CN105925727A - Primer and kit for detecting foot and mouth disease virus type A and type O mixed infection and use method and application of kit - Google Patents
Primer and kit for detecting foot and mouth disease virus type A and type O mixed infection and use method and application of kit Download PDFInfo
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Abstract
The invention discloses a primer for detecting foot and mouth disease virus type A and type O mixed infection. The primer is characterized by comprising a primer pair FMDA and a primer pair FMDO; the primer pair FMDA comprises an upstream primer FMDAF which is shown in 5'-GCAACCCAGATGTTGAT-3', and a downstream primer FMDAR which is shown in 5'-CCACATGGACTATGGAA-3'; the primer pair FMDO comprises an upstream primer FDMOF which is shown in 5'-TGGCCTCAAAGACTCTCG-3' and a downstream primer FMDOR which is shown in 5'-CAAGCTACAGATCACTTTAC-3'. The use method of a kit includes the steps of acquisition of a fresh sample of a diseased animal, nucleic acid extraction, conduction of a reverse transcriptase polymerase chain reaction and agarose gel electrophoresis detection. The method is high in specificity and sensitivity and easy and convenient to operate, labor and time are saved, and the detection accuracy and specificity are improved. A foot and mouth disease virus type A and type O strain or type A and type O mixed infection can be rapidly and conveniently detected, the primer and the kit can be used for epidemiological investigation and analysis of foot and mouth disease viruses, and wide market application prospects and great economic benefits are achieved.
Description
Technical field
The present invention relates to a kind of for detect foot and mouth disease virus A type and the primer of O type mixed infection, test kit,
Using method and application thereof, belong to biological technical field.
Background technology
Foot and mouth disease (aphthae epizooticae；Foot and mouth disease, FMD) it is by foot and mouth disease virus
Acute, hot, the high degree in contact sexually transmitted disease of a kind of Zoonosis caused, it faces, and to examine feature be in oral cavity
Mucosa, extremity lower end and breast etc. skin forms vesicle and speck.This disease is propagated rapidly, popular wide, becomes
Year, animal took optimum process more, and young animal is many, and because of myocardial damage, mortality rate is higher.Foot and mouth disease is widely current
In all over the world, cause huge economic loss, national governments and international organization and pay much attention to, be listed in
World community decides through consultation the No.1 Notifiable disease put out jointly.Along with the increase increasingly of international trade, material is handed over
Stream and international association are more and more frequent, and prevention and control to foot and mouth disease add difficulty, therefore countries in the world
All spend a large amount of human and material resources and financial resources to study, control and eliminate foot and mouth disease.
Foot and mouth disease virus (Foot-and-Mouth disease virus) belongs to the foot and mouth disease in microRNA Viraceae
Tobamovirus, is minimum in RNA viruses one.Foot and mouth disease virus has pleiomorphism, the feature of easily variation.
According to its serological characteristic, 7 serotypes, i.e. A, O, C, SAT1 (South Africa I), SAT2 can be divided into
(South Africa II type), SAT3 (South Africa type III) and Asia 1 type (Type Asia 1).Without handing between each serotype
Fork immunological phenomena.Each serotype comprises again several hypotypes, also only has part between each hypotype of homotype
Cross-immunity.This characteristic of this virus brings many difficulties to the anti-system of foot and mouth disease.
Foot and mouth disease virus content in vesicle liquid, vesicle skin, lymph fluid and the pyrogenic stage blood of infected animal
The highest, next to that each histoorgan, secretions, Excreta, can long-term existence outside toxin expelling, after bringing down a fever
Virus can come across in breast, excrement, urine, tear, saliva and each internal organs.Foot and mouth disease virus environment to external world
Resistance is very strong, resistance to dry.Under field conditions (factors), toxic tissue and the feedstuff of pollution, drinking-water, forage grass,
The contained virus such as fur and soil still has infectivity within a few days or even several weeks.Foot and mouth disease virus is to bronsted lowry acids and bases bronsted lowry
The most especially sensitive, in the buffer of pH3.0 and more than pH9.0, viral infection by pop-off, 2%～4%
Sodium hydroxide, 3%～5% formalin solution, 5% ammonia, 0.2%～0.5% peracetic acid or 5% sodium hypochlorite
The disinfectant good etc. being foot and mouth disease virus.
Foot and mouth disease is the infectious disease that a kind of infectiousness is extremely strong, once occurring often in popularity, in pastoral area, many
Presenting and be very popular, epidemic situation once occurs, and can the flowing of follower spread rapidly, epidemic situation after the regular period
Just die away.Foot and mouth disease can infect many animals, and artiodactylous animals susceptibility is the highest, susceptibility height
Order is followed successively by cattle, milch cow, yak, Babalus bubalis L., pig, sheep, camel.
Preventing and treating for foot and mouth disease there is no specific medicament therapy at present, mainly takes comprehensive measures for prevention and control and suits the medicine to the illness
Therapy.The most basic way is to eliminate infected animal, band poison animal and thorough disinfection, cuts off route of transmission.
Therefore should strengthen quarantine and foot and mouth disease is monitored, in case foot and mouth disease diffusion.
At present, cut open inspection feature according to epidemiology, clinical symptom and case and can make tentatively examining of foot and mouth disease
Disconnected, but make a definite diagnosis and need to carry out laboratory diagnosis.The separation of virus is that laboratory is examined with qualification and serodiagnosis
Disconnected conventional method, but relatively time consuming arduously, the longest.
Summary of the invention
The technical problem to be solved is to provide a kind of mixed for detecting foot and mouth disease virus A type and O type
Close primer, test kit, using method and the application thereof infected, use the test kit of the present invention, it is possible to three
Make diagnosis in individual hour, provide a kind of effective point for foot and mouth disease clinical diagnosis and Molecule Epidemiology Investigation
Sub-biometric diagnostic method.
To achieve these goals, the technical solution adopted in the present invention is: one is used for detecting hoof-and-mouth disease
Poison A type and the primer of O type mixed infection, described primer be primer to FMDA and FMDO, wherein,
Primer is to FMDA:
Forward primer FMDAF:5 '-GCAACCCAGATGTTGAT-3 '
Downstream primer FMDAR:5 '-CCACATGGACTATGGAA-3 '
Primer is to FMDO:
Forward primer FDMOF:5 '-TGGCCTCAAAGACTCTCG-3 '
Downstream primer FMDOR:5 '-CAAGCTACAGATCACTTTAC-3 '.
A kind of for detecting foot and mouth disease virus A type and the test kit of O type mixed infection, described test kit bag
Include primer to FMDA and FMDO, wherein,
Primer is to FMDA:
Forward primer FMDAF:5 '-GCAACCCAGATGTTGAT-3 '
Downstream primer FMDAR:5 '-CCACATGGACTATGGAA-3 '
Primer is to FMDO:
Forward primer FDMOF:5 '-TGGCCTCAAAGACTCTCG-3 '
Downstream primer FMDOR:5 '-CAAGCTACAGATCACTTTAC-3 '.
Described test kit also includes 5 × RT Buffer, dNTPs, Taq enzyme, RNase inhibitor, reversion
Record enzyme, DEPC water, lysate and flushing liquor.
Described test kit also includes positive control and negative control.
The component of described lysate is: hydroxyquinoline 3～5% (mass fraction), trichloroethyl phosphate
3.2～5.2 μMs, phenyl isothiocyanate 1.5～3.5 μMs, polyvinyl pyrrolidone 1.3～3.5% (mass fraction),
Sodium chloride 0.3～0.5M and sodium citrate 0.6～0.9M.
The component of described flushing liquor is: trishydroxymethylaminomethane 10 μMs, ethylenediaminetetraacetic acid 1 μM, second
Alcohol 60～80% (volume fraction)；pH 7.0.
A kind of using method of the test kit for detecting foot and mouth disease virus A type and O type mixed infection, including
Following steps:
(1) collection of sample
(2) extraction of sample RNA
(3)RT-PCR
Sample RNA is carried out RT-PCR amplification, reaction system 25 μ l:5 × RT Buffer 5 μ l, 12.5 μMs
DNTPs 5 μ l, 125 μMs of forward primer FMDAF and 125 μMs of each 1 μ l of downstream primer FMDAR, 125 μMs
Forward primer FMDOF and 125 μMs of each 1 μ l of downstream primer FMDOR, 25U/ μ l Taq enzyme 0.5 μ l, 2.5U/ μ l
RNase inhibitor 1 μ l, 2.5U/ μ l reverse transcription 1 μ l, DEPC water 3.5 μ l and 5ng/ μ l RNA 5 μ l；
RT-PCR amplification program is: 50 DEG C of 30min, 94 DEG C of 2min；94 DEG C of 1min, 55 DEG C of 45s, 72 DEG C
1min, totally 30 circulations；72℃3min；
(4) detection of RT-PCR amplified production
Pcr amplification product is carried out agarose gel electrophoresis, observed result under gel imaging system.
The collection of described sample is divided into gathering or the collection of liquid animal tissue of solid, shaped animal tissue；
Wherein,
The acquisition method of solid, shaped animal tissue is: aseptic collection animal tissue pathological material of disease 0.1-100mg, loads nothing
RNase pollute centrifuge tube in standby；
The collection of liquid animal tissue is divided into the collection of juice or the collection of blood, the collection side of juice
Method is: gather nasopharynx liquid by Nasopharyngeal swabs, loads in the centrifuge tube without RNase pollution standby；Adopting of blood
Diversity method is: take whole blood, serum or blood plasma 10-100 μ l, adds in the centrifuge tube without RNase pollution standby.
The extraction of described sample RNA, method particularly includes:
(1) animal tissue of solid, shaped first carrying out milled processed, liquid animal tissue is directly used in and carries
Take；
(2) become in animal tissue 20-100mg or the liquid animal tissue 20-100 μ l of pasty state after grinding,
Adding lysate 500 μ l, 12000rpm is centrifuged 30s, isolates supernatant；
(3) supernatant is proceeded in centrifuge tube, adds dehydrated alcohol 500 μ l, forward on RNA purification column by several times,
12000rpm is centrifuged 30s, the RNA slightly carried；
(4) rinsing RNA purification column with flushing liquor 500 μ l, 12000rpm is centrifuged 30s；
(5) forwarding RNA purification column to another centrifuge tube, add DEPC water 50 μ l, 12000rpm is centrifuged 30s,
Obtain virus and the RNA of tissue gene group, save backup in-20 DEG C.
A kind of test kit for detecting foot and mouth disease virus A type and O type mixed infection is detection foot and mouth disease virus
Application in terms of A type and O type mixed infection.
Beneficial effects of the present invention
1, nucleic acid extraction process is improved by the present invention, i.e. uses the cracking with available reagent box different formulations
Liquid and flushing liquor, substantially reduce the nucleic acid extraction time, makes the extraction time of nucleic acid by contracting in original 1-2 hour
Short is 2 minutes, saves the time of nucleic acid extraction, thus improves the service efficiency of test kit, makes reagent
The inspection of box is more quick, economical.Wherein, the lysate of the present invention can make sample crack rapidly, release
Go out RNA.Flushing liquor can effectively remove protein, salt and impurity etc., makes extracted RNA purer
Only.
2, the present invention is using foot and mouth disease virus 3D genome sequence as genes of interest region, is designed to detection
Other serotypes of foot and mouth disease can not be expanded under A type and the specific primer of O type, equal conditions, have the highest
Specificity.Meanwhile, the test kit of the present invention has the highest sensitivity, and lowest detection is limited to 0.1pg/ μ l.
3, the invention provides a kind of for detecting foot and mouth disease virus A type and O type mixed infection test kit,
This test kit can detect foot and mouth disease virus A type and O type mixed infection strain fast and accurately.Can boil on the nape opposite the mouth
A type and O type single sample that fever aphthous infects detect, it is possible to A type in clinical case and O type mixed infection
Detection, can be used for foot and mouth disease virus cause of disease generaI investigation, Molecule Epidemiology Investigation and vaccine screening and monitoring.
4, the method for the present invention is built upon on molecular biology mechanism, detection provides negative control and
Positive control, substantially increases the accuracy of detection, reduces false-positive occurrence probability, and specificity is relatively strong,
Time saving and energy saving, the detection time is foreshortened to 3 hours by this test kit, substantially increases work efficiency.
5, the invention is particularly suited to the quick diagnosis of a large amount of clinical samples of mouth disease virus infection, Ke Yiwei
In clinic and scientific research, hoof-and-mouth disease preventing and treating and treatment provide scientific basis, and to raising foot and mouth disease virus cause of disease
Detection, monitoring, vaccine development etc. significant.
Accompanying drawing explanation
Fig. 1 is RT-PCR product agarose gel electrophoresis testing result figure；Wherein, swimming lane M represents DL 2000
DNA Marker (is followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp) from top to bottom,
Swimming lane 1,2 is foot and mouth disease virus negative control, and 3 is that foot and mouth disease virus A type is right with the O type mixed infection positive
According to, 4 is foot and mouth disease virus A type and O type mixed infection sample, and 5 is foot and mouth disease virus O type positive control,
6 is foot and mouth disease virus O type positive, and 7 is foot and mouth disease virus A type positive control, and 8 is foot and mouth disease virus
A type positive.
Fig. 2 is RT-PCR product agarose gel electrophoresis testing result figure；Wherein, swimming lane M represents DL 2000
DNA Marker (is followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp) from top to bottom,
Swimming lane 1 represents foot and mouth disease virus A type and the sample of O type mixed infection；Swimming lane 2-6 represents foot and mouth disease respectively
Virus c-type, SAT1 (South Africa I), SAT2 (South Africa II type), SAT3 (South Africa type III) and Asia 1
Type (Type Asia 1) sample.
Detailed description of the invention
Below in conjunction with embodiment, the detailed description of the invention of the present invention is described in further detail.
The relative molecular weight of the polyvinyl pyrrolidone used by the present invention is 40000.
Hydroxyquinoline used by the present invention is 2-hydroxyquinoline.
Embodiment 1 is for detecting foot and mouth disease virus A type and the test kit of O type mixed infection
1, the design of primer
According to foot and mouth disease virus A type and the gene order of O type mixed infection, design primer to FMDA and
FMDO, wherein,
Primer is to FMDA:
Forward primer FMDAF:5 '-GCAACCCAGATGTTGAT-3 ' (SEQ ID NO.1)
Downstream primer FMDAR:5 '-CCACATGGACTATGGAA-3 ' (SEQ ID NO.2)
Primer is to FMDO:
Forward primer FDMOF:5 '-TGGCCTCAAAGACTCTCG-3 ' (SEQ ID NO.3)
Downstream primer FMDOR:5 '-CAAGCTACAGATCACTTTAC-3 ' (SEQ ID NO.4).
2, the extraction of RNA
To make a definite diagnosis the cattle carrying foot and mouth disease virus A type and O type mixed infection as laboratory animal, extract sample
RNA, comprises the following steps:
(1) collection of sample
Gather cattle whole blood 100 μ l, add in the centrifuge tube without RNase pollution standby.
(2) extraction of sample RNA
A (), in the 100 μ l whole bloods gathered, adds lysate 500 μ l, 12000rpm is centrifuged 30s, isolates
Supernatant；
C supernatant is proceeded in centrifuge tube by (), add dehydrated alcohol 500 μ l, forwards on RNA purification column by several times,
12000rpm is centrifuged 30s, the RNA slightly carried；
D () rinses RNA purification column with flushing liquor 500 μ l, 12000rpm is centrifuged 30s；
E () forwards RNA purification column to another centrifuge tube, add DEPC water 50 μ l, and 12000rpm is centrifuged 30s,
Obtain virus and the RNA of tissue gene group, save backup in-20 DEG C.
The component of lysate is: hydroxyquinoline 3% (mass fraction), trichloroethyl phosphate 3.2 μMs, different sulfur
Phenyl-cyanate 3.5 μMs, polyvinyl pyrrolidone 1.3% (mass fraction), sodium chloride 0.3M and citric acid
Sodium 0.9M.
The component of flushing liquor is: trishydroxymethylaminomethane 10 μMs, ethylenediaminetetraacetic acid 1 μM, ethanol 60%
(volume fraction)；pH 7.0.
3、RT-PCR
With sample RNA as template, carry out RT-PCR amplification, simultaneously with swine vesicular disease virus as negative control,
With foot and mouth disease virus A type and O type mixed infection sample as positive control, extract negative control and positive control
The RNA of sample, RT-PCR expand, reaction system 25 μ l:5 × RT Buffer 5 μ l, 12.5 μMs of dNTPs 5 μ l,
125 μMs of forward primer FMDAF and 125 μMs of each 1 μ l of downstream primer FMDAR, 125 μMs of forward primer
FMDOF and 125 μMs of each 1 μ l of downstream primer FMDOR, 25U/ μ l Taq enzyme 0.5 μ l, 2.5U/ μ l RNA
Enzyme inhibitor 1 μ l, 2.5U/ μ l reverse transcription 1 μ l, DEPC water 3.5 μ l and 5ng/ μ l RNA 5 μ l.
RT-PCR amplification program is: 50 DEG C of 30min, 94 DEG C of 2min；94 DEG C of 1min, 55 DEG C of 45s, 72 DEG C
1min, totally 30 circulations；72℃3min.
4, the detection of RT-PCR amplified production
Take RT-PCR amplified production 5 μ l and Loading Buffer 5 μ l mixing, be added to 1.0% containing EB
In agarose gel, it is simultaneously introduced negative control, the RT-PCR amplified production of positive control and DL2000
DNA Marker, voltage is 120V, electrophoresis 25min, then observed result (result in gel imaging instrument
See Fig. 1).
Embodiment 2 is for detecting foot and mouth disease virus A type and the test kit of O type mixed infection
The test kit of embodiment 2 is essentially identical with the test kit of embodiment 1, and difference is: this enforcement
The component of the lysate of example is hydroxyquinoline 5% (mass fraction), trichloroethyl phosphate 4 μMs, isothiocyanic acid
Phenyl ester 2.5 μMs, polyvinyl pyrrolidone 2% (mass fraction), sodium chloride 0.5M and sodium citrate 0.6M.
The component of flushing liquor is trishydroxymethylaminomethane 10 μMs, ethylenediaminetetraacetic acid 1 μM, ethanol 80% (body
Fraction)；pH 7.0.
The laboratory animal of the present embodiment is the cattle made a definite diagnosis and carry foot and mouth disease virus A type, with swine vesicular disease virus
For negative control, with foot and mouth disease virus A pattern product as positive control, the agarose of RT-PCR amplified production coagulates
Glue testing result is shown in Fig. 1 (detection method is with embodiment 1).
Embodiment 3 is for detecting foot and mouth disease virus A type and the test kit of O type mixed infection
The test kit of embodiment 3 is essentially identical with the test kit of embodiment 1, and difference is: this enforcement
The component of the lysate of example is hydroxyquinoline 4% (mass fraction), trichloroethyl phosphate 5.2 μMs, different sulfur cyanogen
Acid phenenyl ester 1.5 μMs, polyvinyl pyrrolidone 3.5% (mass fraction), sodium chloride 0.4M and sodium citrate
0.8M.The component of flushing liquor is trishydroxymethylaminomethane 10 μMs, ethylenediaminetetraacetic acid 1 μM, ethanol 70%
(volume fraction)；pH 7.0.
The laboratory animal of the present embodiment is the cattle made a definite diagnosis and carry foot and mouth disease virus O type, with swine vesicular disease virus
For negative control, with foot and mouth disease virus O pattern product as positive control, the agarose of RT-PCR amplified production coagulates
Glue testing result is shown in Fig. 1 (detection method is with embodiment 1).
The test kit using the embodiment of the present invention 1 extracts the geneome RNA of different sample, to the gene extracted
Group RNA concentration and purity are measured, and result see table.
The geneome RNA concentration of table test kit of the present invention extraction and purity testing result
| Sample number into spectrum | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| The RNA concentration extracted | 19.39 | 21.89 | 20.19 | 21.82 | 19.97 | 21.82 | 20.73 |
| Absorbance ratio | 1.81 | 1.92 | 1.95 | 1.83 | 1.91 | 1.87 | 1.82 |
Note: RNA concentration unit is ng/ μ l；Absorbance ratio is RNA suction at 260nm and 280nm
Luminosity ratio (260/280), judges the purity of RNA by absorbance ratio, as can be seen from the above table,
Absorbance, between 1.8～2.0, illustrates that the RNA purity extracted is preferable.
In table, sample 1,2,3,4,5,6,7 respectively uses this test kit to extract about from 100 μ l serum
0.97 μ g, 1.09 μ g, 1.00 μ g, 1.09 μ g, 1.00 μ g, 1.09 μ g, 1.04 μ g geneome RNAs.
Experimental example
1, for detecting foot and mouth disease virus A type and the test kit specificity experiments of O type mixed infection
To make a definite diagnosis the cattle carrying foot and mouth disease virus A type and O type mixed infection as laboratory animal, verify this
The specificity of the RT-PCR kit of bright detection foot and mouth disease virus A type and O type mixed infection, including following
Step:
(1) collection of sample
Gather cattle whole blood 100 μ l, add in the centrifuge tube without RNase pollution standby.
(2) extraction (formula of lysate and flushing liquor is with embodiment 1) of sample RNA
A (), in the 100 μ l whole bloods gathered, adds lysate 500 μ l, 12000rpm is centrifuged 30s, isolates
Supernatant；
C supernatant is proceeded in centrifuge tube by (), add dehydrated alcohol 500 μ l, forwards on RNA purification column by several times,
12000rpm is centrifuged 30s, the RNA slightly carried；
D () rinses RNA purification column with flushing liquor 500 μ l, 12000rpm is centrifuged 30s；
E () forwards RNA purification column to another centrifuge tube, add DEPC water 50 μ l, and 12000rpm is centrifuged 30s,
Obtain virus and the RNA of tissue gene group, save backup in-20 DEG C.
(3)RT-PCR
With sample RNA as template, carry out RT-PCR amplification, simultaneously with c-type, SAT1 (South Africa I),
SAT2 (South Africa II type), SAT3 (South Africa type III) and Asia1 type (Type Asia 1) sample are comparison, carry
Take the RNA of control sample, RT-PCR amplification, reaction system 25 μ l:5 × RT Buffer 5 μ l, 12.5 μMs
DNTPs 5 μ l, 125 μMs of forward primer FMDAF and 125 μMs of each 1 μ l of downstream primer FMDAR, 125 μMs
Forward primer FMDOF and 125 μMs of each 1 μ l of downstream primer FMDOR, 25U/ μ l Taq enzyme 0.5 μ l, 2.5U/ μ l
RNase inhibitor 1 μ l, 2.5U/ μ l reverse transcription 1 μ l, DEPC water 3.5 μ l and 5ng/ μ l RNA 5 μ l.
RT-PCR amplification program is: 50 DEG C of 30min, 94 DEG C of 2min；94 DEG C of 1min, 55 DEG C of 45s, 72 DEG C
1min, totally 30 circulations；72℃3min.
(4) detection of RT-PCR amplified production
Take RT-PCR amplified production 5 μ l and Loading Buffer 5 μ l mixing, be added to 1.0% containing EB
In agarose gel, it is simultaneously introduced RT-PCR amplified production and the DL2000DNA Marker of comparison, electricity
Pressure is 120V, electrophoresis 25min, then observed result (result is shown in Fig. 2) in gel imaging instrument.
Agarose gel electrophoresis result shows, the RT-PCR amplified production of inventive samples RNA is at RT-PCR
There is band at 1350bp and 860bp in amplified production, is consistent with expection, it was demonstrated that RT-PCR of the present invention expands
Product is foot and mouth disease virus A type and O type mixed infection purpose fragment.Meanwhile, the c-type of comparison, SAT1
(South Africa I), SAT2 (South Africa II type), SAT3 (South Africa type III) and Asia1 type (Type Asia 1) sample
There is not specific band in product RT-PCR amplification, illustrates that the RT-PCR kit of the present invention has good spy
The opposite sex.
2, for detecting foot and mouth disease virus A type and the test kit sensitivity experiment of O type mixed infection
The RNA of extraction is taken 1 μ g/ μ l make 10 times of serial dilutions, take respectively 1 μ g/ μ l, 100ng/ μ l, 10ng/ μ l,
1ng/ μ l, 100pg/ μ l, 10pg/ μ l, 1pg/ μ l, 0.1pg/ μ l, 0.01pg/ μ l, 0.001pg/ μ l RNA are carried out
RT-PCR one-step method expands, and result shows the detection foot and mouth disease virus A type of the present invention and O type
Low detection limit is 0.1pg/ μ l, and this exceeds 10 than the sensitivity (1pg/ μ l) of common RT-PCR two-step method
Times.
Claims (10)
1. one kind is used for detecting foot and mouth disease virus A type and the primer of O type mixed infection, it is characterised in that
Described primer be primer to FMDA and FMDO, wherein,
Primer is to FMDA:
Forward primer FMDAF:5 '-GCAACCCAGATGTTGAT-3 '
Downstream primer FMDAR:5 '-CCACATGGACTATGGAA-3 '
Primer is to FMDO:
Forward primer FDMOF:5 '-TGGCCTCAAAGACTCTCG-3 '
Downstream primer FMDOR:5 '-CAAGCTACAGATCACTTTAC-3 '.
2. one kind is used for detecting foot and mouth disease virus A type and the test kit of O type mixed infection, it is characterised in that
Described test kit include primer to FMDA and FMDO, wherein,
Primer is to FMDA:
Forward primer FMDAF:5 '-GCAACCCAGATGTTGAT-3 '
Downstream primer FMDAR:5 '-CCACATGGACTATGGAA-3 '
Primer is to FMDO:
Forward primer FDMOF:5 '-TGGCCTCAAAGACTCTCG-3 '
Downstream primer FMDOR:5 '-CAAGCTACAGATCACTTTAC-3 '.
The most according to claim 2 for detecting foot and mouth disease virus A type and the examination of O type mixed infection
Agent box, it is characterised in that described test kit also includes 5 × RT Buffer, dNTPs, Taq enzyme, RNA
Enzyme inhibitor, reverse transcription, DEPC water, lysate and flushing liquor.
The most according to claim 3 for detecting foot and mouth disease virus A type and the examination of O type mixed infection
Agent box, it is characterised in that described test kit also includes positive control and negative control.
The most according to claim 3 for detecting foot and mouth disease virus A type and the examination of O type mixed infection
Agent box, it is characterised in that the component of described lysate is: hydroxyquinoline 3～5% (mass fraction), phosphorus
Acid trichloro ethyl ester 3.2～5.2 μMs, phenyl isothiocyanate 1.5～3.5 μMs, polyvinyl pyrrolidone 1.3～3.5%
(mass fraction), sodium chloride 0.3～0.5M and sodium citrate 0.6～0.9M.
The most according to claim 3 for detecting foot and mouth disease virus A type and the examination of O type mixed infection
Agent box, it is characterised in that the component of described flushing liquor is: trishydroxymethylaminomethane 10 μMs, ethylenediamine
Tetraacethyl 1 μM, ethanol 60～80% (volume fraction)；pH 7.0.
7. one kind as claimed in claim 3 for detecting foot and mouth disease virus A type and O type mixed infection
The using method of test kit, it is characterised in that comprise the following steps:
(1) collection of sample
(2) extraction of sample RNA
(3)RT-PCR
Sample RNA is carried out RT-PCR amplification, reaction system 25 μ l:5 × RT Buffer 5 μ l, 12.5 μMs
DNTPs 5 μ l, 125 μMs of forward primer FMDAF and 125 μMs of each 1 μ l of downstream primer FMDAR, 125 μMs
Forward primer FMDOF and 125 μMs of each 1 μ l of downstream primer FMDOR, 25U/ μ l Taq enzyme 0.5 μ l, 2.5U/ μ l
RNase inhibitor 1 μ l, 2.5U/ μ l reverse transcription 1 μ l, DEPC water 3.5 μ l and 5ng/ μ l RNA5 μ l；
RT-PCR amplification program is: 50 DEG C of 30min, 94 DEG C of 2min；94 DEG C of 1min, 55 DEG C of 45s, 72 DEG C
1min, totally 30 circulations；72℃3min；
(4) detection of RT-PCR amplified production
Pcr amplification product is carried out agarose gel electrophoresis, observed result under gel imaging system.
The most according to claim 7 for detecting foot and mouth disease virus A type and the examination of O type mixed infection
The using method of agent box, it is characterised in that the collection of described sample is divided into the collection of solid, shaped animal tissue
Or the collection of liquid animal tissue；Wherein,
The acquisition method of solid, shaped animal tissue is: aseptic collection animal tissue pathological material of disease 0.1-100mg, loads nothing
RNase pollute centrifuge tube in standby；
The collection of liquid animal tissue is divided into the collection of juice or the collection of blood, the collection side of juice
Method is: gather nasopharynx liquid by Nasopharyngeal swabs, loads in the centrifuge tube without RNase pollution standby；Adopting of blood
Diversity method is: take whole blood, serum or blood plasma 10-100 μ l, adds in the centrifuge tube without RNase pollution standby.
The most according to claim 8 for detecting foot and mouth disease virus A type and the examination of O type mixed infection
The using method of agent box, it is characterised in that the extraction of described sample RNA, method particularly includes:
(1) animal tissue of solid, shaped first carrying out milled processed, liquid animal tissue is directly used in and carries
Take；
(2) become in animal tissue 20-100mg or the liquid animal tissue 20-100 μ l of pasty state after grinding,
Adding lysate 500 μ l, 12000rpm is centrifuged 30s, isolates supernatant；
(3) supernatant is proceeded in centrifuge tube, adds dehydrated alcohol 500 μ l, forward on RNA purification column by several times,
12000rpm is centrifuged 30s, the RNA slightly carried；
(4) rinsing RNA purification column with flushing liquor 500 μ l, 12000rpm is centrifuged 30s；
(5) forwarding RNA purification column to another centrifuge tube, add DEPC water 50 μ l, 12000rpm is centrifuged 30s,
Obtain virus and the RNA of tissue gene group, save backup in-20 DEG C.
10. being used for as described in any one of claim 2-6 detects foot and mouth disease virus A type and O type mixes
Close the test kit infected application in terms of detection foot and mouth disease virus A type and O type mixed infection.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610343265.0A CN105925727A (en) | 2016-05-23 | 2016-05-23 | Primer and kit for detecting foot and mouth disease virus type A and type O mixed infection and use method and application of kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610343265.0A CN105925727A (en) | 2016-05-23 | 2016-05-23 | Primer and kit for detecting foot and mouth disease virus type A and type O mixed infection and use method and application of kit |
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| CN105925727A true CN105925727A (en) | 2016-09-07 |
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| CN201610343265.0A Pending CN105925727A (en) | 2016-05-23 | 2016-05-23 | Primer and kit for detecting foot and mouth disease virus type A and type O mixed infection and use method and application of kit |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106435022A (en) * | 2016-09-23 | 2017-02-22 | 中国农业科学院兰州兽医研究所 | A type and O type foot and mouth disease virus specific primer and kit |
| US20200327995A1 (en) * | 2017-12-29 | 2020-10-15 | Shenzhen Mindray Bio-Medical Electronics Co., Ltd. | Method for identifying anesthetic drug, and method and device for processing anesthesia electroencephalogram signal |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102230029A (en) * | 2011-06-17 | 2011-11-02 | 河南省动物疫病预防控制中心 | Ternary RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent and method of type O, type A and type Asial foot and mouth disease viruses |
-
2016
- 2016-05-23 CN CN201610343265.0A patent/CN105925727A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102230029A (en) * | 2011-06-17 | 2011-11-02 | 河南省动物疫病预防控制中心 | Ternary RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent and method of type O, type A and type Asial foot and mouth disease viruses |
Non-Patent Citations (2)
| Title |
|---|
| 曹丽娟等: "O型口蹄疫病毒3D基因荧光定量PCR方法的建立及应用", 《中国畜牧兽医》 * |
| 邹克琴: "《基因工程原理和技术》", 31 January 2009 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106435022A (en) * | 2016-09-23 | 2017-02-22 | 中国农业科学院兰州兽医研究所 | A type and O type foot and mouth disease virus specific primer and kit |
| US20200327995A1 (en) * | 2017-12-29 | 2020-10-15 | Shenzhen Mindray Bio-Medical Electronics Co., Ltd. | Method for identifying anesthetic drug, and method and device for processing anesthesia electroencephalogram signal |
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