CN114540544A - Primer-probe combination for detecting respiratory viruses, kit and application thereof - Google Patents
Primer-probe combination for detecting respiratory viruses, kit and application thereof Download PDFInfo
- Publication number
- CN114540544A CN114540544A CN202111602922.6A CN202111602922A CN114540544A CN 114540544 A CN114540544 A CN 114540544A CN 202111602922 A CN202111602922 A CN 202111602922A CN 114540544 A CN114540544 A CN 114540544A
- Authority
- CN
- China
- Prior art keywords
- seq
- primer
- probe
- detecting
- virus
- 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 202
- 241000700605 Viruses Species 0.000 title claims abstract description 105
- 230000000241 respiratory effect Effects 0.000 title claims abstract description 23
- 238000001514 detection method Methods 0.000 claims abstract description 45
- 108020004414 DNA Proteins 0.000 claims description 56
- 208000002606 Paramyxoviridae Infections Diseases 0.000 claims description 32
- 238000006243 chemical reaction Methods 0.000 claims description 31
- 241000725643 Respiratory syncytial virus Species 0.000 claims description 21
- 230000003321 amplification Effects 0.000 claims description 21
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 21
- 241000709661 Enterovirus Species 0.000 claims description 18
- 108020004707 nucleic acids Proteins 0.000 claims description 15
- 150000007523 nucleic acids Chemical class 0.000 claims description 15
- 102000039446 nucleic acids Human genes 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 13
- 241000701161 unidentified adenovirus Species 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 102000004190 Enzymes Human genes 0.000 claims description 12
- 108090000790 Enzymes Proteins 0.000 claims description 12
- 239000003153 chemical reaction reagent Substances 0.000 claims description 12
- 102100034343 Integrase Human genes 0.000 claims description 11
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims description 11
- 241000124740 Bocaparvovirus Species 0.000 claims description 10
- 238000004925 denaturation Methods 0.000 claims description 10
- 230000036425 denaturation Effects 0.000 claims description 10
- 239000000243 solution Substances 0.000 claims description 10
- 241001428935 Human coronavirus OC43 Species 0.000 claims description 9
- 238000012408 PCR amplification Methods 0.000 claims description 9
- 239000012295 chemical reaction liquid Substances 0.000 claims description 9
- 241000342334 Human metapneumovirus Species 0.000 claims description 8
- 241001500351 Influenzavirus A Species 0.000 claims description 8
- 241000711573 Coronaviridae Species 0.000 claims description 5
- 241000711467 Human coronavirus 229E Species 0.000 claims description 5
- 241001500350 Influenzavirus B Species 0.000 claims description 5
- 238000000137 annealing Methods 0.000 claims description 5
- 238000012257 pre-denaturation Methods 0.000 claims description 5
- 238000003908 quality control method Methods 0.000 claims description 5
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 3
- 241000713196 Influenza B virus Species 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 2
- 238000010790 dilution Methods 0.000 claims description 2
- 102000016911 Deoxyribonucleases Human genes 0.000 claims 1
- 108010053770 Deoxyribonucleases Proteins 0.000 claims 1
- 102100021519 Hemoglobin subunit beta Human genes 0.000 claims 1
- 108091005904 Hemoglobin subunit beta Proteins 0.000 claims 1
- 239000000463 material Substances 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 42
- 238000010353 genetic engineering Methods 0.000 abstract 1
- 230000015572 biosynthetic process Effects 0.000 description 45
- 238000003786 synthesis reaction Methods 0.000 description 45
- 238000002474 experimental method Methods 0.000 description 24
- 238000012360 testing method Methods 0.000 description 24
- 238000012216 screening Methods 0.000 description 18
- 239000013612 plasmid Substances 0.000 description 13
- 238000010586 diagram Methods 0.000 description 8
- 238000007403 mPCR Methods 0.000 description 7
- 241000726041 Human respirovirus 1 Species 0.000 description 5
- 241000712003 Human respirovirus 3 Species 0.000 description 5
- 241001559187 Human rubulavirus 2 Species 0.000 description 5
- 241000351643 Metapneumovirus Species 0.000 description 5
- 206010057190 Respiratory tract infections Diseases 0.000 description 5
- 238000013461 design Methods 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 241001559186 Human rubulavirus 4 Species 0.000 description 4
- 238000012136 culture method Methods 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 238000003753 real-time PCR Methods 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 101100519164 Arabidopsis thaliana PCR8 gene Proteins 0.000 description 3
- 238000003912 environmental pollution Methods 0.000 description 3
- 244000052769 pathogen Species 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- 241001647372 Chlamydia pneumoniae Species 0.000 description 2
- 101000899111 Homo sapiens Hemoglobin subunit beta Proteins 0.000 description 2
- 241000202934 Mycoplasma pneumoniae Species 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- UDGUGZTYGWUUSG-UHFFFAOYSA-N 4-[4-[[2,5-dimethoxy-4-[(4-nitrophenyl)diazenyl]phenyl]diazenyl]-n-methylanilino]butanoic acid Chemical compound COC=1C=C(N=NC=2C=CC(=CC=2)N(C)CCCC(O)=O)C(OC)=CC=1N=NC1=CC=C([N+]([O-])=O)C=C1 UDGUGZTYGWUUSG-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000606161 Chlamydia Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000204031 Mycoplasma Species 0.000 description 1
- 206010036790 Productive cough Diseases 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 208000036142 Viral infection Diseases 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
- 238000003149 assay kit Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 125000006853 reporter group Chemical group 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 210000003802 sputum Anatomy 0.000 description 1
- 208000024794 sputum Diseases 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
-
- 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)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of genetic engineering, in particular to a primer probe combination, a kit and application thereof for detecting respiratory viruses, wherein the primer probe combination comprises 15 pairs of primers and probes corresponding to the primers; the kit comprises the primer probe combination and is used for detecting and/or detecting the respiratory viruses in an auxiliary way. The sensitivity of the primer probe combination of the invention to the detection of the corresponding respiratory viruses is higher than that of the existing primer probe combination; the kit can be divided into 4 groups, each group has 4 channels, 15 respiratory viruses can be detected simultaneously, and the detection method has the advantages of simple operation, high sensitivity, good specificity, short time and good clinical application value.
Description
Technical Field
The invention relates to a primer probe combination and application thereof, in particular to a primer probe combination for detecting respiratory viruses, a kit and application thereof.
Background
Respiratory tract infection is one of the most common infectious diseases, and the main pathogens include bacteria, viruses, mycoplasma, chlamydia, fungi and the like, wherein the main pathogens are mainly viral infection. Respiratory pathogens often cause acute respiratory infections in children, and the acute respiratory infections are one of the most major threats of children, are frequently generated in children, the elderly and people with low immune function, especially in common diseases and frequently encountered diseases in pediatrics, and are caused by respiratory virus infections in more than 90 percent of the children.
Clinically common viruses that can cause respiratory infections include respiratory syncytial virus a (rsva), respiratory syncytial virus b (rsvb), influenza virus a (flua), influenza virus b (flub), coronavirus (OC43), coronavirus (229E), human Metapneumovirus (MPV), bocavirus (HBOV), enterovirus (HEV), parainfluenza virus 1(HPIV1), parainfluenza virus 2(HPIV2), parainfluenza virus 3(HPIV3), parainfluenza virus 4(HPIV4), rhinovirus (HRV), Adenovirus (ADV), and the like.
Currently, the gold standard for clinical virus detection is still the virus culture method. However, the method has long time consumption, low sensitivity and high requirement on experimental operation. The virus antigen antibody detection can be used as infection evidence, the detection is rapid, but the sensitivity is low, and false positive and false negative results are easy to occur.
In view of the above, there is a need for a method for detecting respiratory viruses that has high sensitivity, good specificity, simple experimental procedures, and short time consumption.
Disclosure of Invention
Aiming at the problems, the invention provides a primer probe combination for detecting respiratory viruses, a kit and application thereof.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a primer probe combination for detecting respiratory syncytial virus A, wherein a forward primer for detecting the respiratory syncytial virus A is shown as SEQ ID NO: 1, and the reverse primer is shown as SEQ ID NO: 2, the probe is shown as SEQ ID NO: 3 is shown in the specification;
the forward primer for detecting the respiratory syncytial virus B is shown as SEQ ID NO: 4, the reverse primer is shown as SEQ ID NO: 5, the probe is shown as SEQ ID NO: 6 is shown in the specification;
the forward primer for detecting the influenza virus A is shown as SEQ ID NO: 7, the reverse primer is shown as SEQ ID NO: 8, the probe is shown as SEQ ID NO: 9 is shown in the figure;
the forward primer for detecting the influenza B virus is shown as SEQ ID NO: 10, and the reverse primer is shown as SEQ ID NO: 11, the probe is shown as SEQ ID NO: 12 is shown in the specification;
the forward primer for detecting the bocavirus is shown as SEQ ID NO: 13, the reverse primer is shown as SEQ ID NO: 14, the probe is shown as SEQ ID NO: 15 is shown in the figure;
the forward primer for detecting coronavirus OC43 is shown as SEQ ID NO: 16, and the reverse primer is shown as SEQ ID NO: 17, the probe is shown as SEQ ID NO: 18 is shown in the figure;
the forward primer for detecting coronavirus 229E is set forth in SEQ ID NO: 19, the reverse primer is shown as SEQ ID NO: 20, the probe is shown as SEQ ID NO: 21 is shown in the figure;
the forward primer for detecting parainfluenza virus 1 is shown as SEQ ID NO: 22, and the reverse primer is shown as SEQ ID NO: 23, the probe is shown as SEQ ID NO: shown at 24;
the forward primer for detecting parainfluenza virus 2 is shown as SEQ ID NO: 25, the reverse primer is shown as SEQ ID NO: 26, the probe is shown as SEQ ID NO: 27 is shown;
the forward primer for detecting parainfluenza virus 3 is shown as SEQ ID NO: 28, the reverse primer is shown as SEQ ID NO: 29, the probe is shown as SEQ ID NO: 30 is shown in the figure;
the forward primer for detecting parainfluenza virus 4 is shown as SEQ ID NO: 31, and the reverse primer is shown as SEQ ID NO: 32, and the probe is shown as SEQ ID NO: 33;
the forward primer for detecting the human metapneumovirus is shown as SEQ ID NO: 34, and the reverse primer is shown as SEQ ID NO: 35, the probe is shown as SEQ ID NO: 36 is shown;
the forward primer for detecting the enterovirus is shown as SEQ ID NO: 37, and the reverse primer is shown as SEQ ID NO: 38, and the probe is shown as SEQ ID NO: 39;
the forward primer for detecting adenovirus is shown as SEQ ID NO: 40, and the reverse primer is shown as SEQ ID NO: 41, the probe is shown as SEQ ID NO: 42 is shown;
the forward primer for detecting rhinovirus is shown as SEQ ID NO: 43, and the reverse primer is shown as SEQ ID NO: 44, the probe is shown as SEQ ID NO: shown at 45.
A kit comprising the primer-probe combination for detecting the respiratory viruses.
Further, the detection of the kit is divided into 4 groups, each group has 4 channels, which are respectively:
the first group detects respiratory syncytial virus A, coronavirus OC43, respiratory syncytial virus B and bocavirus;
a second set of detection parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3 and parainfluenza virus 4;
the third group detects influenza virus A, influenza virus B, human metapneumovirus and enterovirus;
and the fourth group detects coronavirus, rhinovirus, adenovirus and internal reference, wherein the internal reference is human beta-globin.
Further, the reagent in the kit comprises: 4 groups of primer probe combination liquid, 2 XPCR premixed reaction liquid, Taq DNA enzyme, reverse transcriptase and DEPC water.
Furthermore, the primer probe combination solution in any one of the 4 groups contains 500nM of any primer and 250nM of any probe.
An application of the kit in detecting/detecting respiratory viruses in an auxiliary way.
Further, the specific steps of the application are that a sample to be detected is respectively mixed with reagents in the kit to prepare 4 groups of reaction systems, then the reaction systems are respectively added into nucleic acid amplification reaction liquid for dilution, PCR amplification reaction is carried out to obtain 4 groups of amplification curve graphs and CT values, and then the sample to be detected is judged to be positive/negative according to the 4 groups of amplification curve graphs and the CT values.
Furthermore, a single group of the reaction system comprises 2 mu L of a sample to be detected, 10 mu L of 2 XPCR premixed reaction liquid, 0.5 mu L of Taq DNA enzyme, 0.5 mu L of reverse transcriptase and 2 mu L of single group of primer probe combined liquid, and DEPC water is supplemented to 20 mu L.
Furthermore, in a single group of the reaction system, the final concentration of any primer is 500nM, and the final concentration of any probe is 250 nM.
Furthermore, in the application process, a positive quality control substance and a negative quality control substance are required to be detected;
the PCR amplification reaction conditions are 50 ℃ pre-denaturation for 15min, 95 ℃ denaturation for 5min, 1 cycle, 95 ℃ denaturation for 10s, 58 ℃ annealing extension and fluorescence collection for 50s, and 40 cycles.
The primer probe combination, the kit and the application thereof for detecting the respiratory viruses have the beneficial effects that:
the sensitivity of the primer probe combination of the invention to the detection of the corresponding respiratory viruses is higher than that of the existing primer probe combination;
the kit can be divided into 4 groups, 15 respiratory viruses can be detected simultaneously, and the detection method is simple and convenient to operate, high in sensitivity, good in specificity, short in time and good in clinical application value;
the kit is added with the reference gene, and whether the sample nucleic acid is successfully extracted can be judged according to the normality of the reference gene, for example, if the reference gene in the sample PCR result has no CT value, the sample nucleic acid extraction or the sample nucleic acid extraction process has problems, the sample nucleic acid extraction or the sample re-acquisition is needed because the nucleic acid is not extracted, and the false negative result is avoided;
the kit can realize multiple detection of samples and can carry out batch detection on the samples.
Drawings
FIG. 1 is a diagram showing the results of a single PCR screening experiment using the RSVA primer probe combination in example 1 of the present invention;
FIG. 2 is a diagram showing the results of a single PCR screening experiment using the RSVB primer probe combination in example 1 of the present invention;
FIG. 3 is a diagram showing the results of a FluA primer probe combination single PCR screening experiment in example 1 of the present invention;
FIG. 4 is a diagram showing the results of a FluB primer probe combination single PCR screening experiment in example 1 of the present invention;
FIG. 5 is a graph showing the results of the HBOV primer probe combination single PCR screening experiment in example 1 of the present invention;
FIG. 6 is a graph showing the results of the single PCR screening experiment using the combination of the OC43 primer probe in example 1;
FIG. 7 is a graph showing the results of 229E primer probe combination singleplex PCR screening experiments in example 1 of the present invention;
FIG. 8 is a graph showing the results of the HPIV1 primer probe combination single PCR screening experiment in example 1 of the present invention;
FIG. 9 is a diagram showing the results of the HPIV2 primer probe combination single PCR screening experiment in example 1 of the present invention;
FIG. 10 is a graph showing the results of the HPIV3 primer probe combination single PCR screening experiment in example 1 of the present invention;
FIG. 11 is a diagram showing the results of a single PCR screening experiment using the HPIV4 primer probe combination in example 1;
FIG. 12 is a diagram showing the results of a single PCR screening experiment using the MPV primer probe combination in example 1 of the present invention;
FIG. 13 is a graph showing the results of a single PCR screening experiment using the HEV primer probe combination in example 1;
FIG. 14 is a diagram showing the results of the ADV primer probe combination single PCR screening experiment in example 1 of the present invention;
FIG. 15 is a graph showing the results of a single PCR screening experiment using HRV primer probe combinations in example 1 of the present invention;
FIG. 16 is a graph of multiplex PCR amplification with a first set of primer probe combinations in example 1 of the present invention;
FIG. 17 is a graph of multiplex PCR amplification with the second set of primer-probe combinations in example 1 of the present invention;
FIG. 18 is a graph showing the multiplex PCR amplification with the third set of primer probe combinations in example 1 of the present invention;
FIG. 19 is a graph of multiplex PCR amplification with a fourth set of primer probe combinations in example 1 of the present invention;
FIG. 20 is a graph showing the results of the sensitivity test of the reagent kit for RSVA virus in example 3 of the present invention;
FIG. 21 is a graph showing the results of the sensitivity test of the reagent kit to RSVB virus in example 3 of the present invention;
FIG. 22 is a graph showing the results of the sensitivity test of the reagent kit for FluA virus in example 3 of the present invention;
FIG. 23 is a graph showing the results of the sensitivity test of the reagent kit for FluB virus in example 3 of the present invention;
FIG. 24 is a graph showing the results of the test of the sensitivity of the reagent kit to HBOV virus in example 3 of the present invention;
FIG. 25 is a graph showing the results of the sensitivity test of the kit for OC43 virus in example 3 of the present invention;
FIG. 26 is a graph showing the results of the sensitivity test of the kit for 229E virus in example 3 of the present invention;
FIG. 27 is a graph showing the results of the sensitivity test of the kit for HPIV1 virus in example 3 of the present invention;
FIG. 28 is a graph showing the results of the sensitivity test of the kit for HPIV2 virus in example 3 of the present invention;
FIG. 29 is a graph showing the results of the sensitivity test of the kit for HPIV3 virus in example 3 of the present invention;
FIG. 30 is a graph showing the results of the sensitivity test of the kit for HPIV4 virus in example 3 of the present invention;
FIG. 31 is a graph showing the results of the sensitivity test of the kit for MPV virus in example 3 of the present invention;
FIG. 32 is a graph showing the results of the sensitivity test of the kit for HEV virus in example 3 of the present invention;
FIG. 33 is a graph showing the results of the sensitivity test of the reagent kit for ADV virus in example 3 of the present invention;
FIG. 34 is a graph showing the results of the sensitivity test of the reagent kit for HRV virus in example 3 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Example 1 design and screening of primer Probe combinations
One) design of primer-probe combination
1) Extraction of viral genomic DNA
And collecting nasopharyngeal swab or sputum samples of the patients to be detected by adopting a commercially available viral genome DNA/RNA extraction kit.
2) Primer probe combination design
Through literature review and market research, 15 target viruses for respiratory tract infection and specific genes thereof are determined, and the specific genes of the 15 target viruses are downloaded through NCBI inquiry to obtain corresponding DNA sequences of Respiratory Syncytial Virus A (RSVA), Respiratory Syncytial Virus B (RSVB), influenza virus A (FluA), influenza virus B (FluB), coronavirus OC43, coronavirus 229E, human Metapneumovirus (MPV), bocavirus (HBOV), enterovirus (HEV), parainfluenza virus 1(HPIV1), parainfluenza virus 2(HPIV2), parainfluenza virus 3(HPIV3), parainfluenza virus 4(HPIV4), rhinovirus (HRV) and Adenovirus (ADV). The corresponding DNA sequences of the obtained 15 viruses are introduced into biological software such as Beacon Designer, oligo, Primer 5 and the like, a Primer probe combination is designed by combining a Primer probe design principle and a personal probe design experience, the sequences of the designed Primer probe combination are input to NCBI for specific comparison, and a single Primer probe combination which can amplify the corresponding viruses and does not generate specific amplification with other viruses is screened out, and the specific amplification is shown in Table 1.
Amplification primers and probes for Table 115 respiratory viruses
The probe is labeled with any one of fluorescence reporter groups FAM, JOE, ROX and Cy5 at the 5 'end and fluorescence quencher groups BHQ1 and BHQ2 at the 3' end.
The designed primer probe combinations were each synthesized by general biology companies.
3) Primer probe combination screening
31) Establishment of Single PCR System
The PCR amplification experiments were performed separately for the single primer probe combinations in Table 1, and the specific procedures were as follows:
respectively taking the single primer probe combination in the table 1, and diluting the single primer probe combination to 10 mu mol/L by DEPC water to obtain a single primer probe combination solution of the corresponding virus (the concentration of the forward primer and the reverse primer in the single primer probe combination solution is 5 mu mol/L, and the concentration of the probe is 2.5 mu mol/L);
respectively taking 10 mu L of 2 XPCR premixed reaction liquid, 0.5 mu L of Taq DNA enzyme, 0.5 mu L of reverse transcriptase and 5 mu L of DEPC water, sequentially adding the premixed reaction liquid, the Taq DNA enzyme, the reverse transcriptase and the DEPC water into a PCR8 combined pipe, then adding 2 mu L of single primer probe combined liquid, and finally adding 2 mu L of nucleic acid template (a virus sample corresponding to the primer probe combination) to obtain a corresponding single reaction system; wherein the enzyme mixed solution comprises Taq DNA enzyme and reverse transcriptase.
mu.L of each of the above single reaction systems was taken, and the final concentration of the primer and the final concentration of the probe in the corresponding single reaction system were 500nM and 250nM, respectively.
And respectively putting the single systems into a fluorescence quantitative PCR instrument for PCR amplification reaction under the conditions of 50 ℃ pre-denaturation for 15min, 95 ℃ denaturation for 5min, 1 cycle, 95 ℃ denaturation for 10s, 58 ℃ annealing extension and collection of fluorescence for 50s and 40 cycles, and obtaining detection results shown in the figure 1-15.
Meanwhile, water is used for replacing the nucleic acid template, and detection is carried out according to the method so as to judge whether the false positive problem is caused by environmental pollution and the like.
And (3) screening the quality of the probe according to the CT value and the fluorescence intensity, wherein the smaller the CT value is, the better the sensitivity of the probe is, and the stronger the fluorescence signal value is (namely, the higher the Rn value is), the stronger the binding capacity of the primer probe and the template is. As can be seen from FIGS. 1 to 15, the sensitivity and binding capacity of the single primer probe combination corresponding to 1c in FIG. 1 are optimal, the sensitivity and binding capacity of the single primer probe combination corresponding to 2c in FIG. 2 are optimal, the sensitivity and binding capacity of the single primer probe combination corresponding to 3c in FIG. 3 are optimal, the sensitivity and binding capacity of the single primer probe combination corresponding to 4a in FIG. 4 are optimal, the sensitivity and binding capacity of the single primer probe combination corresponding to 5a in FIG. 5 are optimal, the sensitivity and binding capacity of the single primer probe combination corresponding to 6c in FIG. 6 are optimal, the sensitivity and binding capacity of the single primer probe combination corresponding to 7a in FIG. 7 are optimal, the sensitivity and binding capacity of the single primer probe combination corresponding to 8c in FIG. 8 are optimal, and the sensitivity and binding capacity of the single primer probe combination corresponding to 9a in FIG. 9 are optimal, the sensitivity and binding capacity of the single primer probe combination corresponding to 10a in fig. 10 is optimal, the sensitivity and binding capacity of the single primer probe combination corresponding to 11b in fig. 11 is optimal, the sensitivity and binding capacity of the single primer probe combination corresponding to 12b in fig. 12 is optimal, the sensitivity and binding capacity of the single primer probe combination corresponding to 13a in fig. 13 is optimal, the sensitivity and binding capacity of the single primer probe combination corresponding to 14a in fig. 14 is optimal, the sensitivity and binding capacity of the single primer probe combination corresponding to 15b in fig. 15 is optimal, and 15 primer probe combinations for detecting corresponding single viruses, namely forward primers for detecting respiratory syncytial virus a such as SEQ ID NO: 1, and the reverse primer is shown as SEQ ID NO: 2, and the probe is shown as SEQ ID NO: 3 is shown in the figure; the forward primer for detecting the respiratory syncytial virus B is shown as SEQ ID NO: 4, the reverse primer is shown as SEQ ID NO: 5, the probe is shown as SEQ ID NO: 6 is shown in the specification; the forward primer for detecting the influenza virus A is shown as SEQ ID NO: 7, and the reverse primer is shown as SEQ ID NO: 8, the probe is shown as SEQ ID NO: 9 is shown in the figure; the forward primer for detecting the influenza B virus is shown as SEQ ID NO: 10, and the reverse primer is shown as SEQ ID NO: 11, the probe is shown as SEQ ID NO: 12 is shown in the specification; the forward primer for detecting the bocavirus is shown as SEQ ID NO: 13, the reverse primer is shown as SEQ ID NO: 14, the probe is shown as SEQ ID NO: 15 is shown in the figure; the forward primer for detecting coronavirus OC43 is shown as SEQ ID NO: 16, and the reverse primer is shown as SEQ ID NO: 17, the probe is shown as SEQ ID NO: 18 is shown in the figure; the forward primer for detecting coronavirus 229E is set forth in SEQ ID NO: 19, the reverse primer is shown as SEQ ID NO: 20, the probe is shown as SEQ ID NO: 21 is shown in the figure; the forward primer for detecting parainfluenza virus 1 is shown as SEQ ID NO: 22, and the reverse primer is shown as SEQ ID NO: 23, the probe is shown as SEQ ID NO: shown at 24; the forward primer for detecting parainfluenza virus 2 is shown as SEQ ID NO: 25, the reverse primer is shown as SEQ ID NO: 26, the probe is shown as SEQ ID NO: 27 is shown; the forward primer for detecting parainfluenza virus 3 is shown as SEQ ID NO: 28, and the reverse primer is shown as SEQ ID NO: 29, the probe is shown as SEQ ID NO: 30 is shown in the figure; the forward primer for detecting parainfluenza virus 4 is shown as SEQ ID NO: 31, and the reverse primer is shown as SEQ ID NO: 32, and the probe is shown as SEQ ID NO: 33; the forward primer for detecting the human metapneumovirus is shown as SEQ ID NO: 34, and the reverse primer is shown as SEQ ID NO: 35, the probe is shown as SEQ ID NO: 36 is shown; the forward primer for detecting the enterovirus is shown as SEQ ID NO: 37, and the reverse primer is shown as SEQ ID NO: 38, and the probe is shown as SEQ ID NO: 39; the forward primer for detecting adenovirus is shown as SEQ ID NO: 40, and the reverse primer is shown as SEQ ID NO: 41, the probe is shown as SEQ ID NO: 42 is shown; the forward primer for detecting rhinovirus is shown as SEQ ID NO: 43, and the reverse primer is shown as SEQ ID NO: 44, the probe is shown as SEQ ID NO: shown at 45.
32) Establishment of multiple PRC systems
Single primer probes corresponding to 15 viruses screened in the construction of the single PCR system are combined and screened by using the multiplex PRC system.
Considering the difficulty that a fluorescent quantitative PCR instrument can only collect 4 types of fluorescence simultaneously, a single group can only distinguish 4 types of viruses once for detection, and different single primer probe combinations are mixed to distinguish and detect different viruses, the grouping detection is performed for multiple times according to the principle that the viruses of the same genus are divided into one group and the viruses of the other genera are randomly grouped to obtain a grouping mode that the primer probe combinations cannot interfere with each other and can be sensitively detected (it needs to be noted here that in the grouping detection process, if the mutual interference or the sensitivity detection limit of the primer probe combinations is not at the same level, the primer probe combinations need to be redesigned to be screened, and only the final result is introduced here), the specific steps are as follows:
the detection of 15 single primer probe combinations is divided into 4 groups, each group has 4 channels, a multiple PRC system is constructed, and the fluorescence channels corresponding to different viruses are specifically grouped, which is shown in the following table:
TABLE 2 packet and channel correspondence for multiple PRC systems
The multiplex PCR amplification experiments were performed according to the groupings in table 2, as follows:
taking corresponding single primer probe combinations according to the groups in the table 2 respectively, and diluting the primer probe combinations by DEPC water until the concentration of any primer in a single group is 5 mu mol/L and the concentration of any probe is 2.5 mu mol/L to obtain single group primer probe combination liquid of corresponding groups (4 groups are prepared by single group primer probe combination liquid);
sequentially adding 10 mu L of 2 XPCR premixed reaction liquid, 0.5 mu L of Taq DNA enzyme, 0.5 mu L of reverse transcriptase and 5 mu L of DEPC water into a PCR8 joint calandria, then adding 2 mu L of single-group primer probe combination liquid, and finally adding 2 mu L of nucleic acid template (containing nucleic acid templates of all virus samples corresponding to the single-group primer probe combination) to obtain a corresponding single-group primer probe reaction system; wherein, a single group of primer probe reaction system prepares 4 groups in total, and the enzyme mixed solution comprises Taq DNA enzyme and reverse transcriptase.
Respectively taking 20 mu L of single-group primer probe reaction systems, wherein the final concentration of any primer in the obtained single-group system is 500nM, and the final concentration of any probe is 250 nM; any one single-group primer probe reaction system in the 4 groups is diluted according to the method to obtain 4 single-group systems.
And respectively taking 4 groups of single-group systems, putting the single-group systems into a fluorescent quantitative PCR instrument, carrying out PCR amplification reaction under the reaction conditions of 50 ℃ pre-denaturation for 15min, 95 ℃ denaturation for 5min, 1 cycle, 95 ℃ denaturation for 10s, 58 ℃ annealing extension and fluorescence collection for 50s and 40 cycles, wherein the CT value is obtained and is shown in a table 3, and the multiple PCR amplification curve chart is shown in a table 16-19.
Meanwhile, water is respectively used for replacing the nucleic acid templates, and detection is carried out according to the method so as to judge whether the false positive problem caused by environmental pollution and the like exists.
TABLE 3 grouping and channel for single primer probe set
After the reaction is finished, analyzing the detection result according to the amplification curve graph and the Ct value, judging that the fluorescence curve is S-shaped curve and the CT is less than or equal to 37 in FAM, JOE, ROX and CY5 channels, and judging that the fluorescence curve is positive; no typical S-type amplification or CT is more than 39, and the CT of the internal standard is less than or equal to 40, and the internal standard is judged to be negative; if the CT value is between 37 and 39, retesting, and if the fluorescence curve is S-shaped and CT is more than or equal to 37 and less than or equal to 39, judging the result to be positive, otherwise, judging the result to be negative. As can be seen from Table 3, 4 sets of primer probe combinations can better detect corresponding viruses, the 15 primer probe combinations are finally selected, and the 15 primer probe combinations are divided into the following 4 sets:
the first group detects Respiratory Syncytial Virus A (RSVA), coronavirus OC43(OC43), Respiratory Syncytial Virus B (RSVB) and bocavirus (HBOV);
a second set of assays for parainfluenza virus 1(HPIV1), parainfluenza virus 2(HPIV2), parainfluenza virus 3(HPIV3), and parainfluenza virus 4(HPIV 4);
a third group for detecting influenza virus A (FluA), influenza virus B (FluB), human Metapneumovirus (MPV) and enterovirus (HEV);
the fourth group detects coronavirus 229E (229E), rhinovirus (HRV), Adenovirus (ADV), and internal reference, which is a human β -globin (IC).
3) Preparation and application of kit
The kit is prepared by utilizing the primer probe combination determined in the establishment of the multiple PRC system and grouping, and the prepared kit comprises 4 groups of primer probe combination liquids (wherein the concentration of any primer contained in any single group of primer probe combination liquid is 5 mu mol/L, and the concentration of any probe is 2.5 mu mol/L, namely the concentration of the primer for detecting respiratory syncytial virus A, coronavirus OC43, respiratory syncytial virus B and bocavirus contained in the first group of primer probe combination liquid is 5 mu mol/L, and the concentration of the probe for detecting respiratory syncytial virus A, coronavirus OC43, respiratory syncytial virus B and bocavirus is 2.5 mu mol/L), 2 x PCR premixed reaction liquid, enzyme mixed liquid, reverse transcriptase and DEPC water.
During detection, 10 mu L of 2 XPCR premixed reaction liquid, 0.5 mu L of Taq DNA enzyme, 0.5 mu L of reverse transcriptase and 5 mu L of DEPC water are sequentially added into a PCR8 combined pipe, 2 mu L of single-group primer probe combined liquid is added, finally 2 mu L of a sample to be detected is added to obtain a single-group reaction system (note that 4 groups of reaction systems containing different single-group primer probe combined liquids are required to be prepared for one-time sample detection), the final concentration of any primer in the obtained corresponding single-group reaction system is 500nM and the final concentration of any probe is 250nM, the single-group reaction system is put into a fluorescence quantitative PCR instrument for PCR amplification reaction under the conditions of 50 ℃ pre-denaturation for 15min, 95 ℃ denaturation for 5min, 1 cycle, 95 ℃ denaturation for 10s, 58 ℃ for annealing extension and collection of fluorescence for 50s and 40 cycles to obtain 4 amplification group graphs and CT values, the detection results are respectively analyzed according to the 4 groups of amplification and Ct values, the fluorescence curve in FAM, JOE, ROX and CY5 channels is S-shaped curve and CT is less than or equal to 37, and the fluorescence curve is judged to be positive; no typical S-type amplification or CT is more than 39, and the CT of the internal standard is less than or equal to 40, and the internal standard is judged to be negative; if the CT value is between 37 and 39, retesting, judging that the fluorescence curve is S-shaped curve and CT is more than or equal to 37 and less than or equal to 39, otherwise, judging that the fluorescence curve is positive; and when the judgment result is positive, the sample to be detected contains the virus to be detected.
Meanwhile, in the detection process, a positive quality control substance and a negative quality control substance are used for replacing samples to be detected respectively, and detection is carried out according to the method so as to judge whether the problem of false negative or false positive caused by reasons of substandard reaction reagents or environmental pollution exists. And when the false negative or false positive problem exists, the corresponding virus detection is carried out on the sample to be detected again.
Example 2 specificity test of primer Probe combination
In the implementation, cross experiments are carried out on various respiratory virus plasmid standard products to verify the specificity of the respiratory virus plasmid standard products, and the specificity is as follows:
taking the first group of primer probe combinations as an example, the first group of primer probe combinations are respectively added into the wells of the same row of the 96-well plate, that is, 4 single primer probe combinations of the first group are added into each tube of the first row, and by this round, the first group to the fourth group of primer probe combinations are respectively and correspondingly added into each tube of the 1 st to 4 th rows, which are 4 rows in total. And then adding the same virus positive plasmids into the same tube hole, namely adding 4 virus positive plasmids corresponding to the first group of primer probe combinations into the first tube hole, and so on, and adding 4 virus positive plasmids corresponding to the first group to the fourth group of primer probe combinations into each tube in the 1 st to 4 th rows respectively, so as to ensure that each primer probe and any one of the 15 virus positive plasmids have independent contact opportunities, and counting each CT value through such cross experiments to verify the specificity of the primer probe combination. In addition, each tube in the fifth row is added with 5 other virus positive plasmids of coronavirus (HKU1), enterovirus E71 (E71), enterovirus CA16 (CA16), Mycoplasma Pneumoniae (MP) and Chlamydia Pneumoniae (CP), and cross experiments are carried out with the primer probe combination of the invention to further verify the specificity of the primer probe combination of the invention, the specific multiplex PCR amplification experiment method is the same as the multiplex PCR amplification experiment method established by the multiplex PRC system in example 1, and the experiment results are shown in the following table:
TABLE 4 summary of specificity test results for primer probe combinations
As can be seen from the results in Table 4, the single primer probe combination of any one of the 4 groups of the present invention only amplified with the corresponding virus-positive plasmid, while the other virus-positive plasmids did not amplify, indicating that the primer probe combination of the present invention has good specificity.
EXAMPLE 3 sensitivity test of primer Probe combination
The 15 kinds of virus-positive plasmids were mixed in groups in accordance with the virus grouping method obtained by establishing the multiplex PRC system in example 1, and then diluted ten-fold, each group was diluted to a concentration of 102～106copies/mL yielded 5 concentrations of virus-positive plasmid solutions (10 concentrations each)2copies/mL、103copies/mL、104copies/mL、105copies/mL and 106copies/mL) of the virus-positive plasmid, amplifying the virus-positive plasmid solutions with 5 concentrations in different groups by using the preparation and application of the kit in example 1, to obtain the detection sensitivity corresponding to 15 viruses, wherein the specific results are shown in FIGS. 20-34, from which the standard curve and R for detecting 15 viruses can be obtained2The values are shown in Table 5, and the detection limit results are shown in tables 6-7.
TABLE 5 Standard Curve and R2 values for the detection by the kit of the invention
TABLE 6 stability of detection limits of the test kit of the present invention
TABLE 7 stability of detection limits of the test kit of the present invention
From tables 5 to 7, it can be seen that the sensitivity of the kit of the present invention can reach 102copies/mL, superior to the kit of patent application 201910677694.5 and the vast majority of products on the market.
Example 4 accuracy test of primer Probe combination
Randomly selecting 5 cases of 15 virus samples, detecting by using the kit and the detection method prepared in the embodiment 1, sequencing the amplified samples, and performing Blast comparison on the obtained sequences to obtain sequencing results shown in the following table:
TABLE 8 sequencing results summary of the test kits of the invention
As can be seen from Table 8, the coincidence rate of the detection result of the kit of the present invention for viruses and the sequencing result is 100%.
EXAMPLE 5 practical clinical sample testing of primer Probe combinations
In this example, 784 clinical samples were collected from nasopharyngeal swab samples collected by hospitals in the corridor city (principle that collectors voluntarily), viral nucleic acids were extracted from the nasopharyngeal swab samples, and the detection was performed by using the kit and the detection method of the present invention, and the positive rate of the detection was 46.8% by comparing with the internal control and the positive control, and the time spent was 1.5 hours.
The virus nucleic acid extracted from the nasopharyngeal swab sample is detected by adopting a traditional virus culture method, the positive rate of the detection is 46.8 percent, and the use time is 7 days.
The virus nucleic acid extracted from the nasopharyngeal swab sample is detected by the kit in the patent application 201910677694.5, the positive rate of the detection is 46.8%, and the use time is 2 hours.
The actual clinical sample detection result of the kit is consistent with the detection result of the traditional virus culture method and the detection result of the kit in patent application 201910677694.5, but the time for using the kit is obviously shorter than the time for detecting the kit in the traditional virus culture method and the time for detecting the kit in patent application 201910677694.5.
The specific test results of the actual clinical samples are shown in the following table:
TABLE 9 summary of test results of actual clinical specimens
It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Sequence listing
<110> Gallery norway Zhongke medical laboratory Co., Ltd
<120> primer probe combination for detecting respiratory viruses, kit and application thereof
<130> 2021-11
<141> 2021-12-24
<160> 45
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> Artificial Synthesis ()
<400> 1
actgcaatca yacaagatgc aac 23
<210> 2
<211> 20
<212> DNA
<213> Artificial Synthesis ()
<400> 2
<210> 3
<211> 28
<212> DNA
<213> Artificial Synthesis ()
<400> 3
ccagatcaag aacacaaccc carcatac 28
<210> 4
<211> 19
<212> DNA
<213> Artificial Synthesis ()
<400> 4
<210> 5
<211> 20
<212> DNA
<213> Artificial Synthesis ()
<400> 5
<210> 6
<211> 23
<212> DNA
<213> Artificial Synthesis ()
<400> 6
accaaaggca gaacctctac tcc 23
<210> 7
<211> 21
<212> DNA
<213> Artificial Synthesis ()
<400> 7
gaccaatcyt gtcacctctg a 21
<210> 8
<211> 24
<212> DNA
<213> Artificial Synthesis ()
<400> 8
gggcattytg gacaaagcgt ctac 24
<210> 9
<211> 23
<212> DNA
<213> Artificial Synthesis ()
<400> 9
tgcagtcctc gctcactggg cac 23
<210> 10
<211> 19
<212> DNA
<213> Artificial Synthesis ()
<400> 10
tgcaagggtt tccatgttc 19
<210> 11
<211> 19
<212> DNA
<213> Artificial Synthesis ()
<400> 11
<210> 12
<211> 20
<212> DNA
<213> Artificial Synthesis ()
<400> 12
<210> 13
<211> 19
<212> DNA
<213> Artificial Synthesis ()
<400> 13
agaggctcgg gctcatatc 19
<210> 14
<211> 21
<212> DNA
<213> Artificial Synthesis ()
<400> 14
cacttggtct gaggtcttcg a 21
<210> 15
<211> 28
<212> DNA
<213> Artificial Synthesis ()
<400> 15
aggaacaccc aatcagccac ctatcgtc 28
<210> 16
<211> 19
<212> DNA
<213> Artificial Synthesis ()
<400> 16
gatctacttc gcgcacatc 19
<210> 17
<211> 20
<212> DNA
<213> Artificial Synthesis ()
<400> 17
<210> 18
<211> 23
<212> DNA
<213> Artificial Synthesis ()
<400> 18
aattggctct actacgcgat cct 23
<210> 19
<211> 18
<212> DNA
<213> Artificial Synthesis ()
<400> 19
<210> 20
<211> 18
<212> DNA
<213> Artificial Synthesis ()
<400> 20
<210> 21
<211> 22
<212> DNA
<213> Artificial Synthesis ()
<400> 21
aacattccaa tagcctataa gc 22
<210> 22
<211> 19
<212> DNA
<213> Artificial Synthesis ()
<400> 22
<210> 23
<211> 24
<212> DNA
<213> Artificial Synthesis ()
<400> 23
ccaggtaata atgagatatt gggg 24
<210> 24
<211> 25
<212> DNA
<213> Artificial Synthesis ()
<400> 24
tgtcccgtag gagaacccct actga 25
<210> 25
<211> 21
<212> DNA
<213> Artificial Synthesis ()
<400> 25
agcgggatct atcatctagg c 21
<210> 26
<211> 22
<212> DNA
<213> Artificial Synthesis ()
<400> 26
gagttaccgg cacaggttat gt 22
<210> 27
<211> 26
<212> DNA
<213> Artificial Synthesis ()
<400> 27
agtgggactc cttcctacaa caagca 26
<210> 28
<211> 20
<212> DNA
<213> Artificial Synthesis ()
<400> 28
<210> 29
<211> 18
<212> DNA
<213> Artificial Synthesis ()
<400> 29
<210> 30
<211> 26
<212> DNA
<213> Artificial Synthesis ()
<400> 30
actattgctc tgggagtggc aacctc 26
<210> 31
<211> 20
<212> DNA
<213> Artificial Synthesis ()
<400> 31
<210> 32
<211> 22
<212> DNA
<213> Artificial Synthesis ()
<400> 32
cccagccaga tcttggatta at 22
<210> 33
<211> 25
<212> DNA
<213> Artificial Synthesis ()
<400> 33
aagttgatgc aatagacccg cagga 25
<210> 34
<211> 20
<212> DNA
<213> Artificial Synthesis ()
<400> 34
<210> 35
<211> 24
<212> DNA
<213> Artificial Synthesis ()
<400> 35
gcattgttct gttaatatcc caca 24
<210> 36
<211> 24
<212> DNA
<213> Artificial Synthesis ()
<400> 36
atgctaatgc tgctgataaa gact 24
<210> 37
<211> 17
<212> DNA
<213> Artificial Synthesis ()
<400> 37
<210> 38
<211> 19
<212> DNA
<213> Artificial Synthesis ()
<400> 38
<210> 39
<211> 23
<212> DNA
<213> Artificial Synthesis ()
<400> 39
<210> 40
<211> 20
<212> DNA
<213> Artificial Synthesis ()
<400> 40
<210> 41
<211> 21
<212> DNA
<213> Artificial Synthesis ()
<400> 41
gccscagtgg gcgtacatgc a 21
<210> 42
<211> 23
<212> DNA
<213> Artificial Synthesis ()
<400> 42
tgcaccagac ccggrctcag gta 23
<210> 43
<211> 19
<212> DNA
<213> Artificial Synthesis ()
<400> 43
<210> 44
<211> 20
<212> DNA
<213> Artificial Synthesis ()
<400> 44
caaagtagtc ggtcccatcc 20
<210> 45
<211> 18
<212> DNA
<213> Artificial Synthesis ()
<400> 45
Claims (10)
1. A primer probe combination for detecting respiratory viruses is characterized in that,
the forward primer for detecting the respiratory syncytial virus A is shown as SEQ ID NO: 1, and the reverse primer is shown as SEQ ID NO: 2, the probe is shown as SEQ ID NO: 3 is shown in the figure;
the forward primer for detecting the respiratory syncytial virus B is shown as SEQ ID NO: 4, the reverse primer is shown as SEQ ID NO: 5, the probe is shown as SEQ ID NO: 6 is shown in the specification;
the forward primer for detecting the influenza virus A is shown as SEQ ID NO: 7, the reverse primer is shown as SEQ ID NO: 8, the probe is shown as SEQ ID NO: 9 is shown in the figure;
the forward primer for detecting the influenza B virus is shown as SEQ ID NO: 10, and the reverse primer is shown as SEQ ID NO: 11, the probe is shown as SEQ ID NO: 12 is shown in the specification;
the forward primer for detecting the bocavirus is shown as SEQ ID NO: 13, the reverse primer is shown as SEQ ID NO: 14, the probe is shown as SEQ ID NO: 15 is shown in the figure;
the forward primer for detecting coronavirus OC43 is shown as SEQ ID NO: 16, and the reverse primer is shown as SEQ ID NO: 17, the probe is shown as SEQ ID NO: 18 is shown in the figure;
the forward primer for detecting coronavirus 229E is set forth in SEQ ID NO: 19, the reverse primer is shown as SEQ ID NO: 20, the probe is shown as SEQ ID NO: 21 is shown in the figure;
the forward primer for detecting parainfluenza virus 1 is shown as SEQ ID NO: 22, and the reverse primer is shown as SEQ ID NO: 23, the probe is shown as SEQ ID NO: shown at 24;
the forward primer for detecting parainfluenza virus 2 is shown as SEQ ID NO: 25, the reverse primer is shown as SEQ ID NO: 26, the probe is shown as SEQ ID NO: 27 is shown;
the forward primer for detecting parainfluenza virus 3 is shown as SEQ ID NO: 28, and the reverse primer is shown as SEQ ID NO: 29, the probe is shown as SEQ ID NO: 30 is shown in the figure;
the forward primer for detecting parainfluenza virus 4 is shown as SEQ ID NO: 31, and the reverse primer is shown as SEQ ID NO: 32, and the probe is shown as SEQ ID NO: 33;
the forward primer for detecting the human metapneumovirus is shown as SEQ ID NO: 34, and the reverse primer is shown as SEQ ID NO: 35, the probe is shown as SEQ ID NO: 36 is shown;
the forward primer for detecting the enterovirus is shown as SEQ ID NO: 37, and the reverse primer is shown as SEQ ID NO: 38, and the probe is shown as SEQ ID NO: 39;
the forward primer for detecting adenovirus is shown as SEQ ID NO: 40, and the reverse primer is shown as SEQ ID NO: 41, the probe is shown as SEQ ID NO: 42 is shown;
the forward primer for detecting rhinovirus is shown as SEQ ID NO: 43, and the reverse primer is shown as SEQ ID NO: 44, the probe is shown as SEQ ID NO: shown at 45.
2. A kit comprising the primer-probe combination for detecting a respiratory virus of claim 1.
3. The kit according to claim 2, wherein the detection of the kit is divided into 4 groups, each of which is:
the first group detects respiratory syncytial virus A, coronavirus OC43, respiratory syncytial virus B and bocavirus;
a second set of detection parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3 and parainfluenza virus 4;
the third group detects influenza virus A, influenza virus B, human metapneumovirus and enterovirus;
and the fourth group detects coronavirus, rhinovirus, adenovirus and internal reference, wherein the internal reference is humanized beta-globin.
4. The kit of claim 3, wherein the reagents in the kit comprise: 4 groups of primer probe combination liquid, 2 XPCR premixed reaction liquid, Taq DNA enzyme, reverse transcriptase and DEPC water.
5. The kit according to claim 4, wherein the primer probe combination solution in any one of the 4 groups contains any primer at a concentration of 500nM and any probe at a final concentration of 250 nM.
6. Use of a kit according to any one of claims 2 to 5 for the detection/co-detection of respiratory viruses.
7. The application of claim 6, wherein the application comprises the specific steps of mixing a sample to be tested with reagents in a kit to prepare 4 groups of reaction systems, adding the reaction systems into a nucleic acid amplification reaction solution for dilution, performing PCR amplification reaction to obtain 4 groups of amplification curve graphs and CT values, and judging whether the sample to be tested is positive or negative according to the 4 groups of amplification curve graphs and the CT values.
8. The application of claim 7, wherein the single set of reaction system comprises 2 μ L of sample to be tested, 10 μ L of 2 XPCR premixed reaction solution, 0.5 μ L of Taq DNase, 0.5 μ L reverse transcriptase, 2 μ L of single set of primer probe combined solution, and 20 μ L of DEPC water.
9. The use of claim 7 or 8, wherein the final concentration of each primer and the final concentration of each probe in a single set of the reaction system are 500nM and 250nM, respectively.
10. The use of claim 7 or 8, wherein the positive and negative quality control materials are further tested during the application process;
the PCR amplification reaction conditions are 50 ℃ pre-denaturation for 15min, 95 ℃ denaturation for 5min, 1 cycle, 95 ℃ denaturation for 10s, 58 ℃ annealing extension and fluorescence collection for 50s, and 40 cycles.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111602922.6A CN114540544A (en) | 2021-12-24 | 2021-12-24 | Primer-probe combination for detecting respiratory viruses, kit and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111602922.6A CN114540544A (en) | 2021-12-24 | 2021-12-24 | Primer-probe combination for detecting respiratory viruses, kit and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114540544A true CN114540544A (en) | 2022-05-27 |
Family
ID=81670284
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111602922.6A Pending CN114540544A (en) | 2021-12-24 | 2021-12-24 | Primer-probe combination for detecting respiratory viruses, kit and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114540544A (en) |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009049007A2 (en) * | 2007-10-10 | 2009-04-16 | Magellan Biosciences, Inc. | Compositions, methods and systems for rapid identification of pathogenic nucleic acids |
| CN102433393A (en) * | 2011-12-15 | 2012-05-02 | 上海市公共卫生临床中心 | Primers, probe and method for detecting various respiratory viruses |
| KR101380414B1 (en) * | 2013-10-16 | 2014-04-02 | 주식회사 현일바이오 | Methods for simultaneously detecting multiple respiratory viruses and uses thereof |
| CN105734168A (en) * | 2015-10-28 | 2016-07-06 | 南京美宁康诚生物科技有限公司 | Multiplex PCR detection kit for nucleic acids of twelve respiratory viruses |
| CN107058622A (en) * | 2017-03-30 | 2017-08-18 | 德必碁生物科技（厦门）有限公司 | A kind of kit of multiple fluorescence PCR method joint-detection respiratory pathogen |
| CN107090519A (en) * | 2017-04-05 | 2017-08-25 | 苏州协云基因科技有限公司 | The multiple RT PCR Polymorphism chip inspecting reagent units of respiratory tract common causative |
| CN107475446A (en) * | 2017-08-24 | 2017-12-15 | 复旦大学附属儿科医院 | Multi-PCR detection method and its probe groups and kit a kind of while that detect various respiratory road virus |
| CN107760799A (en) * | 2017-09-28 | 2018-03-06 | 苏州旷远生物分子技术有限公司 | A kind of multiple single tube reverse transcriptase polymerase chain reaction detection throat swab infection of the upper respiratory tract virus detection kit |
| CN110358865A (en) * | 2019-07-25 | 2019-10-22 | 廊坊诺道中科医学检验实验室有限公司 | For detecting kit and its application of Respirovirus |
| CN110468234A (en) * | 2019-08-09 | 2019-11-19 | 厦门安普利生物工程有限公司 | Multiple fluorescence quantitative PCR kit for 19 kinds of human respiratory viral diagnosis |
| CN112391496A (en) * | 2020-11-27 | 2021-02-23 | 魏尔啸实验室科技(杭州)有限公司 | Novel coronavirus and respiratory virus combined detection primer and probe combination, detection kit and application |
-
2021
- 2021-12-24 CN CN202111602922.6A patent/CN114540544A/en active Pending
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009049007A2 (en) * | 2007-10-10 | 2009-04-16 | Magellan Biosciences, Inc. | Compositions, methods and systems for rapid identification of pathogenic nucleic acids |
| CN102433393A (en) * | 2011-12-15 | 2012-05-02 | 上海市公共卫生临床中心 | Primers, probe and method for detecting various respiratory viruses |
| KR101380414B1 (en) * | 2013-10-16 | 2014-04-02 | 주식회사 현일바이오 | Methods for simultaneously detecting multiple respiratory viruses and uses thereof |
| CN105734168A (en) * | 2015-10-28 | 2016-07-06 | 南京美宁康诚生物科技有限公司 | Multiplex PCR detection kit for nucleic acids of twelve respiratory viruses |
| CN107058622A (en) * | 2017-03-30 | 2017-08-18 | 德必碁生物科技（厦门）有限公司 | A kind of kit of multiple fluorescence PCR method joint-detection respiratory pathogen |
| CN107090519A (en) * | 2017-04-05 | 2017-08-25 | 苏州协云基因科技有限公司 | The multiple RT PCR Polymorphism chip inspecting reagent units of respiratory tract common causative |
| CN107475446A (en) * | 2017-08-24 | 2017-12-15 | 复旦大学附属儿科医院 | Multi-PCR detection method and its probe groups and kit a kind of while that detect various respiratory road virus |
| CN107760799A (en) * | 2017-09-28 | 2018-03-06 | 苏州旷远生物分子技术有限公司 | A kind of multiple single tube reverse transcriptase polymerase chain reaction detection throat swab infection of the upper respiratory tract virus detection kit |
| CN110358865A (en) * | 2019-07-25 | 2019-10-22 | 廊坊诺道中科医学检验实验室有限公司 | For detecting kit and its application of Respirovirus |
| CN110468234A (en) * | 2019-08-09 | 2019-11-19 | 厦门安普利生物工程有限公司 | Multiple fluorescence quantitative PCR kit for 19 kinds of human respiratory viral diagnosis |
| CN112391496A (en) * | 2020-11-27 | 2021-02-23 | 魏尔啸实验室科技(杭州)有限公司 | Novel coronavirus and respiratory virus combined detection primer and probe combination, detection kit and application |
Non-Patent Citations (2)
| Title |
|---|
| DENG J等: "Respiratory virus multiplex RT-PCR assay sensitivities and influence factors in hospitalized children with lower respiratory tract infections", VIROL SIN, vol. 28, no. 02, pages 97 - 102, XP037068937, DOI: 10.1007/s12250-013-3312-y * |
| 隋竑弢等: "呼吸道病毒多病原检测技术研究进展", 病毒学报, vol. 29, no. 02, pages 238 - 244 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111020064B (en) | Novel coronavirus ORF1ab gene nucleic acid detection kit | |
| WO2021196498A1 (en) | Primer, probe and kit for detecting novel coronavirus | |
| CN108060269B (en) | DPO primer group for detecting porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine rotavirus and application thereof | |
| CN111500776A (en) | Novel coronavirus 2019-nCoV fluorescent RPA detection primer, probe, kit and method | |
| CN116555497B (en) | Kit and method for detecting novel coronaviruses | |
| CN115052996A (en) | Novel coronavirus 2019-nCoV nucleic acid detection kit | |
| CN113462820A (en) | Multiplex RT-PCR primer probe set for real-time fluorescent quantitative detection of four porcine diarrhea viruses, kit and detection method thereof | |
| CN111733291A (en) | Method and kit for detecting novel coronavirus nucleic acid by digital PCR (polymerase chain reaction) | |
| CN111763766B (en) | Primer pair, taqMan probe and method for detecting canine diarrhea virus by one-step method and application | |
| CN112725475A (en) | Mycobacterium tuberculosis detection primer, probe composition, kit and application | |
| CN117363767A (en) | Probe combination, primer set and kit for real-time fluorescence PCR detection of target genes and application of probe combination and primer set and kit | |
| CN114317786A (en) | Primer-probe combination for detecting 14 respiratory tract infection pathogenic bacteria, kit and application thereof | |
| CN113930546A (en) | RT-RAA fluorescence detection primer pair, kit and detection method for J subtype avian leukosis virus gp85 gene | |
| CN111471800B (en) | Kit for detecting novel coronavirus and amplification primer composition thereof | |
| CN112195278A (en) | Six respiratory tract virus nucleic acid detection kit and use method thereof | |
| CN112176109A (en) | Influenza A and B virus nucleic acid detection kit and use method thereof | |
| CN112410465A (en) | Novel coronavirus SARS-CoV-2ORF1ab and N gene constant temperature amplification primer group and kit | |
| CN116814859A (en) | Primer probe composition, kit and method for identifying African swine fever virus genes I and II | |
| CN111926114A (en) | Multiplex-time PCR (polymerase chain reaction) kit for detecting parainfluenza virus, method and application | |
| EP1546413A1 (en) | Method and kit for quantitative and qualitative determination of human papillomavirus | |
| CN108424977B (en) | Primer probe for enterovirus typing identification and identification method | |
| CN114540544A (en) | Primer-probe combination for detecting respiratory viruses, kit and application thereof | |
| CN115449563A (en) | Multiple fluorescence detection primer probe set and kit for new coronavirus Onckrozen variant strain | |
| CN112266978A (en) | Primer-probe combination, detection kit and application thereof | |
| CN112126713A (en) | Coronavirus and influenza virus combined detection product and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |