CN106498094A - A kind of for detecting that foot and mouth disease virus is O-shaped and the primer of Asia1 type mixed infections, kit, using method and its application - Google Patents
A kind of for detecting that foot and mouth disease virus is O-shaped and the primer of Asia1 type mixed infections, kit, using method and its application Download PDFInfo
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Abstract
The invention discloses a kind of for detecting that foot and mouth disease virus is O-shaped and the primer of Asia1 type mixed infections, kit, using method and its application, described primer includes primer pair FMDO and FMDAsia, and wherein, primer pair FMDO includes：Upstream primer FMDOF：5 ' TGGCCTCAAAGACTCTCG 3 ', downstream primer FMDOR：5′‑CAAGCTACAGATCACTTTAC‑3′；Primer pair FMDAsia includes：Upstream primer FMDAsiaF：5 ' CTGGGTTTTACAAACCTGTG 3 ', downstream primer FMDAsiaR：5′‑CTCTGACGTCACAATTGGC‑3′.The using method of kit of the present invention includes the collection of infected animal fresh sample, nucleic acid extraction；Reverse transcriptional PCR, agarose gel electrophoresis are detected.The method high specificity, sensitivity are high, easy to operate, saving of work and time, improve the accuracy and specificity of detection.
Description
Technical Field
The invention relates to a primer and a kit for detecting foot-and-mouth disease virus type O and Asia1 mixed infection, a using method and application thereof, belonging to the technical field of biology.
Background
Foot and Mouth Disease (FMD) is an acute, hot and highly contagious disease of zoonosis caused by foot and mouth disease virus, and is characterized in that blisters and rotten spots are formed on the skin at the oral mucosa, the lower extremities, the breast and the like. The disease is spread rapidly and has wide epidemic range, adult animals mostly take benign processes, and young animals mostly have higher death rate due to myocardial damage. Foot-and-mouth disease widely spreads all over the world, causes huge economic loss, and is listed as the first-number legal infectious disease which is commonly agreed and killed by all countries around the world, with high importance given to governments and international organizations of all countries. With the increasing international trade, the material exchange and the international trade are more and more frequent, which increases the difficulty for the prevention and control of the foot-and-mouth disease, so that countries in the world spend a great deal of manpower, material resources and financial resources to research, control and eliminate the foot-and-mouth disease.
Foot-and-Mouth disease virus (Foot-and-mouse disease virus) belongs to the genus of Foot-and-Mouth disease virus in the family of picornaviridae, the smallest of RNA viruses. Foot-and-mouth disease virus has the characteristics of polymorphism and easy variation. Based on their serological properties, they can be divided into 7 serotypes, A, O, C, SAT1 (south African I), SAT2 (south African II), SAT3 (south African III) and Asia1 (Asia 1). No cross-immunity phenomenon exists among all serotypes. Each serotype comprises several subtypes, and only partial cross-immunity exists among the subtypes of the same type. This characteristic of the virus presents a number of difficulties in the control of foot and mouth disease.
The foot-and-mouth disease virus has the highest content in vesicular fluid, vesicular skin, lymph fluid and blood in fever period of the sick animals, and then each tissue organ, secretion and excrement can exist for a long time and expel toxin outwards, and the virus can appear in milk, feces, urine, tears, saliva and each organ after fever reduction. The foot-and-mouth disease virus has strong resistance to the external environment and is resistant to dryness. Under natural conditions, viruses contained in toxic tissues and contaminated feed, drinking water, forage grass, fur, soil and the like are still infectious for days to weeks. Foot-and-mouth disease virus is particularly sensitive to acid and alkali, virus infectivity disappears instantly in buffer solution with pH3.0 and pH9.0, 2-4% of sodium hydroxide, 3-5% of formalin solution, 5% of ammonia water, 0.2-0.5% of peroxyacetic acid or 5% of sodium hypochlorite and the like are good disinfectants for foot-and-mouth disease virus.
Foot-and-mouth disease is an infectious disease with extremely strong infectivity, once epidemic occurs, the foot-and-mouth disease often presents a pandemic in pasturing areas, once epidemic situation occurs, the foot-and-mouth disease can quickly spread along with the flow of animals, and the epidemic situation gradually subsides after a certain period. The foot-and-mouth disease can infect various animals, and the animals of the order artiodactyla have the highest susceptibility, and the susceptibility is in turn yellow cattle, milk cow, yak, buffalo, pig, sheep and camel.
No specific drug therapy is available for preventing and treating foot and mouth disease at present, and comprehensive prevention measures and symptomatic therapy are mainly adopted. The most fundamental method is to eliminate sick animals and toxic animals and thoroughly disinfect and cut off the transmission path. Therefore, quarantine and foot-and-mouth disease monitoring should be enhanced to prevent spread of foot-and-mouth disease.
At present, the preliminary diagnosis of the foot-and-mouth disease can be made according to epidemiology, clinical symptoms and case examination characteristics, but the laboratory diagnosis is needed for the accurate diagnosis. The isolation and identification of viruses and serological diagnosis are common methods for laboratory diagnosis, but are time-consuming, labor-consuming and time-consuming.
Disclosure of Invention
The invention aims to solve the technical problem of providing a primer, a kit, a use method and application for detecting foot-and-mouth disease virus type O and Asia1 mixed infection.
In order to achieve the purpose, the invention adopts the technical scheme that: a primer for detecting foot-and-mouth disease virus type O and Asia1 mixed infection, the primer comprises primer pair FMDO and FMDAsia, wherein,
primer pair FMDO:
upstream primer FMDOF: 5'-TGGCCTCAAAGACTCTCG-3'
The downstream primer FMDOR: 5'-CAAGCTACAGATCACTTTAC-3'
Primer pair FMDAsia:
the upstream primer FMDAsiaF: 5'-CTGGGTTTTACAAACCTGTG-3'
Downstream primer FMDAsiaaR: 5'-CTCTGACGTCACAATTGGC-3' are provided.
A kit for detecting foot-and-mouth disease virus type O and Asia1 mixed infection, the kit comprises primer pairs FMDO and FMDAsia, wherein,
primer pair FMDO:
upstream primer FMDOF: 5'-TGGCCTCAAAGACTCTCG-3'
The downstream primer FMDOR: 5'-CAAGCTACAGATCACTTTAC-3'
Primer pair FMDAsia:
the upstream primer FMDAsiaF: 5'-CTGGGTTTTACAAACCTGTG-3'
Downstream primer FMDAsiaaR: 5'-CTCTGACGTCACAATTGGC-3' are provided.
The kit also comprises 5 × RT Buffer, dNTPs, Taq enzyme, an RNase inhibitor, reverse transcriptase, ultrapure water, lysate and flushing fluid.
The kit also comprises a positive control and a negative control.
The components of the lysis solution are as follows: 3 to 5 percent of hydroxyquinoline, 3.2 to 5.2 mu M of trichloroethyl phosphate, 1.5 to 3.5 mu M of phenyl isothiocyanate, 1.3 to 3.5 percent of polyvinylpyrrolidone, 0.3 to 0.5M of sodium chloride and 0.6 to 0.9M of sodium citrate.
The washing liquid comprises the following components: 10 mu M of trihydroxymethyl aminomethane, 1 mu M of ethylene diamine tetraacetic acid and 60-80% (volume fraction) of ethanol; the pH was 7.0.
A method of using a kit for detecting mixed infection of foot and mouth disease virus type O and type Asia1, comprising the steps of:
(1) collection of samples
(2) Extraction of sample RNA
(3)RT-PCR
And (3) carrying out RT-PCR amplification on the sample RNA, wherein the reaction system is 25 μ l: 5 × RT Buffer 5 μ l, 12.5 μ M dNTPs 5 μ l, 125 μ M upstream primer FMDOF and 125 μ M downstream primer FMDOR 1 μ l each, 125 μ M upstream primer FMDAsiaF and 125 μ M downstream primer FMDAsiaR 1 μ l each, 25U/μ l Taq enzyme 0.5 μ l, 2.5U/μ l RNase inhibitor 1 μ l, 2.5 μ M reverse transcriptase 1 μ l, ultrapure water 3.5 μ l and 5ng/μ l RNA 5 μ l;
the RT-PCR amplification program is as follows: 30min at 50 ℃ and 3min at 94 ℃; 35 cycles of 94 ℃ for 40s, 55 ℃ for 45s and 72 ℃ for 30 s; 3min at 72 ℃;
(4) detection of RT-PCR amplification products
And (3) carrying out agarose gel electrophoresis on the PCR amplification product, and observing the result under a gel imaging system.
The collection of the sample is divided into the collection of solid animal tissues or the collection of liquid animal tissues; wherein,
the solid animal tissue collection method comprises the following steps: aseptically collecting 0.1-100mg animal tissue pathological material, and placing into a centrifuge tube without RNA enzyme pollution for later use;
the collection of the liquid animal tissue is divided into collection of secretion or collection of blood, and the collection method of the secretion comprises the following steps: collecting nasopharyngeal fluid with nasopharyngeal swab, and filling into centrifuge tube without RNA enzyme pollution; the blood collection method comprises the following steps: taking 10-100 μ l of whole blood, serum or plasma, and adding into a centrifuge tube without RNase pollution for standby.
The specific method for extracting the sample RNA comprises the following steps:
(1) grinding solid animal tissue, and directly extracting liquid animal tissue;
(2) adding 500 μ l lysate into 20-100mg of ground pasty animal tissue or 20-100 μ l of liquid animal tissue, centrifuging at 12000rpm for 30s, and separating supernatant;
(3) transferring the supernatant into a centrifuge tube, adding 500 mu l of absolute ethyl alcohol, transferring the supernatant onto an RNA purification column in batches, and centrifuging the column at 12000rpm for 30s to obtain crude RNA;
(4) washing the RNA purification column with 500. mu.l of washing solution, and centrifuging at 12000rpm for 30 s;
(5) the RNA purification column is transferred to another centrifuge tube, 50 mul of ultrapure water is added, centrifugation is carried out at 12000rpm for 30s, the RNA of the virus and the tissue genome is obtained, and the RNA is preserved at the temperature of minus 20 ℃ for standby.
An application of a kit for detecting foot-and-mouth disease virus type O and Asia1 mixed infection in the aspect of detecting foot-and-mouth disease virus type O and Asia1 mixed infection.
The invention has the advantages of
1. The invention improves the nucleic acid extraction process, namely, lysate and flushing fluid with different formulas from the prior kit are adopted, so that the nucleic acid extraction time is greatly shortened, the nucleic acid extraction time is shortened from the original 1-2 hours to 2 minutes, and the nucleic acid extraction time is saved, thereby improving the use efficiency of the kit and ensuring that the test of the kit is quicker and more economic. The lysis solution can quickly lyse a sample and release RNA. The washing liquid can effectively remove protein, salt, impurities and the like, so that the extracted RNA is purer.
2. The invention takes the foot-and-mouth disease virus 3D genome sequence as a target gene region, designs specific primers capable of detecting O type and Asia1 type, can not amplify other serotypes of foot-and-mouth disease under the same condition, and has high specificity. Meanwhile, the kit has high sensitivity, and the minimum detection limit is 0.1 pg/mu l.
3. The invention provides a kit for detecting foot-and-mouth disease virus type O and Asia1 mixed infection, which can quickly and accurately detect foot-and-mouth disease virus type O and Asia1 mixed infection strains. The kit can be used for detecting single samples of O type and Asia1 type infected foot-and-mouth disease, can also be used for detecting mixed infection of O type and Asia1 type in clinical cases, and can be used for general investigation of foot-and-mouth disease virus pathogens, molecular epidemiological investigation and vaccine screening and monitoring.
4. The method is based on molecular biology, negative control and positive control are provided in detection, the accuracy of detection is greatly improved, the probability of false positive is reduced, the specificity is strong, time and labor are saved, the detection time is shortened to 3 hours by the kit, and the working efficiency is greatly improved.
5. The invention is particularly suitable for the rapid diagnosis of a large number of clinical samples infected by the foot-and-mouth disease virus, can provide scientific basis for the prevention and treatment of the foot-and-mouth disease in clinic and scientific research, and has important significance for improving the detection, monitoring, vaccine development and the like of the foot-and-mouth disease virus pathogen.
Drawings
FIG. 1 is a diagram showing the result of agarose gel electrophoresis detection of RT-PCR products; wherein, the lane M represents DL2000 DNAmarker (2000 bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom), the lanes 1 and 2 are foot-and-mouth disease virus negative control, the lane 3 is foot-and-mouth disease virus O type and Asia1 type mixed infection positive control, and the lane 4 is foot-and-mouth disease virus O type and Asia1 type mixed infection sample.
FIG. 2 is a diagram showing the result of agarose gel electrophoresis detection of RT-PCR products; wherein, Lane M represents DL2000 DNAmarker (2000 bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom), Lane 1 in a represents a foot-and-mouth disease virus O type and Asia1 type mixed infection sample, Lane 2 represents a foot-and-mouth disease virus O type sample, and Lane 3 represents a foot-and-mouth disease virus Asia1 type sample; in b, lane 1 represents the positive control of mixed infection of foot and mouth disease virus type O and Asia1, lane 2 represents the positive control of foot and mouth disease virus type O, and lane 3 represents the positive control of foot and mouth disease virus type Asia 1.
FIG. 3 is a diagram showing the result of agarose gel electrophoresis detection of RT-PCR products; wherein, Lane M represents DL2000 DNAmarker (2000 bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom), Lane 1 represents the sample of foot-and-mouth disease virus type O and Asia1 mixed infection; lanes 2-6 represent foot and mouth disease virus type A, C, SAT1 (south African I), SAT2 (south African II) and SAT3 (south African III) samples, respectively.
Detailed Description
The following examples further illustrate the embodiments of the present invention in detail.
The polyvinylpyrrolidone used in the present invention had a relative molecular weight of 40000.
The hydroxyquinoline used in the present invention is 2-hydroxyquinoline.
Example 1 kit for detecting foot and mouth disease Virus type O and Asia1 Mixed infection
1. Design of primers
Designing primer pairs FMDO and FMDAsia according to the 3D genome sequence of the foot-and-mouth disease virus, wherein the primer pairs FMDO:
upstream primer FMDOF: 5'-TGGCCTCAAAGACTCTCG-3' (SEQ ID NO.1)
The downstream primer FMDOR: 5'-CAAGCTACAGATCACTTTAC-3' (SEQ ID NO.2)
Primer pair FMDAsia:
the upstream primer FMDAsiaF: 5'-CTGGGTTTTACAAACCTGTG-3' (SEQ ID NO.3)
Downstream primer FMDAsiaaR: 5'-CTCTGACGTCACAATTGGC-3' (SEQ ID NO. 4).
2. Extraction of RNA
Taking cattle which are diagnosed to carry mixed infection of foot-and-mouth disease virus type O and Asia1 as experimental animals, extracting sample RNA, comprising the following steps:
(1) collection of samples
100 mul of cattle whole blood is collected and added into a centrifuge tube without RNA enzyme pollution for standby.
(2) Extraction of sample RNA
(a) Adding 500. mu.l of lysate into 100. mu.l of whole blood, centrifuging at 12000rpm for 30s, and separating supernatant;
(c) transferring the supernatant into a centrifuge tube, adding 500 mu l of absolute ethyl alcohol, transferring the supernatant onto an RNA purification column in batches, and centrifuging the column at 12000rpm for 30s to obtain crude RNA;
(d) washing the RNA purification column with 500. mu.l of washing solution, and centrifuging at 12000rpm for 30 s;
(e) the RNA purification column is transferred to another centrifuge tube, 50 mul of ultrapure water is added, centrifugation is carried out at 12000rpm for 30s, the RNA of the virus and the tissue genome is obtained, and the RNA is preserved at the temperature of minus 20 ℃ for standby.
The components of the lysate are: 3 percent of hydroxyquinoline, 3.2 mu M of trichloroethyl phosphate, 3.5 mu M of phenyl isothiocyanate, 1.3 percent of polyvinylpyrrolidone, 0.3M of sodium chloride and 0.9M of sodium citrate.
The components of the flushing liquid are as follows: 10 mu M of trihydroxymethyl aminomethane, 1 mu M of ethylene diamine tetraacetic acid and 60 percent of ethanol (volume fraction); the pH was 7.0.
3、RT-PCR
Performing RT-PCR amplification by taking sample RNA as a template, simultaneously taking the swine vesicular disease virus as a negative control, taking the foot and mouth disease virus O type and Asia1 type mixed infection as a positive control, extracting the RNA of the negative control sample and the positive control sample, performing RT-PCR amplification, and reacting the RNA in a reaction system of 25 mu l: 5 × RT Buffer 5 μ l, 12.5 μ M dNTPs 5 μ l, 125 μ M upstream primer FMDOF and 125 μ M downstream primer FMDOR 1 μ l each, 125 μ M upstream primer FMDAsiaF and 125 μ M downstream primer FMDAsiaR 1 μ l each, 25U/μ l Taq enzyme 0.5 μ l, 2.5U/μ l RNase inhibitor 1 μ l, 2.5 μ M reverse transcriptase 1 μ l, ultrapure water 3.5 μ l and 5ng/μ l RNA 5 μ l.
The RT-PCR amplification program is as follows: 30min at 50 ℃ and 3min at 94 ℃; 35 cycles of 94 ℃ for 40s, 55 ℃ for 45s and 72 ℃ for 30 s; 3min at 72 ℃.
4. Detection of RT-PCR amplification products
And (3) uniformly mixing 5 mu l of RT-PCR amplification product and 5 mu l of Loading Buffer, adding the mixture into 1.0% agarose gel containing EB, simultaneously adding RT-PCR amplification products of negative control and positive control and DL2000DNA Marker, carrying out electrophoresis for 35min at a voltage of 120V, and observing the result in a gel imager (the result is shown in figure 1).
Agarose gel electrophoresis results show that the RT-PCR amplification products of the sample RNA of the invention have bands at 1350bp and 200bp in the RT-PCR amplification products, which are consistent with expectations, and prove that the RT-PCR amplification products of the invention are foot-and-mouth disease virus type O and Asia1 mixed infection target fragments.
Example 2 kit for detecting foot and mouth disease Virus type O and Asia1 Mixed infection
The kit of example 2 is substantially the same as the kit of example 1, except that: the composition of the lysate in this example was 5% (mass fraction) of hydroxyquinoline, 4. mu.M of trichloroethyl phosphate, 2.5. mu.M of phenyl isothiocyanate, 2% (mass fraction) of polyvinylpyrrolidone, 0.5M of sodium chloride and 0.6M of sodium citrate. The components of the flushing fluid are trihydroxymethyl aminomethane 10 MuM, ethylenediamine tetraacetic acid 1 MuM and ethanol 80% (volume fraction); pH 7.0.
In the embodiment, cattle which are diagnosed to carry foot-and-mouth disease virus type O are taken as experimental animals, foot-and-mouth disease virus type O is taken as a positive control, sample RNA is extracted, RT-PCR amplification is carried out, and the agarose gel detection result of the RT-PCR amplification product is shown in figure 2 (the detection method is the same as the embodiment 1).
Example 3 kit for detecting foot and mouth disease Virus type O and AsiaI Mixed infection
The kit of example 3 is substantially the same as the kit of example 1, except that: the composition of the lysate in this example was 4% (mass fraction) of hydroxyquinoline, 5.2. mu.M of trichloroethyl phosphate, 1.5. mu.M of phenyl isothiocyanate, 3.5% (mass fraction) of polyvinylpyrrolidone, 0.4M of sodium chloride, and 0.8M of sodium citrate. The components of the flushing fluid are trihydroxymethyl aminomethane 10 MuM, ethylenediamine tetraacetic acid 1 MuM and ethanol 70% (volume fraction); the pH was 7.0.
In this example, cattle confirmed to carry foot-and-mouth disease virus Asia1 type were used as experimental animals, and foot-and-mouth disease virus Asia1 type was used as a positive control, sample RNA was extracted and subjected to RT-PCR amplification, and the result of agarose gel detection of the RT-PCR amplification product is shown in FIG. 2 (the detection method is the same as in example 1).
The kit of example 1 of the present invention was used to extract genomic RNA from different samples, and the concentration and purity of the extracted genomic RNA were determined, and the results are shown in the following table.
Table results of concentration and purity of genomic RNA extracted by the kit of the present invention
Note: RNA concentration unit is ng/ul; the absorbance ratio is the absorbance ratio (260/280) of the RNA at 260nm and 280nm, the purity of the RNA is judged according to the absorbance ratio, and the absorbance value is 1.8-2.0, which indicates that the purity of the extracted RNA is better.
Samples 1, 2, 3, 4, 5, 6, and 7 in the table are obtained by extracting approximately 1.02. mu.g, 1.09. mu.g, 1.04. mu.g, 1.09. mu.g, 0.99. mu.g, 1.09. mu.g, and 1.04. mu.g of genome from 100. mu.l of serum using the kit, respectively.
Examples of the experiments
1. Kit specificity experiment for detecting foot and mouth disease virus O type and Asia1 type mixed infection
The specificity of the RT-PCR kit for detecting the mixed infection of the foot-and-mouth disease virus type O and the Asia1 is verified by taking cattle which are diagnosed to carry the mixed infection of the foot-and-mouth disease virus type O and the Asia1 as experimental animals, and the method comprises the following steps:
(1) collection of samples
100 mul of cattle whole blood is collected and added into a centrifuge tube without RNA enzyme pollution for standby.
(2) Extraction of sample RNA (formulation of lysate and rinse solution same as in example 1)
(a) Adding 500. mu.l of lysate into 100. mu.l of whole blood, centrifuging at 12000rpm for 30s, and separating supernatant;
(c) transferring the supernatant into a centrifuge tube, adding 500 mu l of absolute ethyl alcohol, transferring the supernatant onto an RNA purification column in batches, and centrifuging the column at 12000rpm for 30s to obtain crude RNA;
(d) washing the RNA purification column with 500. mu.l of washing solution, and centrifuging at 12000rpm for 30 s;
(e) the RNA purification column is transferred to another centrifuge tube, 50 mul of ultrapure water is added, centrifugation is carried out at 12000rpm for 30s, the RNA of the virus and the tissue genome is obtained, and the RNA is preserved at the temperature of minus 20 ℃ for standby.
(3)RT-PCR
RT-PCR amplification was performed using sample RNA as a template, and RNA was extracted from control samples using A-, C-, SAT1 (south African I), SAT2 (south African II) and SAT3 (south African III) samples as controls, and RT-PCR amplification was performed in a reaction system of 25. mu.l: 5 × RTBuffer 5 μ l, 12.5 μ M dNTPs 5 μ l, 125 μ M upstream primer FMDOF and 125 μ M downstream primer FMDOR 1 μ l each, 125 μ M upstream primer FMDAsiaF and 125 μ M downstream primer FMDAsiaR 1 μ l each, 25U/μ l Taq enzyme 0.5 μ l, 2.5U/μ l RNase inhibitor 1 μ l, 2.5 μ M reverse transcriptase 1 μ l, ultrapure water 3.5 μ l and 5ng/μ l RNA 5 μ l.
The RT-PCR amplification program is as follows: 30min at 50 ℃ and 3min at 94 ℃; 35 cycles of 94 ℃ for 40s, 55 ℃ for 45s and 72 ℃ for 30 s; 3min at 72 ℃.
(4) Detection of RT-PCR amplification products
5 mul of RT-PCR amplification product and 5 mul of Loading Buffer are taken and mixed evenly, added into 1.0 percent agarose gel containing EB, added with control RT-PCR amplification product and DL2000DNA Marker at the same time, the voltage is 120V, electrophoresis is carried out for 35min, and then the result is observed in a gel imager (the result is shown in figure 3).
Agarose gel electrophoresis results show that the RT-PCR amplification products of the sample RNA of the invention have bands at 1350bp and 200bp in the RT-PCR amplification products, which are consistent with expectations, and prove that the RT-PCR amplification products of the invention are foot-and-mouth disease virus type O and Asia1 mixed infection target fragments. Meanwhile, specific bands do not appear in the comparative A-type, C-type, SAT1 (south African I), SAT2 (south African II) and SAT3 (south African III) sample RT-PCR amplification, which shows that the RT-PCR kit has good specificity.
2. Kit sensitivity experiment for detecting foot-and-mouth disease virus O type and Asia1 type mixed infection
The extracted RNA is serially diluted by 10 times at1 mu g/mu l, and the RNA at1 mu g/mu l, 100 ng/mu l, 10 ng/mu l, 1 ng/mu l, 100 pg/mu l, 10 pg/mu l, 1 pg/mu l, 0.1 pg/mu l, 0.01 pg/mu l and 0.001 pg/mu l is respectively amplified by an RT-PCR one-step method, and the result shows that the minimum detection limit of the foot-and-mouth disease virus O type and Asia1 type is 0.1 pg/mu l, which is 10 times higher than the sensitivity (1 pg/mu l) of the common RT-PCR two-step method.
SEQUENCE LISTING
<110> institute of zootechnics of academy of agricultural sciences of Henan province
<120> primers, kit and use method for detecting foot-and-mouth disease virus type O and Asia1 mixed infection
And uses thereof
<160>4
<170>PatentIn version 3.5
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<212>DNA
<213> Artificial sequence
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tggcctcaaa gactctcg 18
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<213> Artificial sequence
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caagctacag atcactttac 20
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<213> Artificial sequence
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ctgggtttta caaacctgtg 20
<210>4
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<212>DNA
<213> Artificial sequence
<400>4
ctctgacgtc acaattggc 19
Claims (10)
1. A primer for detecting foot-and-mouth disease virus type O and Asia1 mixed infection, which is characterized in that the primer comprises a primer pair FMDO and FMDAsia, wherein,
primer pair FMDO:
upstream primer FMDOF: 5'-TGGCCTCAAAGACTCTCG-3'
The downstream primer FMDOR: 5'-CAAGCTACAGATCACTTTAC-3'
Primer pair FMDAsia:
the upstream primer FMDAsiaF: 5'-CTGGGTTTTACAAACCTGTG-3'
Downstream primer FMDAsiaaR: 5'-CTCTGACGTCACAATTGGC-3' are provided.
2. A kit for detecting foot-and-mouth disease virus type O and Asia1 mixed infection is characterized in that the kit comprises a primer pair FMDO and FMDAsia, wherein,
primer pair FMDO:
upstream primer FMDOF: 5'-TGGCCTCAAAGACTCTCG-3'
The downstream primer FMDOR: 5'-CAAGCTACAGATCACTTTAC-3'
Primer pair FMDAsia:
the upstream primer FMDAsiaF: 5'-CTGGGTTTTACAAACCTGTG-3'
Downstream primer FMDAsiaaR: 5'-CTCTGACGTCACAATTGGC-3' are provided.
3. The kit for detecting mixed infection of foot-and-mouth disease virus type O and Asia1 according to claim 2, wherein the kit further comprises 5 XTT Buffer, dNTPs, Taq enzyme, RNase inhibitor, reverse transcriptase, ultra pure water, lysate and rinse.
4. The kit for detecting mixed infection of foot-and-mouth disease virus type O and Asia1 according to claim 3, wherein the kit further comprises a positive control and a negative control.
5. The kit for detecting mixed infection of foot-and-mouth disease virus type O and Asia1 according to claim 3, wherein the lysate comprises the following components: 3 to 5 percent of hydroxyquinoline, 3.2 to 5.2 mu M of trichloroethyl phosphate, 1.5 to 3.5 mu M of phenyl isothiocyanate, 1.3 to 3.5 percent of polyvinylpyrrolidone, 0.3 to 0.5M of sodium chloride and 0.6 to 0.9M of sodium citrate.
6. The kit for detecting mixed infection of foot-and-mouth disease virus type O and Asia1 according to claim 3, wherein the washing solution comprises the following components: 10 mu M of trihydroxymethyl aminomethane, 1 mu M of ethylene diamine tetraacetic acid and 60-80% (volume fraction) of ethanol; the pH was 7.0.
7. A method of using the kit for detecting mixed infection of foot-and-mouth disease virus type O and type Asia1 according to claim 3, comprising the steps of:
(1) collection of samples
(2) Extraction of sample RNA
(3)RT-PCR
And (3) carrying out RT-PCR amplification on the sample RNA, wherein the reaction system is 25 μ l: 5 × RT Buffer 5 μ l, 12.5 μ M dNTPs 5 μ l, 125 μ M upstream primer FMDOF and 125 μ M downstream primer FMDOR 1 μ l each, 125 μ M upstream primer FMDAsiaF and 125 μ M downstream primer FMDAsiaR 1 μ l each, 25U/μ l Taq enzyme 0.5 μ l, 2.5U/μ l RNase inhibitor 1 μ l, 2.5 μ M reverse transcriptase 1 μ l, ultrapure water 3.5 μ l and 5ng/μ l RNA 5 μ l;
the RT-PCR amplification program is as follows: 30min at 50 ℃ and 3min at 94 ℃; 35 cycles of 94 ℃ for 40s, 55 ℃ for 45s and 72 ℃ for 30 s; 3min at 72 ℃;
(4) detection of RT-PCR amplification products
And (3) carrying out agarose gel electrophoresis on the PCR amplification product, and observing the result under a gel imaging system.
8. The method for using the kit for detecting the mixed infection of foot-and-mouth disease virus type O and Asia1 as claimed in claim 7, wherein the collection of the sample is divided into the collection of solid animal tissue or the collection of liquid animal tissue; wherein,
the solid animal tissue collection method comprises the following steps: aseptically collecting 0.1-100mg animal tissue pathological material, and placing into a centrifuge tube without RNA enzyme pollution for later use;
the collection of the liquid animal tissue is divided into collection of secretion or collection of blood, and the collection method of the secretion comprises the following steps: collecting nasopharyngeal fluid with nasopharyngeal swab, and filling into centrifuge tube without RNA enzyme pollution; the blood collection method comprises the following steps: taking 10-100 μ l of whole blood, serum or plasma, and adding into a centrifuge tube without RNase pollution for standby.
9. The use method of the kit for detecting foot-and-mouth disease virus type O and Asia1 mixed infection according to claim 8, wherein the method for extracting the sample RNA comprises the following steps:
(1) grinding solid animal tissue, and directly extracting liquid animal tissue;
(2) adding 500 μ l lysate into 20-100mg of ground pasty animal tissue or 20-100 μ l of liquid animal tissue, centrifuging at 12000rpm for 30s, and separating supernatant;
(3) transferring the supernatant into a centrifuge tube, adding 500 mu l of absolute ethyl alcohol, transferring the supernatant onto an RNA purification column in batches, and centrifuging the column at 12000rpm for 30s to obtain crude RNA;
(4) washing the RNA purification column with 500. mu.l of washing solution, and centrifuging at 12000rpm for 30 s;
(5) the RNA purification column is transferred to another centrifuge tube, 50 mul of ultrapure water is added, centrifugation is carried out at 12000rpm for 30s, the RNA of the virus and the tissue genome is obtained, and the RNA is preserved at the temperature of minus 20 ℃ for standby.
10. Use of the kit for detecting mixed infection of foot and mouth disease virus type O and Asia1 according to any one of claims 2-6 for detecting mixed infection of foot and mouth disease virus type O and Asia 1.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102220436A (en) * | 2011-04-06 | 2011-10-19 | 珠海出入境检验检疫局检验检疫技术中心 | Method for detecting FMDV (Foot and Mouth Disease Virus) through real-time fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) |
| 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 |
| CN104195268A (en) * | 2014-09-15 | 2014-12-10 | 中国农业科学院兰州兽医研究所 | Reagent box for detecting aftosa A type, O type and Asia 1 type viruses and preparing method thereof |
| CN105112558A (en) * | 2015-08-06 | 2015-12-02 | 河南省动物疫病预防控制中心 | Foot and mouth disease viruses O, A and Asia I type ternary real time fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection kit |
| CN105506176A (en) * | 2015-11-26 | 2016-04-20 | 洛阳莱普生信息科技有限公司 | Foot and mouth disease virus O-type, A-type, AsiaI-type triple real-time fluorescent RT-PCR detection kit |
-
2016
- 2016-11-08 CN CN201610977701.XA patent/CN106498094A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102220436A (en) * | 2011-04-06 | 2011-10-19 | 珠海出入境检验检疫局检验检疫技术中心 | Method for detecting FMDV (Foot and Mouth Disease Virus) through real-time fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) |
| 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 |
| CN104195268A (en) * | 2014-09-15 | 2014-12-10 | 中国农业科学院兰州兽医研究所 | Reagent box for detecting aftosa A type, O type and Asia 1 type viruses and preparing method thereof |
| CN105112558A (en) * | 2015-08-06 | 2015-12-02 | 河南省动物疫病预防控制中心 | Foot and mouth disease viruses O, A and Asia I type ternary real time fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection kit |
| CN105506176A (en) * | 2015-11-26 | 2016-04-20 | 洛阳莱普生信息科技有限公司 | Foot and mouth disease virus O-type, A-type, AsiaI-type triple real-time fluorescent RT-PCR detection kit |
Non-Patent Citations (1)
| 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》 * |
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