CN109097489A - A kind of the duplex RT-PCR detection primer and kit of quick differentiation TGEV and PReoV - Google Patents
A kind of the duplex RT-PCR detection primer and kit of quick differentiation TGEV and PReoV Download PDFInfo
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Abstract
The invention discloses a kind of duplex RT-PCR detection primers and kit for quickly distinguishing TGEV and PReoV, belong to animal virology and technical field of molecular biology.The kit includes primer sequence shown in ID1 ~ 4 SEQ, further comprises PrimeSTAR HR buffer and aqua sterilisa, cDNA template and aqua sterilisa.The duplex RT-PCR detection kit of TGEV and PReoV provided by the invention are expanded using duplex RT-PCR, two kinds of lesions are similar and are all the viral one-time detection of RNA virus, total time control is detected at 2 hours or so, detection method is easily operated, convenient and efficient, can achieve the purpose that quickly to detect.
Description
Technical field
The invention belongs to animal virologies and technical field of molecular biology, are specially directed to the double RT- of TGEV and PReoV
Detection primer, detection kit and the application of PCR.
Background technique
Transmissible gastro-enteritis virus (porcine transmissible gastroenteritis, TGEV) belongs to coronal
Viraceae coronavirus genus member.Cause with fever, vomiting, dehydration, severe diarrhea, a height of main clinical characteristics of piglet lethality
A kind of acute, high degree in contact enterogastric diseases, transmissible gastroenteritis of swine.The pig at various ages can infection morbidity, and 10
The piglet death rate within age in days is up to 100%.Although the death rate is very low after pig infection more than 5 week old, these pigs are logical
Often become cad pig, the price of deed is low, brings heavy losses to pig breeding industry.The U.S. reports the disease in nineteen forty-six for the first time, then multiple
Countries and regions are reported in succession the occurrence of this disease and prevalence.Late 1950s, China's report have the occurrence of this disease, arrive mesh
Before until China most province (city) have the generation of the disease and popular, and the disease there is no effective therapeutic agent, to pig raising
Industry causes great puzzlement.
The 1950s reovirus discovery for the first time, it was confirmed that dsRNA virus can be used as stable life shape
Formula is present in nature.Reovirus (Reovirus) is Reoviridae (Reoviridea) Orthoreovirus
(Orthoreovirus) member's main infection people and mammal, such as pig, ox, dog, cat, mouse and fowl respiratory tract or disappear
Change road, has to animal extensive pathogenic.
Pig reovirus (Porcine reovirus, PReoV) belongs to Reoviridae Orthoreovirus member,
It is one kind of mammalian reovirus (Mammalian Reovirus, MRV), is segmented diplornavirus, no capsule
Film has characteristic 20 face body symmetrical double layer capsid structure, and complete virion diameter is 76nm or so, and virus is through mouthfeel
It contaminates or through respiratory tract infection.The PReoV reported at present shares 3 serotypes, wherein 1 type is by Kasza and McFerran in 1970
Year, separation was reported respectively；3 types are reported by Robl in separation in 1971；2 types were separated in 1996 in Japan for the first time by Fukutomi
Report；The antibody of 3 kinds of serotype nearly all can be detected in all pigs.China Zeng Zhiyong 2006 is at home for the first time out of pig body
Be isolated to 3 types feed MRV, SC-A plants；Then, one plant of 3 type MRV, MRV-HLJ/2007 are also isolated within Zhang etc. 2007
Strain；45 part of 3 type MRV positive has been detected within Kwon etc. 2011 from regional 78 237 parts of the pig farm diarrhea samples of Korea S, and (recall rate reaches
19%), and from positive pathological material of disease it is isolated to 10 plant of 3 type MRV；It is separated to the U.S. within Narayanappa etc. 2015
PReoV infects newborn piglet, and severe diarrhea occurs in discovery piglet, and the death rate is up to 100%.From with respiratory disease or
The pig of enterogastric diseases, the pig of clinical health, aborted fetus etc. all separate reovirus.In addition, in healthy pig groups
Also there can be pig reovirus.The body temperature raising and diarrhea of pig can be caused by not eating colostrum newborn piglet using PReoV infection
Etc. symptoms.Above-mentioned report explanation, PReoV is popular extensive in swinery, and has a degree of pathogenic effects to pig.
Since 2010, the generation of intractable grice diarrhoea and prevalence are in lasting ascendant trend, it has also become lose most on pig farm
For important one of reason, wherein especially the most serious with the harm of virus diarrhea.And TGEV and PReoV are to cause piglet
The main pathogen of virus diarrhea.Clinically only there is diarrhea of pigs symptom, and when without other performances, it is difficult to differentiation be by TGEV also
It is the diarrhea that PReoV causes pig, or causes the diarrhea of pig by TGEV and PReoV mixed infection, it is therefore necessary to establishes
TGEV and PReoV duplex RT-PCR detection method, for clinically timely and accurately to the diarrhea of pigs cause of disease carry out investigation and monitoring mention
For technical support, thus the effectively generation of prevention and control diarrhea of pigs disease and prevalence.
Summary of the invention
Present invention aim to address above-mentioned technical problems, provide a kind of double RT- for quickly distinguishing TGEV and PReoV
PCR detection primer and kit, the kit can quickly distinguish TGEV and PReoV, sensitive sense height, high specificity, to be
The generation of effective prevention and control TGEV and PReoV virosis and popular offer technical support.Skill used in purpose to realize the present invention
Art scheme are as follows:
A kind of duplex RT-PCR detection primer of quick differentiation TGEV and PReoV, the duplex RT-PCR detection primer include
TGEV RT-PCR detection primer and PReoV RT-PCR detection primer；The TGEV RT-PCR detection primer is by with SEQ ID
The upstream primer of nucleotide sequence shown in NO:1 and with nucleotide sequence shown in SEQ ID NO:2 downstream primer composition；Institute
PReoV RT-PCR detection primer is stated as the upstream primer with nucleotide sequence shown in SEQ ID NO:3 and there is SEQ ID
The downstream primer of nucleotide sequence shown in NO:4 forms.
Preferably, the TGEVRT-PCR detection primer is that the full genome of foundation TGEV is designed, the purpose piece of amplification
Duan great little is 277bp；The PReoV RT-PCR detection primer is that the S2 gene of PReoV is designed, the target fragment of amplification
Size is 974bp.
Preferably, the duplex RT-PCR detection primer is in preparation transmissible gastro-enteritis virus and pig reovirus mirror
The application of other detection kit.
The present invention also provides a kind of duplex RT-PCR detection kit for quickly distinguishing TGEV and PReoV, the double RT-
PCR detection kit includes having the upstream primer of nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3 and having
The downstream primer of nucleotide sequence shown in SEQ ID NO:2, SEQ ID NO:4.
Preferably, the duplex RT-PCR detection kit further includes PrimeSTAR HR buffer, cDNA template and goes out
Bacterium water.
Preferably, in the duplex RT-PCR detection kit, including RT reaction system and PCR reaction system；It is described
RT reaction system are as follows: use 20 μ L systems, 14 μ L, 5 × PrimeScript TM buffer of RNA template, 4 μ L, RT Enzyme
1.0 μ L of Mix 1.0 μ L, reverse transcription primer Oligo18T；The PCR reaction system are as follows: use 25 μ L systems, 7.5 μ of aqua sterilisa
L, PrimeSTAR HR (Premix) 12.5 μ L, 25 μm of each 0.5 μ L of ol/L TGEV and PReoV upstream and downstream primer, 3.0 μ L of template.
Preferably, the optimum annealing temperature of the duplex RT-PCR detection kit is 60 DEG C.
Preferably, RT reaction condition in the duplex RT-PCR detection kit are as follows: 37 DEG C of 20min, 85 DEG C of 30s；PCR
Reaction condition are as follows: 98 DEG C of initial denaturation 3min；98 DEG C of denaturation 10s, 60 DEG C of renaturation 5s, 72 DEG C of extension 1min, 35 recycle；Last 72
DEG C extend 10min.
Preferably, the SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ of the RT-PCR detection kit
Primer shown in ID NO:4 is 25 μm of ol/L using concentration.
The invention has the benefit that
The duplex RT-PCR detection kit of TGEV and PReoV provided by the invention are expanded using duplex RT-PCR, two kinds of lesions
Viral one-time detection that is similar and being all RNA virus, the control of detection total time at 2 hours or so, detection method is easily operated,
It is convenient and efficient, it can achieve the purpose that quickly to detect.The present invention can thoroughly solve TGEV symptom similar with caused by PReoV but cause of disease
Different diagnosis problems is suitble to pig farm quickly to detect, and is also suitable for epidemiological survey.
The present invention provides the specific primer that two pairs are directed to TGEV and PReoV, TGEV-P1/TGEV-P2 is that TGEV is special
Property amplimer group, PReoV-P1/PReoV-P2 is PReoV specificity amplification primer group, utilizes duplex RT-PCR made of it
Detection kit can quickly, specifically detect the hybrid virus of single virus and the two in TGEV and PReoV, and TGEV goes out
There is mono- band of 974bp in existing mono- band of 277bp, PReoV, two kinds of virus mixing samples of TGEV, PReoV occur 277bp,
Two bands of 974bp.
The duplex RT-PCR detection kit of TGEV and PReoV of the invention is to the detectable limit of TGEV
102.55TCID50/ mL, the detectable limit to PReoV are 104.10TCID50/ mL shows that TGEV's and PReoV of the invention is double
RT-PCR detection kit sensitivity with higher when detecting TGEV and PReoV.TGEV and PReoV RT- of the invention
PCR, which identifies detection kit, not only can be shortened Diagnostic Time, has saved diagnostic reagent, but also can reduce use cost.
TGEV with PReoV RT-PCR identification detection kit testing result in 6 months provided by the invention is consistent, table
Bright TGEV and PReoV RT-PCR of the present invention, which identifies detection kit, has very high stability.Field trial proves institute of the present invention
TGEV the and PReoV RT-PCR obtained identifies detection kit practicability and applicability is good.
Detailed description of the invention
Fig. 1 is the RT-PCR amplification of target gene, wherein M:DNA molecular mass standard DL2000；1:TGEV is positive
Tissue samples；2:PReoV assaypositive tissue sample；The bis- assaypositive tissue samples of 3:TGEV/PReoV；4: negative control.
Fig. 2 is duplex RT-PCR detection kit different annealing temperature of the present invention as a result, wherein M:DNA molecular mass standard
DL2000；1:50.0 DEG C；2:50.3 DEG C；3:51.0 DEG C；4:52.0 DEG C；5:53.2 DEG C；6:54.4；7:55.6 DEG C；8:56.8 DEG C；
9:58.0 DEG C；10:59.0 DEG C；11:60 DEG C.
Fig. 3 is duplex RT-PCR detection kit specific test of the present invention as a result, wherein M:DNA molecular mass standard
DL2000；The bis- assaypositive tissue samples of 1:TGEV/PReoV；2:CSFV；3:PRRSV；4:PDCoV；5:PEDV；6:JEV；7:PPV；
8:PRV；9:PCV2；10:TTV；11: negative control.
Fig. 4 is duplex RT-PCR detection kit sensitivity tests of the present invention as a result, wherein M:DNA molecular mass standard
DL2000；The TCID of 1-7:TGEV50Respectively 105.55TCID50/mL、104.55TCID50/mL、103.55TCID50/mL、
102.55TCID50/mL、101.55TCID50/mL、100.55TCID50/mL、10-0.45TCID50/mL；The TCID of 1-7:PReoV50Respectively
It is 107.10TCID50/mL、106.10TCID50/mL、105.10TCID50/mL、104.10TCID50/mL、103.10TCID50/mL、
102.10TCID50/mL、101.10TCID50/mL；8: negative control.
Specific embodiment
The present invention program is described in further detail below with reference to embodiment, following the description is merely to explain this hair
It is bright, its content is not defined.
Embodiment
1 materials and methods
1.1 viruses and clinical sample
Transmissible gastro-enteritis virus (TGEV), pig reovirus (PReoV), pig fourth type coronavirus (PDCoV), pig are popular
Property diarrhea virus (PEDV), swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), Latex agglutination test
(JEV), pig parvoviral (PPV), porcine pseudorabies virus (PRV), porcine circovirus 2 type (PCV2) and the thin circovirus virus of pig
(TTV) it is saved and is provided by this laboratory.Clinical sample picks up from Guangxi various regions pig farm diarrhea of pigs excrement and intestine of young pigs of having loose bowels
Sample, clip tissue sample about 0.5g-1.0g are placed in sterilizing mortar, are fully ground into homogenate, the 1mol/ of sterilizing is added
LPBS solution is made into 1:5 emulsion suspension liquid, and multigelation 3 times.10 000r/min be centrifuged 5min, collect supernatant, be transferred to 1.5mL from
Number is saved backup for RNA extracting or -20 DEG C in heart pipe.
1.2 main agents
Reverse transcription reagent box (PrimeScript RT reagent Kit), PrimeSTAR HR (Premix), DL2000 DNA
Marker is Dalian treasured bioengineering Co., Ltd product；Virus genome RNA/DNA Rapid extraction kit is Axygen
Biotechnology Co., Ltd's product.
The design and synthesis of 1.3 primers
According to the TGEV whole genome sequence and PReoV S2 gene order logged on GenBank, Meg Align (DNA is utilized
Star), 7.0 software of Primer Premier is separately designed for the conservative region of TGEV full-length genome and PReoV S2 gene
A pair of of specific primer TGEV-P1/TGEV-P2 and PReoV-P1/PReoV-P2.TGEV upstream primer TGEV-P1:5 '-
CGTGGTCGGAAGARTAAT AAC A-3 ', TGEV downstream primer TGEV-P2:5 '-CAACCCAGACAACTCCATCTA A-
3 ', PCR amplification target fragment size is 277bp.PReoV upstream primer PReoV-P1:5 '-
TCGCGCTGCGTTCCTATTCAACGT-3 ', PReoV downstream primer PReoV-P2:5 '-
GGAGCTGGTTACCAGCTCTRCCRG-3 ', PCR amplification target fragment size are 974bp.Primer is had by Dalian treasured bioengineering
The synthesis of limit company.
1.4 viral RNAs/DNA extraction
Transmissible gastro-enteritis virus (TGEV), pig reovirus (PReoV), pig fourth type coronavirus (PDCoV), pig are popular
Property diarrhea virus (PEDV), swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), Latex agglutination test
(JEV), pig parvoviral (PPV), porcine pseudorabies virus (PRV), porcine circovirus 2 type (PCV2) and the thin circovirus virus of pig
(TTV) and the tissue pathological material of disease handled well it, is used according to AxyPrep Body Fluid Viral DNA/RNA Miniprep Kit
Specification carries out RNA/DNA extraction, and RT-PCR or PCR of the RNA/DNA obtained for next step react or are placed in -20 DEG C of guarantors
It deposits.
1.5 reverse transcription-polymerase chain reaction (RT-PCR)
Reverse transcription synthesizes cDNA: illustrates according to RT (reverse transcription) kit, using 20 μ L systems, and RNA template 14 μ L, 5 ×
4 μ L, RT Enzyme Mix of PrimeScript TM buffer 1.0 μ L, 1.0 μ L of reverse transcription primer Oligo18T, by following journey
Sequence carries out reverse transcription reaction: 37 DEG C of 20min, 85 DEG C of 30s, 12 DEG C of preservations.
25 μ L of PCR reaction system, 7.5 μ L, PrimeSTAR HR (Premix) of aqua sterilisa 12.5 μ L, 25 μm of ol/L TGEV
With each 0.5 μ L of PReoV upstream and downstream primer, 3.0 μ L of template.It is expanded according to following condition: 98 DEG C of initial denaturation 3min；98 DEG C of changes
Property 10s, 60 DEG C of renaturation 5s, 72 DEG C of extension 1min, 35 circulation；Last 72 DEG C of extensions 10min, PCR total reaction time is about 77
Minute.10 μ L of amplified production electrophoresis in 10g/L Ago-Gel is taken, and observes result.
1.6 duplex RT-PCR detection method specific tests
With the pig fourth type coronavirus (PDCoV), Porcine epidemic diarrhea virus (PEDV), swine fever virus (CSFV), pig prepared
Reproductive and respiratory syndrome virus (PRRSV), Latex agglutination test (JEV), pig parvoviral (PPV), porcine pseudorabies disease
Malicious (PRV), porcine circovirus 2 type (PCV2) and the thin circovirus virus of pig (TTV) cDNA, DNA and TGEV, PReoV and TGEV/
PReoV mixing positive material cDNA is template, is expanded with the method established, to verify the specificity of this method.
1.7 duplex RT-PCR detection method sensitivity tests
1.7.1 the preparation of TGEV, PReoV positive template
By TGEV (106.86TCID50/ mL) virus liquid and PReoV (108.44TCID50/ mL) virus liquid 1:1 mixing, be then for 10 times
Column are diluted to 10-7(i.e. TGEV is diluted to 10-0.45TCID50/mL；1-7:PReoV is diluted to 101.10TCID50/ mL), after each dilution
Various concentration virus liquid according to AxyPrep Body Fluid Viral RNA/DNA Miniprep Kit operation instructions
RNA extraction is carried out, RT-PCR of the RNA obtained for next step reacts.
1.7.2 duplex RT-PCR detection method sensitivity tests
It using above-mentioned resulting RNA as RT-PCR reaction template, is reacted, is surveyed with the duplex RT-PCR detection method established
The sensibility of fixed established duplex RT-PCR detection method.
1.8 duplex RT-PCR repetitive tests
With the duplex RT-PCR detection method of foundation, detection 3 times is repeated to the bis- positive samples of TGEV, PReoV, with verification result
Reliability.
2 results
The amplification of 2.1 duplex RT-PCRs
Using the bis- positive mixing RNA of TGEV positive RNA, PReoV positive RNA, TGEV/PReoV as template, according to the reaction in 1.5
System and response procedures carry out duplex RT-PCR reaction.Electrophoresis result shows the duplex RT-PCR method energy established (see Fig. 1)
It enough detects that TGEV is positive, PReoV is positive and the bis- positive materials of TGEV/PReoV, amplifies TGEV target gene about 277bp, expands
Increase PReoV target gene about 974bp out, is consistent with expected purpose clip size.TGEV the and PReoV PCR amplified is produced
Object is served marine growth Engineering Co., Ltd and is sequenced, by sequencing result be placed on NCBI carry out Blast be determined as TGEV and
PReoV specific fragment.
2.2 duplex RT-PCR detection method optimum annealing temperatures
To obtain the best expanding effect of the reaction, 11 temperature gradients are set within the temperature range of 50-60 DEG C and carry out double RT-
PCR amplification finds that two bands are most obvious at 60 DEG C, it is determined that optimum annealing temperature is 60 DEG C (see Fig. 2).
2.3 duplex RT-PCR detection method specific test results
Test result shows (see Fig. 3), the duplex RT-PCR that this research is established identify detection method can specifically detect TGEV,
PReoV, and pig fourth type coronavirus (PDCoV), Porcine epidemic diarrhea virus (PEDV), swine fever virus (CSFV), pig are bred
With breath syndrome virus (PRRSV), Latex agglutination test (JEV), pig parvoviral (PPV), porcine pseudorabies virus
(PRV), the testing result of porcine circovirus 2 type (PCV2) and the thin circovirus virus of pig (TTV) shows established method without band
With preferable specificity.
2.4 duplex RT-PCR detection method sensitivity tests
The results show that the duplex RT-PCR detection method is respectively 10 to the detectable limit of TGEV, PReoV2.55TCID50/mL、
104.10TCID50/ mL (see Fig. 4).
2.5 duplex RT-PCR detection method repetitive test results
With the duplex RT-PCR detection method of foundation, the bis- positive samples of TGEV, PReoV are detected, is repeated 3 times, as a result exists
At 277bp and 974bp, high-visible purpose band has been obtained, has illustrated that the duplex RT-PCR detection method established has very
Good stability.
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, any
The change or replacement expected without creative work, should be covered by the protection scope of the present invention.Therefore, of the invention
Protection scope should be determined by the scope of protection defined in the claims.
Sequence table
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Claims (9)
1. a kind of duplex RT-PCR detection primer for quickly distinguishing TGEV and PReoV, which is characterized in that the duplex RT-PCR
Detection primer includes TGEV RT-PCR detection primer and PReoV RT-PCR detection primer；The TGEV RT-PCR detection primer
As the upstream primer with nucleotide sequence shown in SEQ ID NO:1 and with nucleotide sequence shown in SEQ ID NO:2
Downstream primer composition；The PReoV RT-PCR detection primer is drawn as the upstream with nucleotide sequence shown in SEQ ID NO:3
Object and with nucleotide sequence shown in SEQ ID NO:4 downstream primer composition.
2. quickly distinguishing the duplex RT-PCR detection primer of TGEV and PReoV according to claim 1, which is characterized in that institute
It states the full genome that TGEV RT-PCR detection primer is foundation TGEV to be designed, the target fragment size of amplification is 277 bp；Institute
It states the S2 gene that PReoV RT-PCR detection primer is PReoV to be designed, the target fragment size of amplification is 974 bp.
3. quickly distinguishing the duplex RT-PCR detection primer of TGEV and PReoV according to claim 1, which is characterized in that institute
It states duplex RT-PCR detection primer and identifies answering for detection kit in preparation transmissible gastro-enteritis virus and pig reovirus
With.
4. a kind of duplex RT-PCR detection kit for quickly distinguishing TGEV and PReoV as described in claim 1, feature exist
In, the duplex RT-PCR detection kit include there is nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3 upper
Swim primer and the downstream primer with nucleotide sequence shown in SEQ ID NO:2, SEQ ID NO:4.
5. quickly distinguishing the duplex RT-PCR detection kit of TGEV and PReoV according to claim 4, which is characterized in that
The duplex RT-PCR detection kit further includes PrimeSTAR HR buffer, cDNA template and aqua sterilisa.
6. the duplex RT-PCR detection kit of quick differentiation TGEV and PReoV according to claim 4 or 5, feature exist
In, in the duplex RT-PCR detection kit, including RT reaction system and PCR reaction system；The RT reaction system are as follows:
Using 20 μ L systems, 14 μ L, 5 × PrimeScript TM buffer of RNA template, 4 μ L, RT Enzyme Mix, 1.0 μ L,
1.0 μ L of reverse transcription primer Oligo18T；The PCR reaction system are as follows: use 25 μ L systems, 7.5 μ L of aqua sterilisa,
PrimeSTAR HR(Premix) 12.5 μ L, 25 μm of each 0.5 μ L of ol/L TGEV and PReoV upstream and downstream primer, template 3.0
µL。
7. the duplex RT-PCR detection kit of quick differentiation TGEV and PReoV according to claim 4 or 5, feature exist
In the optimum annealing temperature of the duplex RT-PCR detection kit is 60 DEG C.
8. the duplex RT-PCR detection kit of quick differentiation TGEV and PReoV according to claim 4 or 5, feature exist
In RT reaction condition in the duplex RT-PCR detection kit are as follows: 37 DEG C of 20 min, 85 DEG C of 30 s；PCR reaction condition
Are as follows: 98 DEG C of 3 min of initial denaturation；98 DEG C of 10 s of denaturation, 60 DEG C of 5 s of renaturation, 72 DEG C of 1 min of extension, 35 recycle；Last 72 DEG C are prolonged
Stretch 10 min.
9. the duplex RT-PCR detection kit of quick differentiation TGEV and PReoV according to claim 4 or 5, feature exist
Shown in the SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 of, the RT-PCR detection kit
Primer is 25 μm of ol/L using concentration.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104611465A (en) * | 2015-02-04 | 2015-05-13 | 四川农业大学 | Fluorogenic quantitative PCR kit for detecting swine transmissible gastroenteritis virus |
| CN105400910A (en) * | 2015-12-31 | 2016-03-16 | 河南农业大学 | Porcine Deltacoronavirus and swine transmissible gastroenteritis virus multiplex RT-PCR detection primer and detection method |
| CN105420412A (en) * | 2015-12-31 | 2016-03-23 | 河南农业大学 | Porcine deltacoronavirus and porcine epidemic diarrhea virus multiple RT-PCR detection primers and detection method therefor |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104611465A (en) * | 2015-02-04 | 2015-05-13 | 四川农业大学 | Fluorogenic quantitative PCR kit for detecting swine transmissible gastroenteritis virus |
| CN105400910A (en) * | 2015-12-31 | 2016-03-16 | 河南农业大学 | Porcine Deltacoronavirus and swine transmissible gastroenteritis virus multiplex RT-PCR detection primer and detection method |
| CN105420412A (en) * | 2015-12-31 | 2016-03-23 | 河南农业大学 | Porcine deltacoronavirus and porcine epidemic diarrhea virus multiple RT-PCR detection primers and detection method therefor |
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| 刘矿等: "多重RT—PCR检测猪传染性胃肠炎病毒和流行性腹泻病毒方法的建立及应用", 《兽医临床》 * |
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