CN109536644B - AS-PCR primer for identifying canine distemper virus wild strain and vaccine strain and application thereof - Google Patents
AS-PCR primer for identifying canine distemper virus wild strain and vaccine strain and application thereof Download PDFInfo
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Abstract
The invention provides an AS-PCR primer for identifying canine distemper virus wild strains and vaccine strains, wherein the AS-PCR primer comprises a forward primer and a reverse primer, and the nucleotide sequence of the forward primer is shown AS SEQ ID NO: 1, and the reverse primer is shown as SEQ ID NO: 2 is shown in the specification; the invention also provides a kit for identifying the canine distemper virus wild strain and the vaccine strain, wherein the kit comprises the AS-PCR primer; the invention also provides a method for identifying the canine distemper virus wild strain and the vaccine strain by adopting the AS-PCR primer; the primer adopts a 3-end mismatch principle to identify the Asia-I type and a vaccine strain: the detection results of Asia-I are positive, the vaccine strains are negative, the Asia-I and the vaccine strains in CDV can be accurately distinguished, and the method has the advantages of strong specificity, high sensitivity, good repeatability and the like.
Description
Technical Field
The invention relates to the technical field of population identification, in particular to an AS-PCR primer for identifying canine distemper virus wild strains and vaccine strains and application thereof.
Background
Canine Distemper Virus (CDV) belongs to a member of morbillivirus of paramyxoviridae, is a single-strand negative-strand non-segmented RNA virus, has a genome of 15690bp, and totally encodes 6 structural proteins, namely a nucleoprotein (N protein), a phosphoprotein (P protein) and a large protein (L protein) which are wound on the surface of viral RNA to form a nucleic acid complex, a hemagglutinin protein (H protein) and a fusion protein (F protein) which promote host cell membrane-viral envelope fusion, and a matrix membrane protein which is related to viral envelope. The gene sequence of each protein of the canine distemper virus genome is as follows: the 3 '-UTR-N-P (C, V) -M-F-H-L-UTR-5' canine distemper virus 2 envelope glycoprotein H proteins and F proteins, wherein the former is responsible for the adhesive binding of the virus and host cell receptor proteins, so that the F protein conformation with membrane fusion activity is changed, the envelope of the virus is fused with an infected cell membrane, and the first step of virus infected cells is started, so that the H gene is one of the determinants of pathogenicity in the process of canine distemper patellar infection. Meanwhile, the H gene is the gene with the lowest gene stability of the canine distemper virus, and according to the H gene sequence phylogenetic tree analysis of CDV of different strains, the CDV has geographical affinity and can be divided into a plurality of genotypes according to different geographical positions, wherein the genotypes comprise: asia type I (Asia-I), Asia type II (Asia-II), American type I (America-I) also known as vaccine type, American type II (America-II), European type (European), African type (Africa), etc.
Canine Distemper (CD) is an acute, highly contagious disease caused by CDV infection of dogs or other carnivores. Clinically, it is mainly characterized by biphase fever, erythra, conjunctivitis and central nervous system damage. Since the first report in 1809, the disease is distributed worldwide, and causes great harm to the canine industry, the fur-bearing animal breeding industry and the wild animal protection industry. The death rate of dogs, minks, foxes and the like caused by CD infection reaches 30-80 percent, and the death rate of ferrets reaches 100 percent. For the prevention and treatment of CD, no specific treatment method is available except for the regular immunization by using the vaccine. However, the main current CD control is attenuated vaccines, and the large-scale application of attenuated vaccines makes it very difficult to distinguish natural infection of Animals from vaccine immunity (DIVA). Therefore, a sensitive and specific detection method capable of identifying and diagnosing the strong and weak wild virus infection of the CDV is imperative.
At present, the CDV detection methods are various and comprise virus isolation culture, animal inoculation tests, serological diagnosis methods, RT-PCR, real-time fluorescent quantitative RT-PCR and the like. The virus separation and animal tests have the defects of long diagnosis period, complex operation, low detection rate and the like in different degrees; conventional serological tests are difficult to identify CDV wild virus infection and vaccination, and the Real time RT-PCR method has high requirements on experimental instruments and is not suitable for clinical detection. At present, the CDV clinical detection method mainly comprises colloidal gold detection and RT-PCR, but wild strains and vaccine strains cannot be distinguished.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides an AS-PCR primer for identifying canine distemper virus wild strains and vaccine strains and application thereof, wherein the primer adopts a 3-terminal mismatch principle to identify Asia-I type and vaccine strains: the detection results of Asia-I are positive, the vaccine strains are negative, the Asia-I and the vaccine strains in CDV can be accurately distinguished, and the method has the advantages of strong specificity, high sensitivity, good repeatability and the like. Has important significance in early detection and rapid diagnosis of canine distemper.
One of the purposes of the invention is to provide an AS-PCR primer for identifying canine distemper virus wild strains and vaccine strains, wherein the AS-PCR primer comprises a forward primer and a reverse primer, and the nucleotide sequence of the forward primer is shown AS SEQ ID NO: 1, and the reverse primer is shown as SEQ ID NO: 2, respectively.
The invention also aims to provide a kit for identifying canine distemper virus wild strains and vaccine strains, wherein the kit comprises the AS-PCR primer.
The invention also aims to provide a method for identifying canine distemper virus wild strains and vaccine strains, which comprises the following steps:
and 3, taking the PCR product, performing agarose gel electrophoresis, observing the result in a gel imaging system, and judging: wild strains are all positive, and vaccine strains are all negative.
Preferably, the PCR reaction system in the step 2 adopts a 50 μ L reaction system: 10 μ M is as shown in SEQ ID NO: 1, 2 μ L of forward primer, 10 μ M is shown as SEQ ID NO: 2, 2 mu L of reverse primer, 45 mu L of Prime gold premixed solution MIX and 1 mu L of template to be detected.
Preferably, the PCR reaction conditions in step 2 are as follows: pre-denaturation at 98 ℃ for 2min before circulation, wherein each circulation comprises denaturation at 98 ℃ for 10s, annealing at 51 ℃ for 10s and extension at 72 ℃ for 10s, 30 cycles are set, and extension at 72 ℃ for 1min is carried out after the circulation is finished.
Preferably, 5ul of PCR products in step 3 are subjected to electrophoresis on a 1% agarose gel.
The invention also aims to provide application of the AS-PCR primer in preparation of a reagent for identifying canine distemper virus wild strains and vaccine strains.
The fifth purpose of the invention is to provide application of the kit in preparation of reagents for identifying canine distemper virus wild strains and vaccine strains.
The invention has the beneficial effects that:
1. the AS-PCR primer for identifying the canine distemper virus wild strain and the vaccine strain adopts a 3-terminal mismatch principle to identify the Asia-I type and the vaccine strain: the detection results of Asia-I are positive, the vaccine strains are negative, the Asia-I and the vaccine strains in CDV can be accurately distinguished, and the method has the advantages of strong specificity, high sensitivity, good repeatability and the like. The primer sequence designed by the invention has important significance in early detection and rapid diagnosis of canine distemper.
2. The method for identifying the canine distemper virus wild strain and the vaccine strain provided by the invention has lower requirements on used instruments and equipment, and can be carried out in a general molecular biology laboratory: the wild strain and the vaccine strain can be well identified only by using the traditional PCR detection method without special instruments.
Drawings
FIG. 1 shows the results of reaction temperature optimization and sensitivity test;
wherein lanes 1-4 in (A) are at a dilution of 2 at 51 deg.C, 54 deg.C, 57 deg.C, and 60 deg.C, respectively0A lower strip; lanes 5-8 are at 51 deg.C, 54 deg.C, 57 deg.C, 60 deg.C at 2 degree of dilution1A lower strip; lanes 9-12 were at 51 deg.C, 54 deg.C, 57 deg.C, 60 deg.C at 2 degree of dilution 2A lower strip; lanes 13-16 are at 51 deg.C, 54 deg.C, 57 deg.C, 60 deg.C at 2 degree of dilution3A lower strip; lanes 17-20 are at 51 deg.C, 54 deg.C, 57 deg.C, 60 deg.C at 2 dilution4A lower strip; m is Marker;
(B) lanes middle 1-4 are at 51 deg.C, 54 deg.C, 57 deg.C, 60 deg.C at 2 degree of dilution5A lower strip; lanes 5-8 are at 51 deg.C, 54 deg.C, 57 deg.C, 60 deg.C at 2 degree of dilution6A lower strip; lanes 9-12 were at 51 deg.C, 54 deg.C, 57 deg.C, 60 deg.C at 2 degree of dilution7A lower strip; lanes 13-16 are at 51 deg.C, 54 deg.C, 57 deg.C, 60 deg.C at 2 degree of dilution8A lower strip; m is Marker;
FIG. 2 shows the results of the specificity test; 1, 2, 3, 4, known sequencing fragments (Asia-type I); 5, 6 are samples that were not sequenced but confirmed to be suffering from canine distemper virus; 7, 8 are parvovirus samples; 9, 10 and 11 are vaccine strains, and M is Marker;
FIG. 3 shows the results of the repeatability tests; 1-3: 3 experiments are respectively carried out by using 3 parts of RNA (Asia-I type) extracted from secretion of the same canine distemper sick animal in different time periods; 4-6: repeating 3 times of experiments with RNA (Asia-I type) extracted from secretion of the same canine distemper diseased animal in the same time period; 7-9: 3 experiments are respectively carried out by using 3 parts of RNA (vaccine strain) extracted from secretion of the same healthy animal at different time periods after vaccine injection; 10-12: dividing RNA (vaccine strain) extracted from secretion of the same healthy animal in the same time period after vaccine injection into 3 parts for 3 times of repeated experiments; and M is Marker.
Detailed Description
EXAMPLE 1 design of AS-PCR primers
1. The wild strains and the vaccine strains are compared and analyzed, and the H proteins of the wild strains and the vaccine strains are found to have obvious difference on average between the nucleotide level and the amino acid water, wherein the difference between the nucleotide level and the amino acid water level is 7-10%, the difference between the amino acid level and the amino acid water level is 8-11%, and the difference between the wild strains in a group is 0-7%. Through collecting canine distemper wild strains 6 and 3 clinical common canine distemper vaccines, sequencing genes of the wild strains 6 and the vaccine strains 3, and typing sequencing results, the 6 vaccine strains are all Asia-I type, and the 3 vaccine strains are America-I type and America-II type.
2. And 427 CDV-H sequences (listed below) were downloaded in GenBank for typing, of which 173 were Asia-I. Combining 173 pieces (the CDV-H sequence of Asia-I type downloaded by GenBank), 6 strains of wild strains and 3 strains of vaccine strains H gene sequencing results. Combining 173 (CDV-H sequences of Asia-I type downloaded by GenBank), 6 strains of wild strains and 3 strains of vaccine strains H gene sequencing results are compared and analyzed: finding a wild strain valine V (GUU) → vaccine strain isoleucine I (AUU) at the 157 th amino acid site of the H gene; and 3-terminal mismatch principle is combined: A/G (primer/template), G/A, C/C, A/A are mismatch types that effectively prevent strand extension reactions. Specifically, the method comprises the following steps:
(1) The CDV-H gene sequence is searched on NCBI, 427 pieces are found, and GenBank is respectively: AF AY EUEU EU EU EU EU EU EU EU EU EU EU EU EU EU EU EU EU EU EU EU EU EU EU EU EU EU EU EU EU EU EU EU EU KF KF KF KF KF KF KF KF KF KF KF KF KF KF KF KF KF KF KF KF KF KF KF KF KF KF KF KF KF KF KF KF KF KF KF KF KF KF KF KF KF KF JX JX JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ JQ DQ DQ DQ DQ DQ DQ DQ DQ HQ AB KP KP KP KP KP KP KM KM KJ AB AB JN JN JN JN JN JN HM HQ GU HQ HQ HQ AB FJ GU HM HQ HM FJ GU HM AY HM FJ FJ FJ GU GU HM HM HM AY HM AF FJ FJ FJ FJ GU GU HM HM HM HM AF AB AB AB AB AB AB DQ KX KX GQ GQ GQ GQ FJ FJ FJ AB AB AB AB FJ FJ FJ FJ FJ AF LC KJ FJ FJ FJ FJ AF FJ HQ AQ GQ GQ GQ FJ FJ FJ FJ FJ FJ FJ AF AQ FJ FJ AF AB AB KT KJ KJ KJ KJ FJ FJ MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF GQ GQ GQ MF HM AY AB KT AB AB KTAB AB KT KJ KJ KJ KT JX FJ FJ FJ FJ MF EU MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF.
(2) And 427 CDV-H are typed and arranged to obtain 176 Asia-I, and the GenBank respectively comprises: EU AY EU KU KU KF KU KU KU KU EU EU EU EU EU KF KF KU KC KF KU DQ EU EU EU EU EU EU EU AB EU EU FJ EU EU EU EU EU EU EU EU EU EU EU EU EF JF KF KF KF KF KF KF AB DQ DQ AB AB LC LC HQ KU HQ KJ KX KX KX KP KP KP KP KP KP KP KP KP LC AB AB AFJ FJ FJ FJ FJ FJ FJ FJ AB AB AB AB EF KP KP KP KP KP KP KP KP KP KP KP KP KP KP KP KP KP LC LC DQ AB XE HM FJ FJ FJ FJ FJ FJ FJ FJ FJ FJ FJ FJ FJ AB EF HM HM HM HM HM AB EU EF HM HM HM HM HM HM HM HM HM MF100808.1 EU716072.1 MF100807.1 MF100805.1 MF100806.1 MF100811.1 MF100801.1 KT341046.1 KT341045.1 KT119347.1 KT341044.1 MF100802.1 KT341047.1 MF100800.1 HM623895.1 FJ848532.1 FJ848534.1 MF100803.1 MF100799.1 MF100804.1 MF100798.1 KP872502.1 KT341048.1 FJ848533.1 JX276746.1 FJ848531.1 FJ848535.1 KT001210.1 KT001209.1 KJ685550.1 KJ 660069.1. 173 sequences in Asia-I of 176 CDV-H are complete, 6 sequenced wild strains and 3 strain vaccine strains (179 Asia-I and 3 strain vaccine strains) are subjected to CDS and base difference alignment and are combined with the 3' end mismatching rule of AS-PCR primers [ A/G (primer/template), G/A, C/C, A/A are mismatching types which effectively prevent chain extension reaction ], a site capable of designing AS-PCR specific primers is obtained, screening is carried out, and finally, on the 157 th amino acid on H gene, vaccine-1, vaccine-2, vaccine-3 and GQ332533.1, GQ332535.1 is isoleucine (I), the other 177 Asia-I are all valine (V), namely Asia-I → vaccine strain is at 157 th amino acid V → I, the base conversion is from G → A at the 469 th base of H gene, because the distinguishing primer can distinguish the primer of the canine distemper virulent and attenuated strains, namely the primer capable of amplifying canine distemper Asia-I strain but not capable of amplifying vaccine strain, the 3' end primer is determined to be G [ Asia-I (G/G) → vaccine strain (G/A) ]. The downstream primer is a conventional primer, namely a part with small difference on the H gene of the canine distemper virus.
The sequence of the AS-PCR primer is AS follows:
a forward primer: 5'-TTAAAATGATTAATGACACTATGTG-3' (shown in SEQ ID NO: 1)
Reverse primer: 5'-CCTGACARGGCAAGAA-3' (shown in SEQ ID NO: 2)
EXAMPLE 2 application of AS-PCR primers
1. Extraction of RNA
Asia-I type CDV wild strains, CDV attenuated vaccine strains and parvovirus samples are all stored in laboratories of Huazhong university of agriculture. The material to be detected is clinical case samples which are submitted from different pet hospitals in central China.
Total RNA of the test strain was extracted according to the RNAioso Plus (Takara, Japan) protocol, as follows:
(1) taking a proper amount of tissues or secretions, adding liquid nitrogen and grinding into powder;
(2) adding trizol to the ground tissue in an amount such that the volume of trizol is 10 times that of the powder;
(3) mixing, standing at room temperature for 5min, adding 1ml homogenate into 1.5ml RNase-free centrifuge tube, centrifuging at 4 deg.C, 12000g for 10min
(4) Adding the supernatant into a new RNase-free 1.5ml centrifuge tube, adding 200 μ L chloroform, shaking and mixing for 15s (no vortex), standing at room temperature for 5min, and allowing to separate;
(5) centrifuging at 4 deg.C for 10min at 12000g, dividing into 3 layers, collecting 500ml of the uppermost colorless aqueous phase, and transferring into a new 1.5ml centrifuge tube without RNase;
(6) Adding 500ml isopropanol, violently reversing and mixing uniformly, standing at room temperature for 10min, centrifuging at 4 ℃, centrifuging at 12000g for 15min, and removing supernatant;
(7) adding 1ml of 75% ethanol (prepared with DEPC water) into the white precipitate, washing the precipitate, gently shaking the centrifuge tube, suspending the precipitate, centrifuging at 4 deg.C for 12000g for 5min, and discarding the supernatant;
(8) repeating the step (7);
(9) drying the precipitate at room temperature for 10 min;
(10) RNA was dissolved in 20-50. mu.L of DEPC water and stored at-80 ℃.
2. Synthesis of cDNA
The Thermo RevertAid First stand cDNA Synthesis Kit was used, and the detailed procedures were referred to the instruction manual.
(1) Reverse transcription reaction: the following mixed solutions were prepared on ice in sterile nuclease-free RCR tubes, as shown in table 1.
TABLE 1
(2) The reaction was carried out on a PCR instrument at 65 ℃ for 5 min. (optional, optimization step)
(3) The mixture of template RNA/primer, etc. was thoroughly mixed by centrifugation for several seconds.
(4) The following reverse transcription reaction solution was added to the PCR tube and the PCR was performed on ice as shown in Table 2:
TABLE 2
(5) The total amount of the above 20. mu.l was mixed, centrifuged briefly, and reverse transcription was performed on a PCR instrument under the following conditions: 5min at 25 ℃; 60min at 42 ℃; 5min at 70 ℃; can be stored at-20 deg.C for one week, and can be stored at-70 deg.C for a long period.
3. PCR amplification
TABLE 3
The PCR reaction system is 50 mu L, wherein 10 mu M is shown as SEQ ID NO: 1, 2 μ L of forward primer, 10 μ M is shown as SEQ ID NO: 2, 2 mu L of reverse primer, 45 mu L of Prime gold premixed solution MIX and 1 mu L of template to be detected.
The reaction conditions were as follows: pre-denaturation at 98 ℃ for 2min before circulation, wherein each circulation comprises denaturation at 98 ℃ for 10s, annealing at 51 ℃ for 10s and extension at 72 ℃ for 10s, 30 cycles are set, and extension at 72 ℃ for 1min is carried out after the circulation is finished.
4. Gel electrophoresis: the 5. mu.L PCR product was electrophoresed through 1% agarose gel and the results were visualized on a gel imaging system. Specific test results: wild strains are positive, and vaccine strains are negative. As shown in fig. 2.
Examples of the experiments
1. Optimizing the reaction temperature:
the premixed primer MIX of the Primulaces gold medal is used in the PCR reaction, only the annealing temperature needs to be optimized, the temperature gradient is designed to be combined with the Tm value of the primer, the primer is firstly searched at the temperature gradients of 51 ℃, 54 ℃, 57 ℃ and 60 ℃, the product is observed in 1% agarose gel electrophoresis, and as shown in figure 1, the optimal temperature is determined to be 51 ℃.
2. Sensitivity test:
the cDNA of the wild strain is sequentially diluted in equal volume, namely 20，21，22，23，24，25，26，27，28Fold dilution, shown in FIG. 1, dilution 128 (2)7) Fragments are still visible.
3. And (3) specific detection:
and (4) cutting and recovering the amplified sample tape, sequencing and verifying. The sequences were analyzed using DNAStar software and BLAST analyzed in GenBank to verify the specificity of the amplified products.
The established detection method has positive detection results on Asia-I, and the vaccine strains are negative, as shown in figure 2, and the Asia-I and the vaccine strains in the CDV can be accurately distinguished.
4. And (3) repeatability test:
3 parts of secretion of the same canine distemper diseased animal in different time periods are used for extracting RNA under the same reaction condition, and after reverse transcription, detection is carried out by the method of example 2 so as to detect batch repeatability. The secretion of the same canine distemper diseased animal in the same time period and the extracted virus RNA are repeatedly tested for 3 times: i.e., after reverse transcription, independent assays were performed using the method of example 2 under the same reaction conditions to examine batch repeatability.
The same healthy animal after vaccine injection secretion extraction of RNA (vaccine strain) in different time period, equal amount of 3 parts, extracted virus RNA, reverse transcription, in the same reaction conditions by example 2 method for independent detection, to detect batch-to-batch repeatability. RNA (vaccine strain) extracted from the secretion of the same healthy animal after injection of the vaccine was repeated 3 times: that is, RNA was extracted, reverse transcribed, and then detected by the method of example 2 to detect the in-batch reproducibility.
As shown in fig. 3, there was no significant difference between the 3 trials repeated within a batch and the 3 trials repeated between batches.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> university of agriculture in Huazhong
<120> AS-PCR primer for identifying canine distemper virus wild strain and vaccine strain and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ttaaaatgat taatgacact atgtg 25
<210> 2
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Claims (10)
1. The AS-PCR primer for non-diagnosis purpose of identifying Asia-I type canine distemper virus wild strains and vaccine strains is characterized by comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown AS SEQ ID NO: 1, and the reverse primer is shown as SEQ ID NO: 2, the sequence shown in SEQ ID NO: 2 is base A or G.
2. A kit for non-diagnostic purposes for identifying Asia-I canine distemper virus wild strains and vaccine strains, said kit comprising AS-PCR primers according to claim 1.
3. The kit of claim 2, wherein the kit further comprises a premix MIX.
4. The kit of claim 2, further comprising a positive control and a negative control, wherein the positive control comprises plasmids containing genes of canine distemper virus wild strains or vaccine strains respectively, and the negative control is RNase-free water.
5. A method for identifying wild strains of Asia-I canine distemper virus from vaccine strains for non-diagnostic purposes, said method comprising the steps of:
step 1, extracting total RNA of a virus strain to be detected, and synthesizing cDNA (complementary deoxyribonucleic acid) by using the extracted RNA as a template through reverse transcription reaction to prepare a template to be detected;
step 2, carrying out PCR amplification on a PCR reaction system consisting of the template to be detected obtained in the step 1, the AS-PCR primer in the claim 1 and the MIX;
and 3, taking the PCR product, performing agarose gel electrophoresis, observing the result in a gel imaging system, and judging: wild strains are all positive, and vaccine strains are all negative.
6. The method of claim 5, wherein the PCR reaction system in the step 2 adopts a 50 μ L reaction system: 10 μ M is as shown in SEQ ID NO: 1, 2 μ L of forward primer, 10 μ M is shown as SEQ ID NO: 2, 2 mu L of reverse primer, 45 mu L of Prime gold premixed solution MIX and 1 mu L of template to be detected.
7. The method of claim 5, wherein the PCR reaction conditions in step 2 are as follows: pre-denaturation at 98 ℃ for 2min before circulation, wherein each circulation comprises denaturation at 98 ℃ for 10s, annealing at 51 ℃ for 10s and extension at 72 ℃ for 10s, 30 cycles are set, and extension at 72 ℃ for 1min is carried out after the circulation is finished.
8. The method of claim 5, wherein 5 μ L of the PCR product in step 3 is subjected to electrophoresis on a 1% agarose gel.
9. Use of the AS-PCR primer of claim 1 in the preparation of a reagent for non-diagnostic purposes for identifying Asia-I canine distemper virus wild strains and vaccine strains.
10. Use of a kit according to any one of claims 2 to 4 in the manufacture of a reagent for non-diagnostic purposes for identifying wild strains of Asia-I canine distemper virus from vaccine strains.
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