CN108504782A - Primer combination for 4 boar infectious disease viruses of synchronous detection and detection kit - Google Patents
Primer combination for 4 boar infectious disease viruses of synchronous detection and detection kit Download PDFInfo
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Abstract
The invention discloses a kind of primer combination for 4 boar infectious disease viruses of synchronous detection and detection kits.Specifically use four pairs of primers, RT PCR simultaneously and rapidly detects the important deadly infectious disease pathogen with examination pig, including one kind in swine fever virus (Classical swine fever virus, CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), Porcine epidemic diarrhea virus (PEDV) and transmissible gastro-enteritis virus (TGEV) or arbitrary several.Have many advantages, such as good specificity, high sensitivity, take short, synchronous detection and screen, is suitable for the monitoring of pig farm epidemic situation and swine disease quick diagnosis.
Description
Technical field
The invention belongs to the detections of pig infectious disease virus, and in particular to one kind is for 4 boar infectious disease virus of synchronous detection
Primer combination and containing the primer combination detection kit.
Background technology
Swine fever virus (CSFV), pig breathing with reproductive syndrome virus (PRRSV), Porcine epidemic diarrhea virus (PEDV) and
Transmissible gastro-enteritis virus (TGEV) is to cause the important infections of pigs such as swine fever, blue otopathy, infectious diarrhea and gastroenteritis respectively
The pathogen of disease, causes to seriously endanger to pig breeding industry；Swine disease occurs and fashion trend is typically the mixing sense of two or more cause of diseases
Dye, collaboration are caused a disease, and atypical symptoms, harm is more serious, and diagnosis and prevention and control are extremely difficult.Promptly and accurately diagnose infectious disease
Cause of disease is effective prevention and control swine disease, promotes the important prerequisite of pig breeding industry sound development.
Swine fever (CSF), also known as hog cholera (hog cholera, HC) are that pig high degree in contact caused by being infected by CSFV causes
Dead sexually transmitted disease is classified as A class infectious diseases by World Organization for Animal Health (OIE).Swine fever is for the first time in 1810 in U.S.'s Tennessee
(Tennessee) state is broken out, and is then propagated in the whole world, because transmission capacity is strong, morbidity and mortality are high, is caused to aquaculture huge
Big economic loss.Phylogenetic analysis shows that CSFV points are strong virus force strain, mesogenic strains and low no virulent strain (vaccine strain)
Deng three kinds of genotype.The usual via intranasal application of CSFV natural infections, oral cavity, conjunctiva or genital mucosa enter pig body.CSFV infected pigs
The incubation period of body is generally 7-10 days, and in infection 4 days or so, conjunctivitis and Neuroleptic Leukocytopenia etc. occurred in infected pigs；When CSFV is strong
When virulent strain acute infection pig, virus first invades tonsillotome epithelial cell, and blood is entered via regional nodes and capillary
Liquid causes viremia virusemia.The a large amount of proliferation of virus in the organ-tissues such as spleen, lymphoid tissue, internal organ lymph node and bone, finally
Cause infected pigs dead.
Porcine reproductive and respiratory syndrome virus (PRRSV) is the pathogen for causing pig blue-ear disease, because of propagated strong, lethality
Height is one of the important pathogen body for seriously endangering global pig breeding industry.Blue otopathy was found in 1987 in southern US first；According to report
Road, the U.S. lose caused by PRRSV is broadcast to pig breeding industry more than 500,000,000 dollars every year.It is broken out for the first time at the bottom of nineteen ninety-five in China
PRRSV epidemic situations cause huge economic losses then in national spread in china.In 2006, it is sudden and violent that second of PRRSV occurs in China
Hair is popular, causes this time pandemic to be highly pathogenic PRRSV variant, unprecedented harm is caused to pig breeding industry.PRRSV master
To pass through respiratory tract horizontal transmission and genital tract vertical transmission diameter.Cause the one of the major reasons of PRRSV prevalences be more after pig,
Subclinical infection pig and susceptible swinery contact with each other caused cross-infection.More pig and subclinical infection pig pass through mouth, nose, eye, the moon after
The number of ways such as road give viral transmission to susceptible swinery.Mosquito is also the communication media of PRRSV.It is stung again after mosquito bite band poison pig
Susceptible swinery is stung, susceptible swinery can be made to infect.It is two kinds of genotype of I type and II type that PRRSV, which is divided to,.The eruption and prevalence is caused to be mainly
II types of PRRSV；PRRSV genomes often morph, and lead to virulence enhancing and antigenic change.The existing vaccine used is to difference
The PRRSV prevalence strain Vaccine effectiveness in area has differences, and incubation period is long after PRRSV infection, and healing can energy band poison and presence the moon
Sexuality dye is easy spread and epidemic, and early stage, accurately viral diagnosis was particularly closed for effectively preventing and controlling PRRSV spread and epidemics
Key.
Porcine epidemic diarrhea virus (PEDV) causes pig high degree in contact enteric infectious disease, has the spy of seasonal outburst
Point was most found earlier than 1978 in Britain.Hereafter PEDV is found in countries such as Germany, Belgium, China, Japan and South Korea.
PEDV belongs to coronavirus, and incubation period, pathological change and the disease degree of infected pigs are because there are significant differences for pig age difference.New newborn
The incubation period of pig is usually 24-36 hours, and growing and fattening pigs are then generally 48 hours or more.After infecting PEDV, pig clinical manifestation is to vomit
It spits, diarrhea, water sample loose stools, lassitude, is become thin at apocleisis, and serious dehydration death is eventually led to.7 age in days piglets are generally occurring
2-4 days internal cause serious dehydrations after diarrhea are dead, and the death rate is up to 50% or more；Sow is cashed as spirit not after infecting PEDV
It shakes, apocleisis, persistent diarrhea, can restore normal after a week, but lactation amount is caused to decline.Growing and fattening pigs show as abdomen after infecting PEDV
It rushes down, the reduction of weight loss, capacity usage ratio, most of pigs restore normally after a week in diarrhea, the death rate about 1%-3%.China
It was separated to PEDV for the first time in 1980；Spring in 2011, southern region of China broke out PEDV epidemic situations, sick pig occur diarrhea,
The symptoms such as vomiting, because dehydration is dead after 1-2 days, the selected swine farms pig death rate causes huge economic losses up to 100%.Due to
PEDV constitutes a serious threat to China's pig breeding industry, and vaccine inoculation preventive effect is still not ideal enough.Accurately early diagnoses and take
Counter-measure, it is popular particularly necessary for effective prevention and control PEDV diffusions.
Transmissible gastro-enteritis virus (TGEV) infection causes pig acute infective gastrointestinal tract scorching.TGEV is most earlier than nineteen forty-six
It is found in the U.S..China found pig acute infective stomach and intestine caused by TGEV in succession in 1956 on Guangzhou Jieyang, Huizhou and other places
It is scorching.Pig is the natural reservoir (of bird flu viruses) of TGEV, and the piglet for being less than 10 ages in days is particularly susceptible to TGEV, and lethality is up to 100%；TGEV infects
Pig body lethalities more than 5 week old is relatively low, but infected pigs' efficiency of feed utilization is low, and growth retardation becomes cad pig, seriously affects pig raising
Benefit.TGEV also belongs to coronavirus.Clinical symptoms are similar caused by bis- kinds of viral infected pigs of TGEV and PEDV, and are often mixed
Infection to the accurate early detection of the two and is screened particularly important to diarrhea of pigs disease prevention and cure.
The common method of detection swine disease virus includes at present：Virus Isolation, immunology detection and molecular biology inspection
Survey etc..Virus Isolation is to detect the goldstandard of virus causing disease, but this method will carry out cell culture, virus infection, be immunized
Fluorescent staining, requires experiment condition and personnel's technology that high, complicated for operation, time-consuming, it is difficult to fast for clinic swine disease
Speed diagnosis；Immunology detection usually uses neutralization test or enzyme-linked immunosorbent assay detection serum specific antibody or virus anti-
It is former.It needs just to can induce within 2-3 weeks body generation special viral antibody after infecting due to virus, viral antigen concentration is relatively low, exempts from
Epidemiology detection is not suitable for Rapid＆Early diagnosis；The complexity such as cell culture, virus infection and detection will also be carried out by neutralizing experiment
Step.Using the molecular biology method of viral nucleic acid in detecting viral nucleic acid as target, directly detection sample, has and need sample
It measures small, detection and takes the advantages that short, sensibility is high, high specificity, have a wide range of applications in swine disease diagnosis.Viral nucleic acid is examined
It surveys and mainly uses PCR (or RT-PCR) and quantitative fluorescent PCR (qPCR) method etc..Currently, existing application PCR or qPCR detections
The report of CSFV, PRRSV, PEDV or TGEV genomic nucleic acids, but these methods are typically to detect one or both disease in sample
Original needs repeated detection for virus mixed infection, exists and needs more sample size, time-consuming length, heavy workload and of high cost etc. lack
It falls into, is promoted and applied in clinic early diagnoses difficult.
Invention content
To improve RT-PCR detection efficiencies, testing cost, these full bases of virus that the present invention is issued according to Genbank are reduced
Because of the conserved sequence of group or partial sequence, universal primer is designed, it is intended to establish a simple, efficient, sensitive quadruple RT-PCR
Detection technique, for quickly detection and screen cause of disease CSFV, PRRSV, PEDV that 4 kinds cause the important potent virus infectious disease of pig and
TGEV provides technical support.
In order to solve the above-mentioned technical problem, the technical solution adopted in the present invention is as follows：
In a first aspect, a kind of primer combination synchronizing 4 boar infectious disease viruses of detection for quadruple RT-PCR is provided, it is described
Primer combination includes the primer pair I of specific amplification swine fever virus (CSFV), the breathing of specific amplification pig and reproductive syndrome disease
The primer pair II of malicious (PRRSV), the primer pair III of specific amplification Porcine epidemic diarrhea virus (PEDV) and specific amplification pig
The primer pair IV of infectious gastroenteritis virus (TGEV), the nucleotide sequence of each primer pair and the segment for corresponding virus amplification
Size such as table 1.
The primer of 1 quadruple RT-PCR detection pig infectious disease viruses of table and its virus of detection
Second aspect, the present invention provide the examination for 4 boar infectious disease viruses of synchronous detection containing the combination of above-mentioned primer
Agent box.Preferably, the kit further include dNTPs, Taq archaeal dna polymerase, one kind in Mg2+, PCR reaction buffer or
It is a variety of.It is highly preferred that the kit further includes standard positive template.
The present invention also provides detect using the combination of above-mentioned primer or detection kit and screen 4 kinds to cause the important strong disease of pig
The method of cause of disease CSFV, PRRSV, PEDV and TGEV of malicious infectious disease, this method are：
1) RNA of extraction detection sample；
2) RNA obtained using step 1), by reverse transcription, obtains cDNA as template；
3) cDNA obtained using step 2) is template, and PCR amplification is carried out to it simultaneously with 4 pairs of primers, wherein 4 pairs of primers
Nucleotide sequence is as shown in Seq ID No.1-8；
4) PCR product gel electrophoresis judges Virus Infection according to target fragment size and positive control；
5) quality control standard：
Negative quality-control product：Without amplified production；
Positive quality control product：All 4 kinds of Virus Standard templates energy specific amplification, band and expected size are completely the same,
Otherwise, this time experiment is considered as invalid.
Preferably, the composition of the inverse transcription reaction liquid (cDNA synthetic systems) of step 2)：5 × RT Buffer, dNTPs
1mM (Invitrogen), M-MLV enzymes 200U (Invitrogen), d (N)9100ng/ μ L, RiboLock RNase
Inhibitor(40U/μL)；The amplification program of reverse transcription is：37 DEG C first reaction 60min, then 95 DEG C of processing 10min, reaction knot
Shu Hou, reaction solution are cDNA templates.
Preferably, the reaction system of step 3) PCR amplification is 1 μ l of 19 μ l of reaction solution and viral cDNA template, overall reaction body
Product is 20 μ l, the composition of the PCR reaction solution：1 × PCR Buffer, Mg2+2mM, dNTPs 0.2mM, Taq archaeal dna polymerase
2.5U, each 0.2 μM of 4 pairs of detection primers；PCR amplification program is：94 DEG C of pre-degeneration 5min；94 DEG C of 30sec, 55 DEG C of 30sec, 72 DEG C
45sec expands 35 cycles, and 72 DEG C of whole ends extend 5min.
Preferably, the positive quality control product is that CSFV, PRRSV, PEDV, TGEV genome are guarded target segment to be cloned into
The recombinant plasmid of pGEM-T vector constructions, is named as pT-CSFV, pT-PRRSV, pT-PEDV and pT-TGEV successively, through 103It is dilute
Release preparation；The feminine gender quality-control product is Sample preservation liquid.
The main technical principle of the present invention is, CSFV, PRRSV, PEDV and TGEV genome are being reported to GenBank
On the basis of sequence alignment analysis, optimization design is directed to 4 kinds of conserved viral target sequence (Seq No respectively:4 pairs of specificity 9-12)
PCR primer；Virus genome RNA is extracted as template using Trizol methods, is added in reverse transcription reaction liquid, in reverse transcription
CDNA templates are synthesized under enzyme effect；CDNA templates are added in PCR reaction solution, synthetic primer specific target segment is expanded.PCR
Amplification makes template nucleic acid be in geometry multiplication by stages, and by agarose gel electrophoresis, extremely micro viral RNA can in clinical sample
It is detected.By optimizing reaction system and PCR amplification condition, 4 pairs of detection primer single tube synchronized specific amplifications are realized.Into
On the basis of the pattern detection verification of row pig farm, provide a kind of for CSFV, PRRSV, PEDV and the viral synchronous inspections of 4 kinds of TGEV
The easy quick diagnosis reagent kit surveyed and screened.
Compared with the prior art, the present invention has the following advantages：
A) single tube synchronized detection and antidiastole can be carried out to 4 kinds of viruses of CSFV, PRRSV, PEDV and TGEV, wherein
Including three kinds of genotype such as the strain of CSFV strong virus forces, mesogenic strains and low no virulent strain (vaccine strain), I types of PRRSV and II type two
Kind genotype is suitable for pig farm epidemic monitoring and disease transmission season to sick pig clinic rapid differential diagnosis；More common detection
The individual event RT-PCR detection efficiencies of single virus improve 4 times.
B) high sensitivity, specificity are good.When detecting sample, virus genome RNA reverse transcription is synthesized into cDNA, then carry out spy
Specific primer PCR amplification can get high copy PCR product.Therefore, this method detection sensitivity is high；By the way that positive quality control is arranged
Product and negative quality-control product, improve the accuracy of detection.Compared to the cross reaction that immunology detection is likely to occur, spy is improved
The opposite sex reduces the generation of false positive.
C) quick, cheap.Pattern detection can be completed in or so 4 hours；Since disposable pcr amplification reaction can detect that
One or more viral mixed infections of any list in tetra- kinds of viruses of CSFV, PRRSV, PEDV and TGEV, have time saving, province in sample
Power and it is cheap outstanding advantages of, be suitable for extensive sample it is quick detect and viruses indentification.
Description of the drawings
The synchronous detection of 4 kinds of viruses such as Fig. 1 CSFV, PRRSV, PEDV and TGEV and single disease in examination kit detection sample
RT-PCR amplified productions existing for poison are observed
M, Marker；1, CSFV (Virus Sample)；2, pT-CSFV (positive quality control products)；3, PRRSV (Virus Samples)；4,
PT-PRRSV (positive quality control product)；5, PEDV (Virus Samples)；6, pT-PEDV (positive quality control products)；7, TGEV (Virus Samples)；
8, pT-TGEV (positive quality control products)；9, negative quality-control product
4 kinds and 2 in the synchronous detection of 4 kinds of viruses such as Fig. 2 CSFV, PRRSV, PEDV and TGEV and examination kit detection sample
RT-PCR amplified productions observation existing for kind various combination virus
M, Marker；1, CSFV/PRRSV；2, CSFV/PEDV；3, CSFV/TGEV；4, PRRSV/PEDV, 5, PRRSV/
TGEV；6, PEDV/TGEV；7, CSFV/PRRSV/PEDV/TGEV
4 kinds and 3 in the synchronous detection of 4 kinds of viruses such as Fig. 3 CSFV, PRRSV, PEDV and TGEV and examination kit detection sample
RT-PCR amplified productions observation existing for kind various combination virus
M, Marker；1, CSFV/PRRSV/PEDV；2, CSFV/PRRSV/TGEV；3, CSFV/PEDV/TGEV；4,
PRRSV/PEDV/TGEV；5, CSFV/PRRSV/PEDV/TGEV
The detection sensitivity of the viral synchronous detection reagent kits of 4 kinds of Fig. 4 CSFV, PRRSV, PEDV and TGEV etc.
M, Marker；A, CSFV；B, PRRSV；C, PEDV；D, TGEV；1-6 is that corresponding viral nucleic acid qRT-PCR is quantitative
Copy number：1,106Copy/μ L；2,105Copy/μ L；3,104Copy/μ L；4,103Copy/μ L；5,102Copy/μ L；6,101It copies
Shellfish/μ L；N, negative control
Specific implementation mode
By following detailed description combination attached drawing it will be further appreciated that the features and advantages of the invention.The implementation provided
Example is only the explanation to the method for the present invention, remaining content without limiting the invention in any way announcement.
In the following examples, the experimental methods for specific conditions are not specified, usually according to Joseph Sambrook et al.《Molecule
Cloning experimentation guide》The third edition (New York:Cold Spring Harbor Laboratory Press, 1989) described in
Condition.
【Embodiment 1】4 kinds of infectious disease viral nucleic acids of pig simultaneously and rapidly detect and screen composition and the preparation of kit
1. reagent forms：Trizol RNA lysates, 5 × RT Buffer, dNTPs, M-MLV enzyme 200U, d (N)9
100ng/ μ L, RiboLock RNase Inhibitor (40U/ μ L) are Invitrogen Products；Taq archaeal dna polymerases
2.5U (Dongsheng Biotech) Co., Ltd's product；Detection primer：Tetra- kinds of virus PCRs of CSFV, PRRSV, PEDV, TGEV
Primer equal proportion blending ingredients；Reverse transcriptase primer：Random primer d (N)9Shanghai Sheng Gong bio-engineering corporations product；CSFV、
Tetra- kinds of virus-positive quality-control product plasmids of PRRSV, PEDV, TGEV are built by inventor laboratory and are preserved；
PCR primer sequence is as follows：
CSFV forward primer sequences：5'GCTCCCTGGGTGGTCTAAGTC 3'
Reverse primer sequences：5'GGGTTAAGGTGTGTCTTGGGC 3'
PRRSV forward primer sequences：5'ACCTCCAGATGCCGTTTGTG 3'
Reverse primer sequences：5'GCTTTTCTGCCACCCAACAC 3'
PEDV forward primer sequences：5'GGTGTCAAGATGGCTATTCTATGG 3'
Reverse primer sequences：5'TGAAGCATTGACTGAACGACCA3'
TGEV forward primer sequences：5'GACAAACTCGCTATCGCATGG 3'
Reverse primer sequences：5'AGTGGTATTTGTGTGTGAACGTGA3'
2. preparation of reagents：
A) RNA lysates：Trizol RNA lysates (100ml/ bottles) are purchased from Invitrogen companies；
B) reverse transcription reaction liquid：5 × RT Buffer, dNTPs 1mM (Invitrogen companies), M-MLV enzymes 200U
(Invitrogen), d (N)9100ng/ μ L, RiboLock RNase Inhibitor (40U/ μ L)；
C) PCR reaction solution：10 × PCR buffer, MgCl22mM, dNTP 0.2mM, Taq archaeal dna polymerase 2.5U
(Dongsheng Biotech), each 0.2 μM of 4 pairs of detection primers；
D) positive quality control product：CSFV, PRRSV, PEDV, TGEV conservative fragments are cloned into pGEM-T vector constructions recombination matter
Grain, is named as pT-CSFV, pT-PRRSV, pT-PEDV and pT-TGEV, through 10 successively3It is prepared by dilution；
E) negative quality-control product：Sample preservation liquid.
F reagent) is provided for oneself：Chloroform, isopropanol, DEPC water, 75% ethyl alcohol prepared with DEPC water.
3. positive recombinant plasmid is built：
Structure for standard plasmid pT-CSFV：
CSFV 5'UTR F：5'GTATACGAGGTTAGTTCATTCTCG 3'
CSFV 5'UTR R：5'GTGCCATGTACAGCAGAGATT 3'
Structure for standard plasmid pT-PRRSV：
PRRSV M F：5'ATGGGGTCGTCTCTAGACGAC3'
PRRSV M R：5'TTATTTGGCATATTTAACAAGGTTT3'
Structure for standard plasmid pT-PEDV：
PEDV M 95F：5'GCTTCAGTATGGCCATTACAAGTAC3'
PEDV M 681R：5'TTTGTCATTAGTAACCCTAAGAGGG3'
Structure for standard plasmid pT-TGEV：
TGEV N 102F：5'CATACCTCTTTCATTCTTCAACCC3'
TGEV N 1134R：5'AACCTGTGTGTCATCAAACACATC3'
Reverse transcriptase primer is d (N)9
All primers are synthesized by Shanghai Sangon Biotech Company.
【Embodiment 2】4 kinds of infectious disease viral nucleic acids of pig simultaneously and rapidly detect and screen kit application method
1. sample process
The sample that sick sample detection is related to includes respiratory tract sample (nose/throat swab, phlegm, nasopharyngeal aspirates, bronchovesicular
Irrigating solution etc.), anal swab, excrement, blood and tissue (liver, kidney, spleen, lymph node, tonsillotome) etc..
It, will if sample liquid contains a small amount of mucus (nose/throat swab, nasopharyngeal aspirates, BAL fluid, blood etc.)
Sample sets in centrifuge 4 DEG C, and 2000rpm centrifuges 20min, to remove impurity.After centrifugation, centrifugation is lightly opened in safety cabinet
It manages, is preserved in Aspirate supernatant to EP pipes, extracted for RNA.
If sample is anal swab and excrement, need suitably to be diluted with PBS buffer solution, in 4 DEG C in centrifuge,
2000rpm centrifuges 20min, removes impurity, centrifuge tube is lightly opened in safety cabinet, is preserved in Aspirate supernatant to EP pipes,
It is extracted for RNA.
If sample is tissue samples, need to be ground tissue, PBS bufferings appropriate are added in process of lapping
Liquid, for lapping liquid in 4 DEG C in centrifuge, 2000rpm centrifuges 20min, removes impurity, centrifuge tube is lightly opened in safety cabinet,
It preserves in Aspirate supernatant to EP pipes, is extracted for RNA.
2. the extraction of tested sample rna
It is test sample number to take the 1.5ml Eppendorf centrifuge tubes of n sterilizing, wherein n；Often pipe is separately added by sample
100 μ L of product are added 1000 μ L Trizol sample lysates, mix well, and stand 5min；200 μ L chloroform (Trizol bodies are added
Long-pending 1/5), gently after mixing, in being stored at room temperature 5min；4 DEG C of 12000g centrifuge 15min；Supernatant is drawn to another centrifugation, is added
Isometric isopropanol, after vibrating mixing, in being stored at room temperature 10min, 4 DEG C of 12000g centrifuge 10min, are abandoned with pipettor suction
Clearly；75% ethyl alcohol (being prepared with DEPC processing water dilutions) of 1mL precoolings is added in precipitation, 4 DEG C of 7500g centrifuge 5min；Use liquid relief
Supernatant is abandoned in device suction, and tube bottom precipitation is placed in air drying 10min, and precipitation gradually becomes translucent by white, and 12.5 μ L are added and go out
The DEPC water dissolutions of bacterium as detect the RNA of sample.
3. reverse transcription reaction synthesizes cDNA
The 12.5 μ L of RNA obtained by 2 steps are taken, 1 μ L random primers d (N) are added9, 70 DEG C are incubated 5min, set rapidly on ice
5 × RT Buffer, 41 μ L, M-MLV enzymes 200U of μ L, dNTPs, 1 μ L, RiboLock RNase Inhibitor are added in 2min
(40U/ μ L) 0.5 μ L, 37 DEG C of 60min, 95 DEG C of 10min.Reaction terminates to obtain cDNA templates.
4.PCR reacts
N branch PCR reaction tubes are taken, often 19 μ L of PCR reaction solution are added in pipe, take each 1 μ of the cDNA template solutions obtained by 3 steps successively
L is separately added into corresponding PCR reaction tubes, pcr amplification reaction is carried out on PCR amplification circulating instrument.Cycling condition is：94℃
Pre-degeneration 5min；94 DEG C of 30sec, 55 DEG C of 30sec, 72 DEG C of 45sec expand 35 cycles, and 72 DEG C of whole ends extend 5min.Then it uses
2% agarose electrophoresis detects PCR results.
5. result judges
PCR amplification after circulation terminates, carries out 2% agarose gel electrophoresis, amplified production band is observed in Ultraviolet Detector
Quantity and size；
6. quality control standard
Negative quality-control product：Without amplified production；
Positive quality control product：All 4 kinds of Virus Standard templates energy specific amplification, band and expected size are completely the same,
Otherwise, this time experiment is considered as invalid.
7. result judgement
Negative findings judge：Without band；
Positive findings judge：There is band, is judged through being compared with positive quality control product amplified production according to stripe size, quantity
Contained viral species in sample.
The result is shown in Figure 1-3.
【Embodiment 3】4 kinds of infectious disease viral nucleic acids of pig simultaneously and rapidly detect the application with examination kit
1. sensitivity test
The standard items (10 being serially diluted again with tetra- kinds of viruses 10 of CSFV, PRRSV, PEDV, TGEV6~101Copy/μ L) be
Template is added in detection kit PCR reaction solution, and amplification reaction condition when by pattern detection carries out expanding in PCR instrument anti-
It answers, pcr amplification product is analyzed (Fig. 4) through 2% agarose gel electrophoresis.
2. specific test
Using 4 kinds of viruses such as CSFV, PRRSV, PEDV and TGEV simultaneously and rapidly detection kit to RSV, H5N1, BVDV and
PCV2 viruses carry out specific test, and the control of negative and positive quality control product, all non-target viral diagnosis results are feminine gender, sun
The property purposeful band of quality-control product.Result above proves that the kit has specificity well in the detection of clinical sample.
3. repetitive test
With a kind in 4 kinds of Virus Samples of identical titre, arbitrary 2 kinds of mixing, arbitrary 3 kinds of mixing and 4 kinds of mixing, extraction
RNA carries out repeating to detect 3 times respectively, and 3 times detection amplified production pillar location is consistent with brightness；It is mixed with 4 kinds of viruses of different titers
Conjunction carry out repeat detect 3 times, 3 times detection amplified production pillar location it is consistent with brightness, it was demonstrated that kit of the invention be used for into
The synchronous detection of 4 kinds of viruses of row has repeatability well.
Sequence table
<110>Wuhan University
<120>Primer combination for 4 boar infectious disease viruses of synchronous detection and detection kit
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
gctccctggg tggtctaagt c 21
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
gggttaaggt gtgtcttggg c 21
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
acctccagat gccgtttgtg 20
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
gcttttctgc cacccaacac 20
<210> 5
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
ggtgtcaaga tggctattct atgg 24
<210> 6
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
tgaagcattg actgaacgac ca 22
<210> 7
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
gacaaactcg ctatcgcatg g 21
<210> 8
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
agtggtattt gtgtgtgaac gtga 24
<210> 9
<211> 116
<212> DNA
<213>Swine fever virus (Classical swine fever virus)
<400> 9
gctccctggg tggtctaagt cctgagtaca ggacagtcgt cagtagttcg acgtgagcag 60
aagcccacct cgagatgcta tgtggacgag ggcatgccca agacacacct taaccc 116
<210> 10
<211> 197
<212> DNA
<213>Pig breathes and reproductive syndrome virus (Porcine respiratory and reproductive
syndrome virus )
<400> 10
acctccagat gccgtttgtg cttgctaggc cgcaagtaca ttctggcccc tgcccaccac 60
gtcgaaagtg ccgcgggctt tcatccgatt gcggcaaatg ataaccacgc atttgtcgtc 120
cggcgtcccg gctccactac ggtcaacggc acattggtgc ccgggttgaa aagcctcgtg 180
ttgggtggca gaaaagc 197
<210> 11
<211> 435
<212> DNA
<213>Porcine epidemic diarrhea virus (porcine epidemic diarrhea virus)
<400> 11
ggtgtcaaga tggctattct atggatactt tggcctcttg tgttagcact gtcacttttt 60
gatgcatggg ctagctttca ggtcaattgg gtcttttttg ctttcagcat ccttatggct 120
tgcatcactc ttatgctgtg gataatgtac tttgtcaata gcattcggtt gtggcgcagg 180
acacattctt ggtggtcttt caatcctgaa acagacgcgc ttctcactac ttctgtgatg 240
ggccgacagg tctgcattcc agtgcttgga gcaccaactg gtgtaacgct aacactcctt 300
agtggtacat tgcttgtaga gggctataag gttgctactg gcgtacaggt aagtcaatta 360
cctaatttcg tcacagtcgc caaggccact acaacaattg tctacggacg tgttggtcgt 420
tcagtcaatg cttca 435
<210> 12
<211> 720
<212> DNA
<213>Transmissible gastro-enteritis virus (transmissible gastro-enteritis virus)
<400> 12
gacaaactcg ctatcgcatg gtgaagggcc aacgtaaaga gcttcctgaa aggtggttct 60
tctactactt aggtactgga cctcatgcag atgccaaatt taaagataaa ttagatggag 120
ttgtctgggt tgccaaggat ggtgccatga acaaaccaac cacgcttggt agtcgtggtg 180
ctaataatga atccaaagct ttgaaattcg atggtaaagt gccaggcgaa tttcaacttg 240
aagttaacca gtcaagggac aattcaaggt cacgctctca atctagatct cggtctagaa 300
acagatctca atctagaggc aggcaacaat ccaataacaa gaaggatgac agtgtagaac 360
aagctgttct tgccgcactt aaaaagttag gtgttgacac agaaaaacaa cagcaacgtt 420
ctcgttctaa atctaaagaa cgtagtaact ctaaaacaag agatactacg cctaagaatg 480
aaaacaaaca cacctggaag agaactgcag gtaaaggtga tgtgacaaga ttttatggag 540
ctagaagcag ttcagccaat tttggtgaca gtgacctcgt tgccaatggg agcagtgcca 600
agcattaccc acaattggct gaatgtgttc catctgtgtc tagcattttg tttggaagct 660
attggacttc aaaggaagat ggcgaccaga tagaagtcac gttcacacac aaataccact 720
<210> 13
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 13
gtatacgagg ttagttcatt ctcg 24
<210> 14
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 14
gtgccatgta cagcagagat t 21
<210> 15
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 15
atggggtcgt ctctagacga c 21
<210> 16
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 16
ttatttggca tatttaacaa ggttt 25
<210> 17
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 17
gcttcagtat ggccattaca agtac 25
<210> 18
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 18
tttgtcatta gtaaccctaa gaggg 25
<210> 19
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 19
catacctctt tcattcttca accc 24
<210> 20
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 20
aacctgtgtg tcatcaaaca catc 24
Claims (4)
1. a kind of primer combination synchronizing 4 boar infectious disease viruses of detection for quadruple RT-PCR, which is characterized in that the primer
Combination includes the primer pair I of specific amplification swine fever virus (CSFV), the breathing of specific amplification pig and reproductive syndrome virus
(PRRSV) the primer pair III of primer pair II, specific amplification Porcine epidemic diarrhea virus (PEDV) and specific amplification pig
The primer pair IV of infectious gastroenteritis virus (TGEV)；
Primer pair I：
CSFV upstream primer sequences：5' GCTCCCTGGGTGGTCTAAGTC 3'（Seq ID No.1）
Downstream primer sequence：5' GGGTTAAGGTGTGTCTTGGGC 3'（Seq ID No.2）；
Primer pair II：
PRRSV upstream primer sequences：5'ACCTCCAGATGCCGTTTGTG 3'（Seq ID No.3）
Downstream primer sequence：5'GCTTTTCTGCCACCCAACAC 3'（Seq ID No.4）；
Primer pair III：
PEDV upstream primer sequences：5'GGTGTCAAGATGGCTATTCTATGG 3'（Seq ID No.5）
Downstream primer sequence：5'TGAAGCATTGACTGAACGACCA 3'（Seq ID No.6）；
Primer pair IV：
TGEV upstream primer sequences：5'GACAAACTCGCTATCGCATGG 3'（Seq ID No.7）
Downstream primer sequence：5'AGTGGTATTTGTGTGTGAACGTGA3'（Seq ID No.8）.
2. containing the kit for 4 boar infectious disease viruses of synchronous detection that primer described in claim 1 combines, feature exists
In the 4 boar infectious disease viruses refer to that swine fever virus (CSFV), pig breathing and reproductive syndrome virus (PRRSV), pig are flowed
Row diarrhea virus (PEDV) and transmissible gastro-enteritis virus (TGEV).
3. kit according to claim 2, which is characterized in that the kit further includes dNTPs, Taq DNA polymerization
Enzyme, Mg2+, it is one or more in PCR reaction buffers.
4. kit according to claim 2 or 3, which is characterized in that the kit further includes standard positive template.
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| CN109762931A (en) * | 2018-09-19 | 2019-05-17 | 天津瑞普生物技术股份有限公司 | Primer, probe and the method for transmissible gastro-enteritis virus fluorescence quantitative RT-RCR detection |
| CN112779352A (en) * | 2019-11-11 | 2021-05-11 | 中国动物疫病预防控制中心(农业农村部屠宰技术中心) | Double digital PCR detection technology for swine fever and African swine fever and special kit thereof |
| CN113801922A (en) * | 2021-09-13 | 2021-12-17 | 青岛农业大学 | On-site rapid high-sensitivity differential diagnosis kit for porcine diarrhea virus pathogens and use method thereof |
| CN114015814A (en) * | 2021-12-17 | 2022-02-08 | 广西壮族自治区动物疫病预防控制中心 | Microdroplet digital PCR kit for ASFV, CSFV and PRRSV and detection method thereof |
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| CN113801922A (en) * | 2021-09-13 | 2021-12-17 | 青岛农业大学 | On-site rapid high-sensitivity differential diagnosis kit for porcine diarrhea virus pathogens and use method thereof |
| CN114015814A (en) * | 2021-12-17 | 2022-02-08 | 广西壮族自治区动物疫病预防控制中心 | Microdroplet digital PCR kit for ASFV, CSFV and PRRSV and detection method thereof |
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