CN108315489A - A kind of primer sets and kit for transmissible gastro-enteritis virus now separation strains and vaccine strain antidiastole - Google Patents
A kind of primer sets and kit for transmissible gastro-enteritis virus now separation strains and vaccine strain antidiastole Download PDFInfo
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- CN108315489A CN108315489A CN201810350653.0A CN201810350653A CN108315489A CN 108315489 A CN108315489 A CN 108315489A CN 201810350653 A CN201810350653 A CN 201810350653A CN 108315489 A CN108315489 A CN 108315489A
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- 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
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- 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]
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Abstract
The invention discloses a kind of primer sets and kit for transmissible gastro-enteritis virus now separation strains and vaccine strain antidiastole, and the primer sets include primer S1, S2 and S3, wherein：S1:5’‑ACTTAGGCTAGCATCACAT‑3’,S2：5 ' CAATAAGCTGAGTCGACCGTATG 3 ', S3：5’‑ATAAGCTTAGTCGACTGAT‑3’.The present invention designs specific primer S1, S2 and S3 according to existing the difference between separation strains and vaccine strain of transmissible gastro-enteritis virus, can now separation strains carry out specific amplification with vaccine strain to transmissible gastro-enteritis virus.The dedicated kit of the present invention has very strong sensibility, specificity, and coincidence rate can be widely applied for clinical transmissible gastro-enteritis virus vaccine and now the antidiastole of separation strains up to 100% compared with the methods of virus purification and IFA.
Description
Technical field
The present invention relates to veterinary biological virus detection techniques fields, and in particular to one kind being used for transmissible gastro-enteritis virus
Now the primer sets and kit of separation strains and vaccine strain antidiastole.
Background technology
Transmissible gastroenteritis of swine is by transmissible gastro-enteritis virus (Transmissible Gastroenteritis of
Swine Virus write a Chinese character in simplified form TGEV) caused by infectious disease, the also known as gastroenteritis of piglet, be a kind of high degree in contact infectiousness, to vomit
Spit, severe diarrhea, dehydration, cause the viral infectious that is characterized of piglet high mortality in two week old.The disease is mainly shown as pig
Vomiting, watery diarrhea and dehydration, are mainly in Winter-Spring cold season, be still at present cause piglet morbidity and dead main epidemic disease it
One, bring huge economic loss to aquaculture.Transmissible gastro-enteritis virus vaccine strain can quickly be distinguished by establishing one kind
With the multiplex PCR and dedicated kit of existing ground separation strains, it can facilitate and occur fast and accurately to examine in early days in epidemic disease
Disconnected epidemic disease, effective reference is provided for the prevention and control of epidemic disease.
Invention content
In view of this, the purpose of the present invention is to provide one kind being used for transmissible gastro-enteritis virus now separation strains and epidemic disease
The primer sets and kit of seedling strain antidiastole, the kit can quickly distinguish transmissible gastro-enteritis virus now separation strains
With vaccine strain.
To achieve the goals above, the technical solution adopted in the present invention is：
A kind of primer sets for transmissible gastro-enteritis virus now separation strains and vaccine strain antidiastole, described draws
Object group includes primer S1, S2, S3, wherein：
S1：5’-ACTTAGGCTAGCATCACAT-3’；
S2：5’-CAATAAGCTGAGTCGACCGTATG-3’；
S3：5’-ATAAGCTTAGTCGACTGAT-3’.
Primer sets described in a kind of in preparation, for transmissible gastro-enteritis virus, now with vaccine strain discriminating examine by separation strains
Application in disconnected kit.
A kind of transmissible gastro-enteritis virus of the primer sets now separation strains and vaccine strain differential diagnosis kit.
The kit further includes nucleic acid extracting reagent, Reverse Transcription, PCR amplification reagent, and/or Ago-Gel
Electrophoresis reagents.
The Reverse Transcription includes 10 × M-MLV buffer solutions, dNTP, M-MLV reverse transcriptase, the inhibition of RNase enzymes
Agent, S2 primers.
The PCR amplification reagent is poly- including 10 × PCR amplification buffer solution, dNTP, S1 primer, S2 primers, Taq DNA
Synthase and distilled water.
PCR amplification system constructed by the PCR amplification reagent is：10 × PCR amplification buffer solution, 5.0 μ L,
2.5mmol/L dNTP8.0 μ L, 1.0 μ L of 10mmol/L S1 primers, 1.0 μ L of 10mmol/L S2 primers, 5U/ μ L Taq DNA
1.0 μ L of polymerase, 3.0 μ L of template, add distilled water to 50 μ L.
A kind of application method of the kit, including nucleic acid extraction, reverse transcription, PCR amplification and Ago-Gel electricity
Swimming.
Further include analyzing the result of agarose gel electrophoresis：When amplified fragments are 768bp, then the pig in sample passes
Metachromia marcy agent is vaccine strain；When amplified fragments size is 378bp, then the transmissible gastro-enteritis virus in sample is existing
Ground separation strains.
The PCR amplification condition is：95 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 57 DEG C of annealing 30s, 72 DEG C extend
90s, totally 35 recycle；Last 72 DEG C of extensions 10min.
Beneficial effects of the present invention：
The present invention is drawn by comparing the difference of transmissible gastro-enteritis virus now separation strains and vaccine strain, design specificity
Object S1, S2, S3, can now separation strains and vaccine strain carry out specific amplification to transmissible gastro-enteritis virus；It is basic herein
On have developed dedicated kit, first extract viral RNA, reuse S2 primed reverse transcriptions synthesis cDNA, and using PCR method differentiate
Transmissible gastro-enteritis virus vaccine strain and now separation strains are detected, it is 768bp that amplification vaccine strain, which is expected amplified fragments size, is expanded
It is 378bp to increase now separation strains to be expected clip size.Compared with prior art, dedicated kit of the invention has very strong
Sensibility, specificity, the amplification to Porcine epidemic diarrhea virus (P-EDV) and porcine rotavirus (PoRV) is feminine gender,
It can be widely applied for clinical transmissible gastro-enteritis virus vaccine strain and now the antidiastole of separation strains.
Description of the drawings
Specific implementation mode
To make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with specific embodiment, to this hair
Bright to be described in further detail, the embodiment is only for explaining the present invention, is not intended to limit the scope of the present invention..
Experimental method used in following embodiments is the conventional method of this field unless otherwise specified, or according to institute of manufactory
It is recommended that condition and implementation steps.
The structure of embodiment 1, differential diagnostic method
1, design of primers
Two specific primers are designed, it is specific as follows:
S1：5’-ACTTAGGCTAGCATCACAT-3’(SEQ ID NO.1)
S2：5’-CAATAAGCTGAGTCGACCGTATG-3’(SEQ ID NO.2)
S3：5’-ATAAGCTTAGTCGACTGAT-3’(SEQ ID NO.3)
PCR amplification is carried out using S1, S2 and S3, it is 768bp that TGEV vaccine strains, which are expected amplified fragments size, and TGEV is now
It is 378bp that separation strains, which are expected amplified fragments size,.
2, the extraction of viral RNA
Take TGEV strain cell culture fluids, after multigelation 3 times, 5000g centrifuges 15min, takes supernatant spare.
(1) 0.2ml chloroforms are taken, isometric supernatant is added, acutely rocks 15s, are placed at room temperature for 3min, then 12,000g,
4 DEG C of centrifugation 5min.Be divided into 3 layers after centrifugation, it is nethermost it is red be phenol-chloroform phase, a middle layer, upper layer is colourless water
Phase, RNA are present in water phase；
(2) upper strata aqueous phase in step (1) is transferred in the clean EP pipes of another, the isopropyl of 0.5ml precoolings is added
Alcohol is stored at room temperature 10min, then 12,000g, 4 DEG C of centrifugation 10min；
(3) it outwells supernatant, 75% ethyl alcohol of 1ml volume fractions is added and washs RNA precipitate, after mixing, 7500g, 4 DEG C of centrifugations
5min；
(4) supernatant is outwelled, 10min is placed at room temperature for, is then added in right amount without RNAse water dissolutions RNA to get to total serum IgE.
3, reverse transcription
The RNA extracted using step 2 carries out reverse transcription as template, obtains cDNA, and reverse transcription reaction system is：
| Template ribonucleic acid | 4.9μL |
| S2 primers (10mmol/L) | 0.3μL |
| RTase enzyme inhibitors (40U/ μ L) | 0.3μL |
| M-MLV reverse transcriptase (200U/ μ L) | 0.5μL |
| dNTP(2.5mmol/L) | 2.0μL |
| 10 × M-MLV buffer solutions | 2.0μL |
Reaction condition is：42 DEG C of heat preservations 60min, 70 DEG C of 10min.
4, PCR (PCR)
4.1 PCR reaction systems
The cDNA obtained using step 3 carries out PCR amplification as template, and PCR reaction systems are：
4.2PCR reaction condition optimization
The PCR reaction conditions of TGEV vaccine strains are optimized, respectively with annealing temperature be 54 DEG C, 55 DEG C, 56 DEG C, 57
DEG C, 58 DEG C, 59 DEG C, 60 DEG C, 61 DEG C, 62 DEG C, 63 DEG C, 64 DEG C carry out PCR reaction amplification TGEV vaccine strains, reaction condition is：95
DEG C pre-degeneration 5min；94 DEG C of denaturation 30s, anneal 30s, 72 DEG C of extension 90s, totally 35 cycles；72 DEG C of extension 10min.It was found that
57 and 62 DEG C of segments that can amplify 768bp sizes, it is in the same size with expection.
The PCR reaction conditions of existing the separation strains of TGEV are optimized, be respectively 52 DEG C with annealing temperature, 53 DEG C, 54
DEG C, 55 DEG C, 56 DEG C, 57 DEG C, 58 DEG C, 59 DEG C, 60 DEG C, 61 DEG C carry out existing the separation strains of PCR reaction amplification TGEV, reaction condition
For：95 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, anneal 30s, 72 DEG C of extension 90s, totally 35 cycles；72 DEG C of extension 10min.
It was found that occur the segment of 378bp sizes at 57 DEG C, it is in the same size with expection.
In summary it reacts, the annealing temperature of composite PCR is finally set as 57 DEG C by the present invention, the reaction condition after optimization
For：95 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 57 DEG C of annealing 30s, 72 DEG C of extension 90s, totally 35 recycle；Last 72 DEG C of extensions
10min.Pcr amplification product is placed in -20 DEG C of preservations.
5, electrophoresis
To PCR product into row agarose gel electrophoresis, voltage 100V, electrophoresis 40min, under DNA gel imaging system instrument
Observe result.
The dedicated kit of the present invention includes nucleic acid extracting reagent, Reverse Transcription, PCR used in above-mentioned detection method
Amplifing reagent, agarose gel electrophoresis reagent.
The differential diagnostic method of existing the separation strains sample of embodiment 2, TGEV vaccine strains and TGEV
Using TGEV vaccine strains and TGEV, now separation strains are detected as sample according to the method in embodiment 1 respectively, knot
Fruit shows that TGEV vaccine strains can obtain about 768bp segments, and now separation strains amplified fragments size is 378bp segments to TGEV, with
It is expected that being consistent.
Embodiment 3, specificity experiments
It is respectively control strain with Porcine epidemic diarrhea virus (P-EDV) and porcine rotavirus (PoRV), with pig transmissible
Marcy agent is to cause the encountered pathogenic of diarrhea as experiment strain, above 3 kinds of virus, and abdomen caused by this 3 kinds of viruses
It rushes down on epidemiology, is clinically extremely similar with pathological change etc., being difficult clinically to distinguish.According in embodiment 1
Method is detected, the results showed that, only now separation strains can respectively obtain 768bp and 378bp for TGEV vaccine strains and TGEV
Segment, other viruses are generated without amplified band.Experimental result confirms that primer of the invention and detection method have very high spy
It is anisotropic.
Embodiment 4, sensitivity experiment
TGEV is showed into ground separation strains virus liquid and carries out 10 times of gradient dilutions, it is 5.4 × 10 to make virus concentration4-5.4 ×10- 4Copies/ μ L, and be detected according to the method for embodiment 1, the results showed that, in the 8th swimming lane a concentration of 5.4 × 10- 3Copies/ μ L it is amplifiable go out expected segment, the minimum detection of nucleic acids amount of primer of the invention and detection method is 5.4 × 10- 3Copies/ μ L, but the 9th swimming lane 5.4 × 10-4Segments of the copies/ μ L without expected size, judges its sensitivity for 5.4 × 10- 3copies/μL。
TGEV vaccine strain virus liquids are subjected to 10 times of gradient dilutions, it is 1.5 × 10 to make virus concentration4-1.0× 10- 4Copies/ μ L, and be detected according to the method for embodiment 1, the results showed that, in the 8th swimming lane a concentration of 1.5 × 10- 3Copies/ μ L it is amplifiable go out expected segment, the minimum detection of nucleic acids amount of primer of the invention and detection method is 1.5 × 10- 3Copies/ μ L, but the 9th swimming lane 1.5 × 10-4Segments of the copies/ μ L without expected size, judges its sensitivity for 1.5 × 10- 3copies/μL。
The above is only presently preferred embodiments of the present invention, is not imposed any restrictions to the present invention, every according to the present invention
Technical spirit changes any simple modification, change and equivalent structure made by above example, still falls within skill of the present invention
In the protection domain of art scheme.
Sequence table
<110>Bai Ling
<120>A kind of primer sets and reagent for transmissible gastro-enteritis virus now separation strains and vaccine strain antidiastole
Box
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
acttaggcta gcatcacat 19
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<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
caataagctg agtcgaccgt atg 23
<210> 3
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
ataagcttag tcgactgat 19
Claims (10)
1. a kind of primer sets for transmissible gastro-enteritis virus now separation strains and vaccine strain antidiastole, feature exists
In, primer sets include S1, S2, S3, wherein：
S1：5’-ACTTAGGCTAGCATCACAT-3’；
S2：5’-CAATAAGCTGAGTCGACCGTATG-3’；
S3：5’-ATAAGCTTAGTCGACTGAT-3’.
2. a kind of primer sets as described in claim 1 are used for transmissible gastro-enteritis virus now separation strains and vaccine in preparation
Application in strain differential diagnosis kit.
3. a kind of transmissible gastro-enteritis virus comprising primer sets as described in claim 1 now with vaccine strain reflect by separation strains
Other diagnostic kit.
4. kit according to claim 3, which is characterized in that the kit further includes nucleic acid extracting reagent, anti-
Transcript reagent, PCR amplification reagent, and/or agarose gel electrophoresis reagent.
5. kit according to claim 4, which is characterized in that the Reverse Transcription is buffered including 10 × M-MLV
Liquid, dNTP, M-MLV reverse transcriptase, RNase enzyme inhibitors, S2 primers.
6. kit according to claim 4, which is characterized in that the PCR amplification reagent is slow including 10 × PCR amplification
Fliud flushing, dNTP, S1 primer, S2 primers, Taq archaeal dna polymerases and distilled water.
7. kit according to claim 6, which is characterized in that the PCR amplification body constructed by the PCR amplification reagent
System is：10 × PCR amplification buffer solution, 5.0 μ L, 2.5mmol/L dNTP8.0 μ L, 1.0 μ L of 10mmol/L S1 primers, 10mmol/L
1.0 μ L of S2 primers, 1.0 μ L of 5U/ μ L Taq archaeal dna polymerases, 3.0 μ L of template, add distilled water to 50 μ L.
8. a kind of application method such as claim 3-7 any one of them kits, which is characterized in that including nucleic acid extraction,
Reverse transcription, PCR amplification and agarose gel electrophoresis.
9. the application method of kit according to claim 8, which is characterized in that further include to agarose gel electrophoresis
As a result it is analyzed：When amplified fragments are 768bp, then the transmissible gastro-enteritis virus in sample is vaccine strain；When amplification piece
Duan great little is 378bp, then the transmissible gastro-enteritis virus in sample is now separation strains.
10. the application method of kit according to claim 8, which is characterized in that the PCR amplification condition is：95
DEG C pre-degeneration 5min；94 DEG C of denaturation 30s, 57 DEG C of annealing 30s, 72 DEG C of extension 90s, totally 35 recycle；Last 72 DEG C of extensions
10min。
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| CN201810350653.0A CN108315489A (en) | 2018-04-18 | 2018-04-18 | A kind of primer sets and kit for transmissible gastro-enteritis virus now separation strains and vaccine strain antidiastole |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108315498A (en) * | 2018-05-02 | 2018-07-24 | 郭庆君 | A kind of primer sets and kit of H3N2 hypotypes swine influenza virus strain and vaccine strain antidiastole |
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| CN102154516A (en) * | 2011-03-22 | 2011-08-17 | 上海交通大学 | Fluorescence quantitative PCR (Polymerase Chain Reaction) detection method for porcine transmissible gastroenteritis virus gene S and primer thereof |
| CN102373301A (en) * | 2011-11-29 | 2012-03-14 | 重庆出入境检验检疫局检验检疫技术中心 | Kit and detection method for quickly detecting porcine transmissible gastroenteritis virus by adopting isothermal amplification technology |
| CN104611465A (en) * | 2015-02-04 | 2015-05-13 | 四川农业大学 | Fluorogenic quantitative PCR kit for detecting swine transmissible gastroenteritis virus |
| CN106435014A (en) * | 2016-08-24 | 2017-02-22 | 广西壮族自治区动物疫病预防控制中心 | Multiple RT-PCR detection kit and primer group of porcine epidemic diarrhea virus virulent strain and vaccine strain |
| CN107475450A (en) * | 2017-09-13 | 2017-12-15 | 河南省农业科学院畜牧兽医研究所 | A kind of primer sets and kit for the strain of Porcine epidemic diarrhea virus novel variant and vaccine strain antidiastole |
-
2018
- 2018-04-18 CN CN201810350653.0A patent/CN108315489A/en not_active Withdrawn
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154516A (en) * | 2011-03-22 | 2011-08-17 | 上海交通大学 | Fluorescence quantitative PCR (Polymerase Chain Reaction) detection method for porcine transmissible gastroenteritis virus gene S and primer thereof |
| CN102373301A (en) * | 2011-11-29 | 2012-03-14 | 重庆出入境检验检疫局检验检疫技术中心 | Kit and detection method for quickly detecting porcine transmissible gastroenteritis virus by adopting isothermal amplification technology |
| CN104611465A (en) * | 2015-02-04 | 2015-05-13 | 四川农业大学 | Fluorogenic quantitative PCR kit for detecting swine transmissible gastroenteritis virus |
| CN106435014A (en) * | 2016-08-24 | 2017-02-22 | 广西壮族自治区动物疫病预防控制中心 | Multiple RT-PCR detection kit and primer group of porcine epidemic diarrhea virus virulent strain and vaccine strain |
| CN107475450A (en) * | 2017-09-13 | 2017-12-15 | 河南省农业科学院畜牧兽医研究所 | A kind of primer sets and kit for the strain of Porcine epidemic diarrhea virus novel variant and vaccine strain antidiastole |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108315498A (en) * | 2018-05-02 | 2018-07-24 | 郭庆君 | A kind of primer sets and kit of H3N2 hypotypes swine influenza virus strain and vaccine strain antidiastole |
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Application publication date: 20180724 |