5'-TATAGCCGCCAAGAGTAAGCGTA-3'；
P(SEQ ID NO:3)：5'-ACTCTTGTTGAACCAGCCCGCATC-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 Yersinia pestis provided by the application is used for detecting caf1 genes of pMT1 plasmids of Yersinia pestis.
Example 2 microfluidic chip kit for detecting Yersinia pestis
(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-drying reagent and an IPC quality control product.
1) The purification reagent comprises 400 mu L of lysate, 850 mu L of eluent and 15 mu L of magnetic beads, wherein 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 of a chip, and the magnetic bead drying method is disclosed in patent 202110222434.6.
2) The freeze-dried reagent system is shown in table 3 below.
Table 3 freeze-dried reagent system
3) The IPC quality control product comprises an IPC plasmid preparation and freeze-drying system.
According to the sequence information of the IPC gene (SEQ ID NO: 7), dsDNA was synthesized by the Optimago Co., ltd and cloned on pUC18 vector to obtain a plasmid carrying the IPC gene.
The IPC quality control lyophilization system is shown in table 4 below.
Table 4 IPC quality control freeze-drying system
| Composition of the components | |
| IPC plasmid | 1×10 5 copies/mL |
| Mannitol | 10％ |
| Trehalose | 10％ |
| Bovine serum albumin BSA | 0.5mg/mL |
| 2-hydroxypropyl-beta-cyclodextrin HP-beta-CD | 0.1％ |
| Defoaming agent SE-15 | 0.009％ |
| Tris-HCl(pH 8.0) | 10mM |
| NaCl | 50mM |
| Tween20 | 0.05％ |
Adding the reagent in the table 3 into a silica gel mold; 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
And (3) putting the freeze-dried sheet and the freeze-dried balls into a microfluidic chip, and assembling the integrated detection kit.
Example 3 methods of Using Yersinia pestis detection kits
The detection of Yersinia pestis according to the present application was performed using Carryon P1000Q, and the specific detection procedure is shown in Table 6 below.
TABLE 6
The real-time quantitative PCR reaction procedure was: 55 ℃ for 10min;95 ℃ for 1min; (95 ℃ C. 10s,60 ℃ C. 20 s), 45cycles.
Experimental example 1 sensitivity detection
According to Yersinia pestis caf1 gene (GenBank: CP045259.1:20820-20969bp;SEQ ID NO:7) sequence information, dsDNA is synthesized by the Optimago Limited company and cloned on pUC18 vector, after sequencing correctly, plasmid concentration is adjusted to 5×10 per reaction 6 copies、5×10 5 copies、5×10 4 copies、5×10 3 copies、5×10 2 The nucleic acid samples were tested according to the system and procedure of examples 1-3 above, the test results of which are shown in Table 7 below, and the test curves are shown in FIG. 1.
TABLE 7
As shown in Table 7, the primer composition and the kit of the application have good sensitivity, and can react 5×10 per reaction 2 Samples of cobies were tested.
Experimental example 2 accuracy test
According to Yersinia pestisSequence information of bacteria caf1 gene (GenBank: CP045259.1:20820-20969bp;SEQ ID NO:7), dsDNA was synthesized by Optimago, inc., cloned into pUC18 vector, and after sequencing was correct, plasmid concentration was adjusted to 1X 10 per reaction 2 The nucleic acid samples were tested according to the system and procedure of examples 1-3 above, and repeated six times, with the test results shown in Table 8 below.
TABLE 8
| Sample of | Yersinia pestis | Internal control Ct value |
| First time | 31.75 | 31.07 |
| Second time | 31.58 | 30.93 |
| Third time | 31.73 | 31.10 |
| Fourth time | 32.23 | 31.65 |
| Fifth time | 31.73 | 30.79 |
| Sixth time | 31.84 | 30.99 |
| Maximum relative deviation | 1.32％ | 1.81％ |
| Coefficient of Variation (CV) | 0.70％ | 0.95％ |
As shown in FIG. 2, it is clear from Table 8 that the primer composition and the kit thereof according to the present application have good accuracy.
Experimental example 3 specificity detection
Staphylococcus aureus (ATCC 25923), pseudomonas aeruginosa (ATCC 27853), escherichia coli (ATCC 11303) were all purchased from the lozenges biotechnology company. Sample concentration was adjusted to 1X 10 per reaction 4 CFU, 200 μL was added to the sample bin of CarryOn P1000Q rapid nucleic acid detection device for nucleic acid detection experiments.
The above samples were tested according to the system and procedure of examples 1-3 above, and the test results are shown in Table 9 below.
TABLE 9
| Sample of | Internal control Ct value | Target detection results |
| Pseudomonas aeruginosa | 31.98 | Negative of |
| Staphylococcus aureus | 30.82 | Negative of |
| Coli bacterium | 30.94 | Negative of |
Wherein, the Ct value of the detection result of the pseudomonas aeruginosa, the staphylococcus aureus and the escherichia coli is an internal reference amplification result. The yersinia pestis primer probe composition disclosed by the application is not amplified to a product. Therefore, the detection result is negative.
As shown in FIG. 3, the primer composition and the kit thereof according to the present application have good specificity as shown in Table 9.
The foregoing is a description of embodiments of the application, which are specific and detailed, but are not to be construed as limiting the scope of the application. 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 application, which are all within the scope of the application.
Sequence listing
<110> Beijing midwifery Instrument science and technology Co., ltd
<120> Yersinia pestis detection primer probe composition and integrated microfluidic chip kit
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 1
atttaactgc aagcaccact 20
<210> 2
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 2
tatagccgcc aagagtaagc gta 23
<210> 3
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 3
actcttgttg aaccagcccg catc 24
<210> 4
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 4
agttgcagtg taaccgtcat gta 23
<210> 5
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<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> 150
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 8
atttaactgc aagcaccact gcaacggcaa ctcttgttga accagcccgc atcactctta 60
catataagga aggcgctcca attacaatta tggacaatgg aaacatcgat acagaattac 120
ttgttggtac gcttactctt ggcggctata 150
Claims (10)
1. A primer probe composition for detecting yersinia pestis, characterized in that: the primer probe composition comprises a primer probe composition for detecting yersinia pestis and a primer probe composition for detecting internal reference;
the primer probe composition for detecting yersinia pestis is Ypst primer probe composition;
the primer probe composition for detecting the internal reference is an IPC primer probe composition;
the Ypst primer probe composition comprises:
F：SEQ ID NO:1：5'-ATTTAACTGCAAGCACCACT-3'；
R：SEQ ID NO:2：5'-TATAGCCGCCAAGAGTAAGCGTA-3'；
P：SEQ ID NO:3：5'-ACTCTTGTTGAACCAGCCCGCATC-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 composition of claim 1 in the preparation of a product for detecting yersinia pestis.
3. The use according to claim 2, characterized in that: the product comprises independent reagents, chips or kits.
4. A product for detecting yersinia pestis, characterized in that: the product comprising the primer probe composition of claim 1.
5. The product according to claim 4, wherein: the product also comprises a purifying reagent, a freeze-drying reagent and an IPC quality control product.
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 freeze-drying agent comprises lipopolysaccharide, carboxypropyl methylcellulose, mannitol, trehalose, pullulan, 2-hydroxypropyl-beta-cyclodextrin, deoxyribonucleic acid polymerase, bovine serum albumin, defoamer, deoxynucleoside triphosphate, KCI, tris-HCI (pH 9.0), mgCl 2 、Tween 20。
8. The product according to claim 5, wherein: the IPC quality control product exists in the micro-fluidic chip in the form of freeze-dried balls;
the artificial synthetic sequence of the IPC quality control product is SEQ ID NO 7;
the IPC quality control product is DNA plasmid;
the freeze-drying system of the IPC quality control product comprises mannitol, trehalose, bovine serum albumin, 2-hydroxypropyl-beta-cyclodextrin, an antifoaming agent, tris-HCl (pH 8.0), naCl and Tween20.
9. A microfluidic chip kit for detecting yersinia pestis is characterized in that: the kit comprises the primer probe composition of claim 1;
the kit also comprises a purification reagent, a freeze-drying reagent and an IPC quality control product;
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 freeze-drying agent comprises lipopolysaccharide, carboxypropyl methylcellulose, mannitol, trehalose, pullulan, 2-hydroxypropyl-beta-cyclodextrin, deoxyribonucleic acid polymerase, bovine serum albumin, defoamer, deoxynucleoside triphosphate, KCI, tris-HCI (pH 9.0), mgCl 2 、Tween 20；
The IPC quality control product exists in the micro-fluidic chip in the form of freeze-dried balls;
the artificial synthetic sequence of the IPC quality control product is SEQ ID NO 7;
the IPC quality control product is DNA plasmid;
the freeze-drying system of the IPC quality control product comprises mannitol, trehalose, bovine serum albumin, 2-hydroxypropyl-beta-cyclodextrin, an antifoaming agent, tris-HCl (pH 8.0), naCl and Tween20;
the kit completes the extraction, purification, amplification and detection of the nucleic acid of the sample on a microfluidic chip.
10. A method for detecting yersinia pestis, said method being a non-disease diagnostic and therapeutic method, characterized by: the method comprises detecting yersinia pestis in the sample to be detected using the primer probe composition of claim 1, the product of any one of claims 4-8, or the kit of claim 9.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210543236.4A CN116904619A (en) | 2022-05-13 | 2022-05-13 | Yersinia pestis detection primer probe composition and integrated microfluidic chip kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210543236.4A CN116904619A (en) | 2022-05-13 | 2022-05-13 | Yersinia pestis detection primer probe composition and integrated microfluidic chip kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116904619A true CN116904619A (en) | 2023-10-20 |
Family
ID=88353603
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210543236.4A Pending CN116904619A (en) | 2022-05-13 | 2022-05-13 | Yersinia pestis detection primer probe composition and integrated microfluidic chip kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116904619A (en) |
-
2022
- 2022-05-13 CN CN202210543236.4A patent/CN116904619A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2021208382A1 (en) | Composition, kit and method for detecting and classifying pathogens causing respiratory tract infections, and application | |
| Mohamed et al. | A sensitive and quantitative single-tube real-time reverse transcriptase-PCR for detection of enteroviral RNA | |
| CN111286559B (en) | Primer, probe and kit for detecting African swine fever virus | |
| WO2019205764A1 (en) | Method and kit for detecting microorganisms | |
| US9650685B2 (en) | Selective detection of human rhinovirus | |
| CN110656163A (en) | Double-label report fluorescent multiple pathogen nucleic acid detection method | |
| Liao et al. | A highly adaptable platform powered by CRISPR-Cas12a to diagnose lumpy skin disease in cattle | |
| RU2360971C1 (en) | Method and test system to detect dna of african swine fever virus by means of specific oligonucleotide primers in polymerase chain reaction | |
| CN111270018A (en) | Influenza virus fluorescent quantitative PCR detection method | |
| CN116904619A (en) | Yersinia pestis detection primer probe composition and integrated microfluidic chip kit | |
| CN114807435A (en) | Kit for detecting respiratory syncytial virus and application thereof | |
| CN116875709A (en) | Bacillus anthracis detection primer probe composition and integrated microfluidic chip kit | |
| CN117305479A (en) | Mycobacterium tuberculosis detection primer probe composition and integrated microfluidic chip kit | |
| CN116904618A (en) | Vibrio cholerae detection primer probe composition and integrated microfluidic chip kit | |
| CN116904446A (en) | Neisseria meningitidis detection primer probe composition and integrated microfluidic chip kit | |
| CN117004765A (en) | Primer probe composition for detecting Marburg virus and integrated microfluidic chip kit | |
| CN116904658A (en) | Smallpox virus detection primer probe composition and integrated microfluidic chip kit | |
| CN116904652A (en) | Primer probe composition for rift valley fever virus nucleic acid detection and integrated microfluidic chip kit | |
| CN116904651A (en) | Monkey pox virus detection primer probe composition and integrated microfluidic chip kit | |
| CN117305297A (en) | Helicobacter pylori nucleic acid detection primer probe composition and integrated microfluidic chip kit | |
| CN116904660A (en) | Primer probe composition for detecting Crimedes-Congo hemorrhagic fever virus and integrated microfluidic chip kit | |
| CN117004766A (en) | Primer probe composition for detecting Ebola virus and integrated microfluidic chip kit | |
| 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 | |
| CN116904617A (en) | Primer probe composition for typhus nucleic acid detection 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 |