CN110592276A - Specific primer and kit for detecting canine influenza virus - Google Patents
Specific primer and kit for detecting canine influenza virus Download PDFInfo
- Publication number
- CN110592276A CN110592276A CN201910610586.6A CN201910610586A CN110592276A CN 110592276 A CN110592276 A CN 110592276A CN 201910610586 A CN201910610586 A CN 201910610586A CN 110592276 A CN110592276 A CN 110592276A
- Authority
- CN
- China
- Prior art keywords
- kit
- probe
- influenza virus
- primer
- canine influenza
- 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
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
- C12Q1/6844—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
- 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
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 belongs to the technical field of biology, and relates to a rapid detection reagent for canine influenza virus based on POCT monomer enzyme and application thereof. The detection reagent comprises reaction buffer solution, dNTPs, dUTP, reverse transcriptase, Taq enzyme, an RNase inhibitor, a specific primer and a probe, a reaction enhancer, an internal reference primer and a probe; the detection kit comprises an extraction reagent and a real-time fluorescence amplification reagent, has the advantages of accurate detection result, simple and convenient operation, no need of liquid preparation (the extracted template is directly added into an amplification system), short detection time, good stability and long storage life, has internal reference as a control (the whole extraction and amplification process is monitored), and can be widely applied to early screening and diagnosis of canine influenza.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a specific primer and a kit for detecting canine influenza virus.
Background
Canine influenza is a disease that infects influenza a virus, causing respiratory symptoms in dogs. Influenza A virus belongs to the genus of influenza A virus of the family of Orthomyxoviridae (Orthomyxoviridae), and is mostly spherical, enveloped, single-stranded negative-strand RNA virus. The viral genome consists of 8 minus-stranded single-stranded RNA fragments, encoding a total of 10 viral proteins. Based on the difference in antigenicity between the surface nucleoproteins Hemagglutinin (HA) and Neuraminidase (NA), the HA subtypes and the NA subtypes are 16 and 9. In 2004, H3N8 subtype influenza virus transmission occurred in the U.S. florida dog population; in 2007, it was reported in korea that influenza virus subtype a H3N2 causes canine respiratory disease; in 2006, H3N2 subtype influenza virus was also found in southern China; in 2015, an H3N2 subtype canine influenza virus epidemic occurred in chicago in the united states; beginning in the new year of 2018, a canadian veterinarian detected influenza virus subtype H3N2 in dogs, and further found that influenza viruses subtype a H1N1, H5N1, H5N2, H9N2, H3N1 also infected dogs. Influenza A not only infects dogs, but also causes death of dogs, thereby greatly threatening the health of dogs. Meanwhile, influenza a can also infect humans, so canine influenza a is threatening public health and hygiene.
The current diagnostic techniques for detecting canine influenza virus include chick embryo isolation culture, hemagglutination and hemagglutination inhibition tests, agar diffusion tests, neuraminidase inhibition tests, virus neutralization tests, enzyme-linked immunosorbent assays, immunofluorescence techniques, RT-PCR techniques and the like. The methods and the kit with low maturity generally have the problems of low sensitivity, poor specificity, time consumption and the like, and can not provide powerful technical support for rapidly and effectively detecting the canine influenza epidemic situation in China. The method further enhances the monitoring of influenza viruses in dog groups, researches and develops a detection reagent for the canine influenza viruses, and establishes a detection kit and a rapid detection system which are simple and convenient to operate, rapid, good in sensitivity, strong in specificity, good in stability and long in storage life.
Disclosure of Invention
Aiming at the problems, the invention develops a specific primer and a kit for detecting the canine influenza virus, further develops and obtains a rapid detection reagent for the canine influenza virus based on the POCT monomer enzyme based on the specific primer, and establishes the rapid detection kit for the canine influenza virus based on the POCT monomer enzyme based on the detection reagent. The detection kit and the detection system have the advantages of simple operation, high detection speed, good sensitivity and strong specificity. The detection kit and the detection system can be used for detecting the canine influenza virus in various places such as laboratories, pet hospitals, canine breeding bases and the like in real time, and can accurately and quickly obtain a detection result.
The technical scheme of the invention is as follows:
the specific primer for detecting the canine influenza virus is characterized in that the nucleotide sequences of the upstream primer and the downstream primer are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2.
The kit for detecting the canine influenza virus is characterized by comprising the primer and the probe.
The nucleotide sequence of the probe is shown as SEQ ID NO. 3.
The 5 'end of the nucleotide sequence of the probe is connected with a fluorescence reporter group FAM label, and the 3' end of the nucleotide sequence of the probe is connected with a non-luminous fluorescence quenching group with a Minor Groove Binder (MGB);
preferably, the fluorescence emitting group is selected from one of FAM, VIC, HEX, JOE, NED, TAMRA, CY3, ROX or CY 5.
The kit further comprises conventional reagents for performing PCR;
preferably, said conventional reagents for performing PCR are selected from: reaction buffer solution, dNTPs, dUTP, reverse transcriptase, Taq enzyme, RNase inhibitor, reaction enhancer, internal reference primer and probe;
further preferably, the reaction enhancer is selected from the group consisting of: ammonium sulfate, formamide or betaine, glycerol, Tween-20, BSA, DTT, or gelatin; the internal reference primer is selected from commercial reagents, preferably Xeno IPC.
A method of making a kit for detecting canine influenza virus, comprising: specific primers were synthesized according to the nucleotide sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2.
The preparation method further comprises the following steps: synthesizing a probe according to the nucleotide sequence shown in SEQ ID NO. 3;
preferably, the preparation method further comprises: placing the specific primers and the probes into a packaging box marked with the application of detecting the canine influenza virus;
preferably, the preparation method further comprises: placing conventional reagents for performing PCR into a packaging box for detecting canine influenza virus according to the following nucleotide sequences;
further preferably, said conventional reagents for performing PCR are selected from: reaction buffer solution, dNTPs, dUTP, reverse transcriptase, Taq enzyme, RNase inhibitor, reaction enhancer, internal reference primer and probe;
further preferably, the reaction enhancer is selected from the group consisting of: ammonium sulfate, formamide or betaine, glycerol, Tween-20, BSA, DTT, or gelatin; the internal reference primer is selected from commercial reagents, preferably Xeno IPC.
A kit for rapidly detecting canine influenza virus is characterized in that the kit is a monomer enzyme amplification composition containing specific primers and probes; the specific primer is the specific primer; the nucleotide sequence of the probe is shown as SEQ ID NO. 3; the monomer-based enzyme amplification composition is described in patent application 201810794915.2.
The monomeric enzyme amplification composition is divided into several parts.
The monomeric enzyme amplification composition is stored in a PCR amplification tube; storing 1 part of the monomer system enzyme amplification composition in each PCR amplification tube;
preferably, the PCR amplification tube is a Cepheid tubes reaction tube; each monomeric enzyme amplification composition was 15. mu.L.
A rapid detection reagent for dog influenza virus based on POCT monomer enzyme is characterized in that: comprises reaction buffer solution, dNTPs, dUTP, reverse transcriptase, Taq enzyme, RNase inhibitor, specific primer and probe, reaction enhancer, internal reference primer and probe.
The nucleotide sequence of the specific primer is shown as SEQ ID NO. 1 and SEQ ID NO. 2, the nucleotide sequence of the probe is shown as SEQ ID NO. 3, the 5 'end of the nucleotide sequence of the probe is connected with a fluorescence reporter group FAM label, and the 3' end of the nucleotide sequence of the probe is connected with a non-fluorescence quenching group and an MGB modifying group. Canine influenza viruses defined in the prior art are typically H3 subtype viruses, and therefore HA subtype genes are typically detected; the canine influenza virus defined by the invention is a canine influenza virus as long as influenza viruses detected on dogs are all called as canine influenza viruses, and comprises subtypes such as H5N2, H9N2, H7N9, H3N2 and H3N8, the site where the specific primer designed by the invention is located is not reported, all the subtypes can be covered, namely, the specific primer can detect whether a sample contains the canine influenza viruses of the subtypes such as H5N2, H9N2, H7N9, H3N2 and H3N8, and the detection accuracy is up to 100%.
The detection reagent is applied to the preparation of a canine influenza virus detection kit.
A rapid detection kit for dog influenza virus based on POCT monomer enzyme is characterized in that the kit consists of an extraction reagent and an amplification reaction system tube.
The amplification reaction system tube comprises reaction liquid and a reaction tube, namely reaction buffer solution, dNTPs, dUTP, reverse transcriptase, Taq enzyme, RNase inhibitor, specific primer and probe, reaction enhancer, internal reference primer and probe are mixed according to a proportion, each 15 mu L of the mixture is packaged into a Cepheid tubes.
The extraction reagent of the detection kit is a nucleic acid extraction reagent and comprises lysis solution, binding solution, washing solution, eluent, a collecting pipe and a centrifugal column.
A rapid detection system based on POCT monomer system enzyme is characterized in that: the detection system comprises a detection kit, a rapid fluorescence PCR instrument, a special centrifuge, a cup opener and a special ice box.
In a first aspect, the invention provides a rapid detection reagent for canine influenza virus based on POCT monomer enzyme, which comprises a specific primer and a probe for detecting canine influenza virus, wherein the nucleotide sequence of the specific primer is shown as SEQ ID NO. 1 and SEQ ID NO. 2, the nucleotide sequence of the probe is shown as SEQ ID NO. 3, the 5 'end of the nucleotide sequence of the probe is connected with a fluorescent reporter group FAM label, and the 3' end of the nucleotide sequence of the probe is connected with a non-fluorescent quencher group and an MGB modifying group.
Further, the invention relates to an application of the detection reagent in preparation of a rapid detection kit for the canine influenza virus based on POCT monomer enzyme.
In a second aspect, the invention provides a rapid detection kit for canine influenza virus based on POCT monosystem enzyme, which comprises an extraction reagent and an amplification reaction system tube; the amplification reaction system tube comprises reaction liquid and a reaction tube, namely reaction buffer solution, dNTPs, dUTP, reverse transcriptase, Taq enzyme, RNase inhibitor, specific primer and probe, reaction enhancer, internal reference primer and probe are mixed according to a proportion, each 15 mu L of the mixture is packaged into a Cepheid tubes. The nucleotide sequence of the specific primer is shown as SEQ ID NO. 1 and SEQ ID NO. 2, the nucleotide sequence of the probe is shown as SEQ ID NO. 3, the 5 'end of the nucleotide sequence of the probe is connected with a fluorescence reporter group FAM label, and the 3' end of the nucleotide sequence of the probe is connected with a non-fluorescence quenching group and an MGB modifying group.
In a third aspect, the present invention provides a rapid detection system based on POCT monosystem enzyme, which is characterized in that: the detection system comprises a nucleic acid extraction kit, a detection kit, a rapid fluorescence PCR instrument, a special centrifuge, a cup opener and a special ice box.
The detection kit and the detection system have the advantages of accurate detection result, simple operation, rapid detection on site, no need of liquid preparation (extracted template is directly added into an amplification system), short detection time, good stability, long storage period, internal reference (monitoring the whole extraction and amplification process), no need of electrophoresis and the like, can be popularized and applied to early screening and diagnosis of canine influenza, have positive influence on prevention and control of canine influenza epidemic situation, and lay a foundation for research and development of vaccines.
Drawings
FIG. 1 shows the results of the specificity test, wherein the numbers are shown below: 1: CPIV; 2: an AIV; 3: CPV (chlorinated polyvinyl chloride); 4: CCV; 5: CAV-1; 6: CAV-2; 7: and (4) CIV.
FIG. 2 shows the results of the sensitivity test, wherein the symbols are listed below: 1: 5X 106Copy/. mu.l; 2: 5X 105Copy/. mu.l; 3: 5X 104Copy/. mu.l; 4: 5X 103Copy/. mu.l; 5: 5X 102Copy/. mu.l; 6: 50 copies/. mu.l.
FIG. 3 shows the results of the sensitivity test, wherein the symbols are listed below: 1: H5N 2; 2: H9N 2; 3: H7N 9; 4: H3N 8; 5: H3N 2; 6: and (5) negative control.
FIG. 4 shows the results of the application test in Experimental example 4, wherein the symbols are as follows: 1-8 is clinical sample detection.
FIG. 5 shows the results of the application test in Experimental example 4, wherein the symbols are as follows: 9-16 are internal references for clinical sample detection.
FIG. 6 is a single sample test result showing that the sample is positive for canine influenza virus.
FIG. 7 is a single sample test result showing that the sample is canine influenza virus negative.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples do not show the specific techniques or conditions, and the techniques or conditions are described in the literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruker et al, Huang Petang et al) or according to the product instructions. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Group 1 example, specific primers for detecting canine influenza virus of the present invention
The embodiment of the group provides a specific primer for detecting canine influenza virus, which is characterized in that the nucleotide sequences of the upstream primer and the downstream primer are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2.
In some specific embodiments, the upstream and downstream primers can be artificially synthesized.
Group 2 example, kit for detecting canine influenza virus of the present invention
The embodiment of the group provides a kit for detecting canine influenza virus, which is characterized by comprising the primer and the probe described in any one of the embodiment of the group 1.
In a specific embodiment, the nucleotide sequence of the probe is shown in SEQ ID NO 3.
In a preferred embodiment, the probe has a nucleotide sequence with a fluorescent reporter FAM label attached to the 5 'end and a non-luminescent fluorescence quencher with Minor Groove Binder (MGB) attached to the 3' end;
preferably, the fluorescence emitting group is selected from one of FAM, VIC, HEX, JOE, NED, TAMRA, CY3, ROX or CY 5.
In a further embodiment, the kit further comprises, conventional reagents for performing PCR;
preferably, said conventional reagents for performing PCR are selected from: reaction buffer solution, dNTPs, dUTP, reverse transcriptase, Taq enzyme, RNase inhibitor, reaction enhancer, internal reference primer and probe;
further preferably, the reaction enhancer is selected from the group consisting of: ammonium sulfate, formamide or betaine, glycerol, Tween-20, BSA, DTT, or gelatin; the internal reference primer is selected from commercial reagents, preferably Xeno IPC.
Group 3 example, preparation method of kit for detecting canine influenza virus of the present invention
The group of embodiments provides a preparation method of a kit for detecting canine influenza virus, comprising: specific primers were synthesized according to the nucleotide sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2.
In a further embodiment, the method of making further comprises: synthesizing a probe according to the nucleotide sequence shown in SEQ ID NO. 3;
preferably, the preparation method further comprises: placing the specific primers and the probes into a packaging box marked with the application of detecting the canine influenza virus;
preferably, the preparation method further comprises: placing conventional reagents for performing PCR into a packaging box for detecting canine influenza virus according to the following nucleotide sequences;
further preferably, said conventional reagents for performing PCR are selected from: reaction buffer solution, dNTPs, dUTP, reverse transcriptase, Taq enzyme, RNase inhibitor, reaction enhancer, internal reference primer and probe;
further preferably, the reaction enhancer is selected from the group consisting of: ammonium sulfate, formamide or betaine, glycerol, Tween-20, BSA, DTT, or gelatin; the internal reference primer is selected from commercial reagents, preferably Xeno IPC.
Group 4 example, kit for rapidly detecting canine influenza virus of the present invention
The embodiment of the group provides a kit for rapidly detecting canine influenza virus, which is characterized in that the kit is a monomer enzyme amplification composition containing specific primers and probes; the specific primer is the specific primer of any one of the group 1 embodiments; the nucleotide sequence of the probe is shown as SEQ ID NO. 3; the monomer-based enzyme amplification composition is described in patent application 201810794915.2.
The entire contents of the description of patent application 201810794915.2 are incorporated herein by reference.
In some embodiments, the monomeric enzyme amplification composition is in several portions.
In other embodiments, the monomeric enzyme amplification composition is deposited in a PCR amplification tube; storing 1 part of the monomer system enzyme amplification composition in each PCR amplification tube;
in a preferred embodiment, the PCR amplification tube is a Cepheid tubes reaction tube; each monomeric enzyme amplification composition was 15. mu.L.
Group 5 example, preparation method of kit for rapidly detecting canine influenza virus of the present invention
The present group of embodiments provides a method for preparing the kit for rapidly detecting canine influenza virus according to any one of the group 4 embodiments, comprising: a monomeric enzyme amplification composition was prepared according to the formulation of monomeric enzyme amplification composition described in patent application 201810794915.2, wherein the specific primers in the monomeric enzyme amplification composition were the specific primers described in any one of examples in group 1.
In a further embodiment, the probe in the monomeric enzyme amplification composition has the nucleotide sequence shown in SEQ ID NO. 3.
In a preferred embodiment, the prepared monomeric enzyme amplification composition is divided into several parts, and each 1 part of monomeric enzyme amplification composition is stored in 1 independent PCR amplification tube; the PCR amplification tube containing 1 part of the monomeric enzyme amplification composition is placed in a packaging box marked with the purpose of detecting the canine influenza virus.
In specific embodiments, the PCR amplification tube is a Cepheid tubes reaction tube; each monomeric enzyme amplification composition was 15. mu.L.
Experimental example 1 Rapid detection kit for Canine influenza Virus
The detection kit comprises an extraction reagent and an amplification reagent.
Wherein, the extraction reagent comprises lysis solution, binding solution, washing solution, eluent, a collecting pipe and a centrifugal column.
The amplification reagent is an amplification reaction system tube, and comprises reaction liquid and a reaction tube, namely reaction buffer solution, dNTPs, dUTP, reverse transcriptase, Taq enzyme, RNase inhibitor, specific primer and probe, reaction enhancer, internal reference primer and probe are mixed according to a proportion, 15 mu L of each part is packaged into a Cepheid tubes reaction tube.
The nucleotide sequences of the specific primers and probes are shown in Table 1 below.
TABLE 1 nucleotide sequences of specific primers and probes
| Name (R) | Sequence (5 '-3') | Length of | Remarks for note | Amplified fragment length |
| Primer 1 | AGGTCGAAACGTACGTTCTCTCTAT | 25 | ||
| Primer 2 | ATTGGTCTTGTCTTTAGCCATTCC | 24 | ||
| Probe 1 | TCAGGCCCCCTCAAAGC | 17 | FAM-MGB | 136bp |
Experimental example 2 Rapid detection System for Canine influenza Virus
The detection system comprises a detection kit, a rapid fluorescence PCR instrument, a special centrifuge, a cup opener and a special ice box.
The detection kit comprises an extraction reagent and an amplification reagent.
The extraction reagent comprises lysis solution, binding solution, washing solution, eluent, a collecting pipe and a centrifugal column.
The amplification reagent is an amplification reaction system tube, and comprises reaction liquid and a reaction tube, namely reaction buffer solution, dNTPs, dUTP, reverse transcriptase, Taq enzyme, RNase inhibitor, specific primer and probe, reaction enhancer, internal reference primer and probe are mixed according to a proportion, 15 mu L of each part is packaged into a Cepheid tubes reaction tube. The nucleotide sequences of the specific primers and probes are shown in table 1.
Experimental example 3, use method of canine influenza virus rapid detection kit
1 sample preparation
1.1 wetting the sterile cotton swab with sterile physiological saline, turning and smearing the swab in a conjunctival sac or a nasal mucosa, and collecting a sample, or dipping the sterile swab in a tracheal lavage sample. And then inserting the swab into sterile normal saline, uniformly mixing the swab by vortex oscillation, then squeezing the swab on the tube wall to dry liquid, putting the liquid into disinfectant, discarding the liquid, and taking 200 mu L of supernatant into a 1.5mL sterile centrifuge tube.
1.2EDTA anticoagulation, cell culture solution, and 200. mu.L of supernatant was put into a 1.5mL sterilized centrifuge tube.
2 extraction of viral RNA
2.1 adding 20 mul RNA internal reference into the processed sample, adding 500 mul lysate, fully reversing and mixing evenly, and standing for 3min at room temperature. The liquid is sucked into the adsorption column (the collection tube is sleeved on the adsorption column, suspended impurities are not sucked as much as possible when the liquid is sucked, so that the adsorption column is not blocked during centrifugation), and the liquid is centrifuged at 13000rpm for 30 s.
2.2 discard the liquid in the collection tube, absorb 500. mu.L of the washing solution and add to the adsorption column, and centrifuge for 30s at 13000 rpm.
2.3 discard the liquid from the collection tube, aspirate 500. mu.L of wash solution into the adsorption column, and centrifuge at 13000rpm for 2min (take out with care to avoid the adsorption column touching the liquid below) to remove the remaining wash solution.
2.4 transferring the adsorption column into a 1.5mL centrifuge tube, adding 50 μ L of eluent into the center of the adsorption column, centrifuging at 13000rpm for 30s, and obtaining the template RNA as the liquid in the centrifuge tube.
3 real-time fluorescent RT-PCR
And (3) melting the reaction tube at room temperature, sucking 5 mu L of template RNA by using a pipette, adding the template RNA into the reaction tube, centrifuging the template RNA for 30s by using a centrifugal machine, putting the template RNA into a machine, and performing amplification detection by using a program CIV.
[ result judgment ]
According to automatic analysis, the Ct value is less than or equal to 36 and a specific amplification curve is positive for the canine influenza virus; when the Ct value is larger than 36, a specific amplification curve appears, the RNA is extracted by sampling again, and the result is judged after the RNA is amplified, if the specific amplification curve still appears, the result can be judged to be positive; some samples that did not show sigmoid curves but had higher background were judged negative.
Experimental example 4 application of the kit of the present invention
1 specificity test
The results of the specificity test are shown in FIG. 1. As can be seen from FIG. 1, the designed primer probe can only amplify the target fragment from the allantoic fluid of canine influenza virus, and the amplification result of other viruses which can infect dogs is negative.
2 sensitivity and sensitivity test results
The results of the sensitivity test are shown in FIG. 2, and the results of the sensitivity test are shown in FIG. 3. As can be seen from FIG. 2, the PCR method of this experiment can be used to obtain a positive amplification result from 5. mu.L of a sample diluted in the positive plasmid of H3N2, with a minimum detection amount of 50 copies/. mu.L. Susceptibility testing As can be seen from FIG. 3, influenza virus subtypes H5N2, H9N2, H7N9, H3N2, H3N8, etc. can be detected by the PCR method of the test, and the specific subtypes can be further determined by sequencing after the subtypes are detected and determined by the kit of the present invention.
| Reference numerals in FIG. 3 | Influenza virus subtypes |
| 1 | H5N2 |
| 2 | H9N2 |
| 3 | H7N9 |
| 4 | H3N2 |
| 5 | H3N8 |
3 results of application test
The results of the application tests are shown in fig. 4 and 5. As can be seen from FIG. 4, 6 positive amplification results were obtained from 8 suspected samples by PCR, and the accuracy was 100%.
FIG. 5 shows the results of the application test internal reference. 9-16 are internal references for clinical sample detection; the specific internal reference primer can be Xeno IPC, and the internal reference can be obtained commercially.
| Reference numerals in FIG. 5 | Sample numbering | Whether the internal reference is detected |
| 9 | Sample 1 | Is that |
| 10 | Sample 2 | Is that |
| 11 | Sample 3 | Is that |
| 12 | Sample No. 4 | Is that |
| 13 | Sample No. 5 | Is that |
| 14 | Sample No. 6 | Is that |
| 15 | Sample 7 | Is that |
| 16 | Sample 8 | Is that |
That is, each blue curve in fig. 4 corresponds to one red curve in fig. 5. In actual detection practice, the detection results of the individual samples are respectively shown in fig. 6 and fig. 7, wherein fig. 6 shows that the sample canine influenza virus is positive, and fig. 7 shows that the sample canine influenza virus is negative. If the red curve does not appear in the graph, the result is invalid and needs to be detected again.
SEQUENCE LISTING
<110> Beijing Shijiheng animal epidemic prevention technology Co., Ltd
<120> specific primer and kit for detecting canine influenza virus
<130> P190479-SJY
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 1
<400> 1
aggtcgaaac gtacgttctc tctat 25
<210> 2
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 2
<400> 2
attggtcttg tctttagcca ttcc 24
<210> 3
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> Probe 1
<400> 3
tcaggccccc tcaaagc 17
Claims (10)
1. The specific primer for detecting the canine influenza virus is characterized in that the nucleotide sequences of the upstream primer and the downstream primer are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2.
2. A kit for detecting canine influenza virus, comprising the primer and probe of claim 1.
3. The kit of claim 2, wherein the nucleotide sequence of the probe is shown in SEQ ID NO 3.
4. The kit according to claim 2 or 3, wherein the probe has a nucleotide sequence with a fluorescent reporter FAM label attached to the 5 'end and a non-luminescent fluorescence quencher with Minor Groove Binder (MGB) attached to the 3' end;
preferably, the fluorescence emitting group is selected from one of FAM, VIC, HEX, JOE, NED, TAMRA, CY3, ROX or CY 5.
5. The kit according to any one of claims 2 to 4, further comprising, conventional reagents for performing PCR;
preferably, said conventional reagents for performing PCR are selected from: reaction buffer solution, dNTPs, dUTP, reverse transcriptase, Taq enzyme, RNase inhibitor, reaction enhancer, internal reference primer and probe;
further preferably, the reaction enhancer is selected from the group consisting of: ammonium sulfate, formamide or betaine, glycerol, Tween-20, BSA, DTT, or gelatin; the internal reference primer is selected from commercial reagents, preferably Xeno IPC.
6. A method of making a kit for detecting canine influenza virus, comprising: specific primers were synthesized according to the nucleotide sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2.
7. The method of manufacturing according to claim 6, further comprising: synthesizing a probe according to the nucleotide sequence shown in SEQ ID NO. 3;
preferably, the preparation method further comprises: placing the specific primers and the probes into a packaging box marked with the application of detecting the canine influenza virus;
preferably, the preparation method further comprises: placing conventional reagents for performing PCR into a packaging box for detecting canine influenza virus according to the following nucleotide sequences;
further preferably, said conventional reagents for performing PCR are selected from: reaction buffer solution, dNTPs, dUTP, reverse transcriptase, Taq enzyme, RNase inhibitor, reaction enhancer, internal reference primer and probe;
further preferably, the reaction enhancer is selected from the group consisting of: ammonium sulfate, formamide or betaine, glycerol, Tween-20, BSA, DTT, or gelatin; the internal reference primer is selected from commercial reagents, preferably Xeno IPC.
8. A kit for rapidly detecting canine influenza virus is characterized in that the kit is a monomer enzyme amplification composition containing specific primers and probes; the specific primer is the specific primer of claim 1; the nucleotide sequence of the probe is shown as SEQ ID NO. 3; the monomer-based enzyme amplification composition is described in patent application 201810794915.2.
9. The kit of claim 8, wherein the monomeric enzyme amplification composition is in several portions.
10. The kit according to claim 8 or 9, characterized in that said monomeric enzyme amplification composition is deposited in a PCR amplification tube; storing 1 part of the monomer system enzyme amplification composition in each PCR amplification tube;
preferably, the PCR amplification tube is a Cepheid tubes reaction tube; each monomeric enzyme amplification composition was 15. mu.L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910610586.6A CN110592276A (en) | 2019-07-08 | 2019-07-08 | Specific primer and kit for detecting canine influenza virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910610586.6A CN110592276A (en) | 2019-07-08 | 2019-07-08 | Specific primer and kit for detecting canine influenza virus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110592276A true CN110592276A (en) | 2019-12-20 |
Family
ID=68852738
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910610586.6A Pending CN110592276A (en) | 2019-07-08 | 2019-07-08 | Specific primer and kit for detecting canine influenza virus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110592276A (en) |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070253981A1 (en) * | 2006-04-06 | 2007-11-01 | Cornell Research Foundation, Inc. | Canine influenza virus |
| CN101253267A (en) * | 2005-04-21 | 2008-08-27 | 佛罗里达大学研究基金公司 | Materials and methods for respiratory disease control in canines |
| CN103409559A (en) * | 2013-08-12 | 2013-11-27 | 中国农业大学 | RT-PCR kit for detection of poultry source pedigree H3N2 subtype canine influenza virus and application thereof |
| CN104131009A (en) * | 2014-07-28 | 2014-11-05 | 中国农业大学 | Primer composition for detecting different influenza viruses and application thereof |
| US20150133329A1 (en) * | 2012-05-03 | 2015-05-14 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of | Methods of detecting influenza virus |
| CN105821158A (en) * | 2016-04-11 | 2016-08-03 | 公安部南京警犬研究所 | RT-LAMP primer combination and kit for detecting canine influenza virus as well as application of RT-LAMP primer combination |
| CN106636475A (en) * | 2017-03-01 | 2017-05-10 | 中国农业大学 | Primer set for detecting American canine influenza virus subtype H3N8 and application thereof |
| CN106939355A (en) * | 2017-03-01 | 2017-07-11 | 苏州系统医学研究所 | A kind of screening of influenza virus attenuated live vaccines strain and authentication method |
| CN108866156A (en) * | 2018-07-19 | 2018-11-23 | 北京世纪元亨动物防疫技术有限公司 | The preparation method and applications of single mass system enzymatic amplification composition for POCT fluorescence RT-PCR system |
| CN109517927A (en) * | 2018-09-03 | 2019-03-26 | 连云港出入境检验检疫局检验检疫综合技术中心 | A kind of A type, influenza B virus rapid typing detection reagent box and its application |
-
2019
- 2019-07-08 CN CN201910610586.6A patent/CN110592276A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101253267A (en) * | 2005-04-21 | 2008-08-27 | 佛罗里达大学研究基金公司 | Materials and methods for respiratory disease control in canines |
| US20070253981A1 (en) * | 2006-04-06 | 2007-11-01 | Cornell Research Foundation, Inc. | Canine influenza virus |
| US20150133329A1 (en) * | 2012-05-03 | 2015-05-14 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of | Methods of detecting influenza virus |
| CN103409559A (en) * | 2013-08-12 | 2013-11-27 | 中国农业大学 | RT-PCR kit for detection of poultry source pedigree H3N2 subtype canine influenza virus and application thereof |
| CN104131009A (en) * | 2014-07-28 | 2014-11-05 | 中国农业大学 | Primer composition for detecting different influenza viruses and application thereof |
| CN105821158A (en) * | 2016-04-11 | 2016-08-03 | 公安部南京警犬研究所 | RT-LAMP primer combination and kit for detecting canine influenza virus as well as application of RT-LAMP primer combination |
| CN106636475A (en) * | 2017-03-01 | 2017-05-10 | 中国农业大学 | Primer set for detecting American canine influenza virus subtype H3N8 and application thereof |
| CN106939355A (en) * | 2017-03-01 | 2017-07-11 | 苏州系统医学研究所 | A kind of screening of influenza virus attenuated live vaccines strain and authentication method |
| CN108866156A (en) * | 2018-07-19 | 2018-11-23 | 北京世纪元亨动物防疫技术有限公司 | The preparation method and applications of single mass system enzymatic amplification composition for POCT fluorescence RT-PCR system |
| CN109517927A (en) * | 2018-09-03 | 2019-03-26 | 连云港出入境检验检疫局检验检疫综合技术中心 | A kind of A type, influenza B virus rapid typing detection reagent box and its application |
Non-Patent Citations (3)
| Title |
|---|
| ZHAO J等: "Influenza A virus segment 7 matrix protein 2(M2) and matrix protein(M1) genes,complete cds", 《NCBI GENBANK》 * |
| 赵明喜等: "广东1株H3N2亚型犬流感病毒的分离与鉴定", 《中国兽医杂志》 * |
| 高晓宇等: "犬流感病毒RT-LAMP快速检测方法的建立", 《中国预防兽医学报》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2021174674A1 (en) | Composition, kit and method for detecting and typing coronaviruses | |
| US20080261198A1 (en) | Diagnostic Primers and Method for Detecting Avian Influenza Virus Subtype H5 and H5n1 | |
| WO2006000140A1 (en) | Primers, probes sequences and methods, useful in the multiple real time fluorescent rt-pcr assay of avian influenza virus subtypes h5, h7 and h9 | |
| JP7064473B2 (en) | How to detect influenza A virus and influenza B virus | |
| CN106636472B (en) | Complete set of reagent and method for detecting avian influenza virus and chicken parvovirus | |
| CN105441586A (en) | A-type H5N6 subtype avian influenza virus dual-channel real-time fluorescence PCR (polymerase chain reaction) detection kit and detection method | |
| Rashid et al. | Multiplex polymerase chain reaction for the detection and differentiation of avian influenza viruses and other poultry respiratory pathogens | |
| US20180265936A1 (en) | Compositions and methods for detection and discrimination of influenza viruses | |
| Agüero et al. | A fully automated procedure for the high-throughput detection of avian influenza virus by real-time reverse transcription–polymerase chain reaction | |
| Sidoti et al. | Development of real time RT-PCR assays for detection of type A influenza virus and for subtyping of avian H5 and H7 hemagglutinin subtypes | |
| CN102321769B (en) | Primer pair for identifying newcastle disease virus and multi-subtype avian influenza virus and application thereof | |
| CN104846121A (en) | Virus triple fluorescence quantitative RT-PCR detection method and virus triple fluorescence quantitative RT-PCR detection kit | |
| CN110656163A (en) | Double-label report fluorescent multiple pathogen nucleic acid detection method | |
| CN106939357A (en) | H4 hypotypes, H6 hypotypes and the triple RT PCR primer combinations of H9 hypotypes AIV, kit and its application | |
| US10329630B2 (en) | Compositions and methods for detection and discrimination of emerging influenza virus subtypes | |
| CN106636473B (en) | Complete set of reagents and method for detecting H5 subtype avian influenza virus and chicken parvovirus | |
| CN101629217A (en) | RT-LAMP detection kit and detection method of swine influenza virus | |
| CN102304591B (en) | PCR (polymerase chain reaction) primer pair for identifying H3 subtype avian influenza virus and application thereof | |
| CN109628640B (en) | RPA-LFD primer, method and kit for rapidly detecting spring viremia of carp virus | |
| JP5561708B2 (en) | Primer set for NA subtype determination of avian influenza virus | |
| CN110669872A (en) | Triple RT-PCR (reverse transcription-polymerase chain reaction) detection primer group, kit and method for H9 and H10 subtype avian influenza viruses | |
| WO2006132601A1 (en) | Diagnostic primers and method for detecting avian influenza virus subtype h5 and h5n1 | |
| Kuriakose et al. | Detection of avian influenza viruses and differentiation of H5, H7, N1, and N2 subtypes using a multiplex microsphere assay | |
| CN110592276A (en) | Specific primer and kit for detecting canine influenza virus | |
| CN104450955B (en) | Eurasian class fowl type H1N1 swine influenza virus fluorescence quantitative RT-PCR detecting kit and its application |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20191220 |
|
| RJ01 | Rejection of invention patent application after publication |