CN103131795B - Primer and kit for identifying marek's disease virus serum 1-type vaccine virus - Google Patents
Primer and kit for identifying marek's disease virus serum 1-type vaccine virus Download PDFInfo
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Abstract
The invention provides a primer and a kit for identifying a marek's disease virus serum 1-type vaccine virus. Nucleotide sequences of the primer for identifying the marek's disease virus serum 1-type vaccine virus are respectively shown by a sequencer identity number one (SEQ ID NO.1) and a sequencer identity number two (SEQ ID NO.2), and the kit contains the primer. The primer and the kit for identifying the marek's disease virus serum 1-type vaccine virus can be used for identifying a marek's disease vaccine virus 814 strain and a marek's disease vaccine virus CVI988 strain. The primer is used for carrying out a polymerase chain reaction (PCR), two bands including a band of 250bp and a band of 750bp can be added to the marek's disease vaccine virus 814 strain, only one band of 250bp is added to the marek's disease vaccine virus CVI988 strain, and other marrk's disease serum 1-type viruses are not provided with purpose bands. The kit for identifying the marek's disease virus serum 1-type vaccine virus has the advantages of being accurate in detection, high in sensitivity, good in specificity, convenient and rapid, and being capable of identifying the marek's disease vaccine virus 814 strain and the marek's disease vaccine virus CVI988 strain of the marek's disease virus serum 1-type vaccine virus.
Description
Technical field
The present invention relates to field of biological product, particularly relate to for differentiating primer and the test kit of marek's disease virus serum 1 type vaccine virus.
Background technology
Marek (Marek ' s disease, MD) have another name called neural lymphomatosis (neurolymphomatosis), that caused by marek's disease virus a kind of has hyperinfection, lymphadenosis tumor disease, take to the single or multiple histoorgan generation monocyte infiltrations of the peripheral nerve of chicken, sexual gland, iris, various internal organs, muscle and skin is feature, causes above-mentioned each Organ and tissue to form tumour.Sick chicken is common to become thin, limb paralysis, and often has acute death.In etiology, can distinguish with other lymph sample neoplastic diseases of chicken.
In China, Marek serum 1 type vaccine virus comprises 814 strains and CVI988 strain at present.814 make no distinction of rank with CVI988 on immune efficacy, but domesticly use clinically 814 and during CVI988, cannot carry out the two service condition to vaccinated flock and effectively distinguish, thereby easily produce and obscure and dispute.Therefore, set up a kind of can distinguish fast 814 with CVI988 vaccine, even distinguish 814 with the simple and quick method of the wild strong poison of other MDV serum 1 types, using and promoting most important the MDV disease vaccine 814 of China's independent research.Meanwhile, be no matter 814 or the use of CVI988, clinically, give always MD really diagnosis broken belt carried out very large difficulty, therefore, MDV vaccine virus is distinguished in research and wild malicious differential diagnosis receives great concern always.For example there is report to have continuous 5 nucleotide deletions for the upstream promoter sequence of the genome PP38 of CVI988, and other strains comprise that 814 do not find such disappearance, therefore for this disappearance, the synthetic two pairs of primers of design, the pair of primers CVI988 that can only increase, pair of primers can increase and comprise other MD serum one C-type virus Cs of 814 in addition, after a PCR, CVI988 and other virus can be distinguished, but cannot distinguish vaccine virus 814 strains or wild poison.Therefore,, in production and scientific research, the chicken group who used 814 vaccines can't be carried out to MDV wild virus infection etiologic diagnosis really.By real time quantitative PCR method, detecting chicken peripheral blood lymphocyte Marek’s disease virus gene group DNA carrying capacity confirms, there is certain difference in the duplicating dynamics of strong and weak strain, can be used as the standard of MD early diagnosis, but more difficult the applying in actual production of this method gone.Further differentiation also needs PCR product to check order.
2010, Wei equality has been developed " discrimination method of a kind of differentiating Marek's disease virus vaccine strain and virulent strain ", the principle of work of the method: the primer that designs a pair of uniqueness, can effectively increase to all MD serum 1 type virus, different virus amplification goes out the segment of different sizes: CVI988 and 814 vaccines, amplify the band of 730 left and right, and other wild malicious MDV amplify more than 930 big bands.Therefore, the method for Wei Ping can be carried out a PCR differentiation the strong poison of the very close vaccine virus of genome and open country, has solved and has failed at present to distinguish fast MDV vaccine virus and a strong malicious difficult problem.But the method still can not be distinguished CVI988 and 814 strains in serum 1 type vaccine virus.
Summary of the invention
The object of the present invention is to provide for differentiating the primer of marek's disease virus serum 1 type vaccine virus.
Another object of the present invention is to be provided for differentiating the test kit of marek's disease virus serum 1 type vaccine virus.
For achieving the above object, by at present disclosed MDV complete sequence carry out in detail relatively and analyze that (reference sequences of the present invention number of logging in NCBI is respectively: CVI988-DQ530348, GA-AF147806, LMS-JQ314003, MD5-AF243438, MD11-AY510475, RB-1B-EF523390, MD814-JF742597, JF742597) design the primer that can differentiate marek's disease virus serum 1 type vaccine virus.
Provided by the invention for differentiating the primer of marek's disease virus serum 1 type vaccine virus, comprise the nucleotide sequence shown in SEQ ID NO.1 ~ 2.
The invention provides the application of the primer shown in SEQ ID NO.1 ~ 2 in differentiating marek's disease virus serum 1 type vaccine virus.
The invention provides a kind of diagnostic reagent of differentiating Marek serum 1 type vaccine strain, comprise the primer shown in SEQ ID NO.1 ~ 2.
The present invention also provides the test kit that contains the primer of nucleotide sequence as shown in SEQ ID NO.1 ~ 2.
Test kit of the present invention, is to adopt the primer of nucleotide sequence as shown in SEQ ID NO.1 ~ 2 to carry out pcr amplification, and the sample that has the amplification of object band is that marek's disease virus serum 1 type vaccine virus is positive, otherwise negative.
Further, adopt above-mentioned primer to carry out after pcr amplification, the sample that only has length and be the amplified production band of 250bp is the strain of marek's disease virus serum 1 type vaccine strain CVI988, and the sample that has length and be 250bp and two special product bands of 750bp is 814 strains of marek's disease virus serum 1 type vaccine virus.
Further, above-mentioned PCR reaction conditions is: 95 ℃ of 3min; 94 ℃ of sex change 30sec, 60 ℃ of annealing 30sec, 72 ℃ of 1min, circulate 40 times; 72 ℃ of 10min.
Further, the present invention also provides the test kit that contains the 2 pair primers of nucleotide sequence as shown in SEQ IDNO.1 ~ 2 and SEQ ID NO.3 ~ 4.
The above-mentioned test kit that contains 2 pairs of primers, its working routine is:
1) primer shown in sample DNA application SEQ ID NO.3 ~ 4 is carried out to PCR reaction, the sample that has length and be the amplified production band of 277bp is that marek's disease virus is positive, otherwise negative;
2) the sample DNA application nucleotide sequence of the step 1) positive is carried out to PCR reaction again as the primer of SEQ IDNO.1 ~ 2, the sample that only has length and be the amplified production band of 250bp is the strain of marek's disease virus serum 1 type vaccine strain CVI988, and the sample that has length and be 250bp and two special product bands of 750bp is 814 strains of marek's disease virus serum 1 type vaccine virus.
Wherein, in step 1), PCR reaction conditions is: 94 ℃ of 3min; 94 ℃ of sex change 30sec, 55 ℃ of annealing 30sec, 72 ℃ of 30s, circulate 35 times; 72 ℃ of 10min.
Beneficial effect of the present invention is: use primer provided by the invention to carry out PCR reaction, can differentiate accurately and rapidly vaccine virus 814 and the CVI988 of Marek serum 1 type virus, and highly sensitive, high specificity, can occur for investigation chicken group reason, investigation chicken group early infection MDV epidemiology, the detection of environment virus and the prevention of MD and the control of poison, disease by force of MDV immuning failure thering is wide market using value.
Accompanying drawing explanation
Fig. 1 is that application specific primer 814-3 and 814-4 carry out PCR method and detect CVI988,814 electrophorograms, M.DL2000DNA marker wherein, 1.814,2.HVT (Fc-126), 3.CVI988,4.SB1,5. strong malicious GA.
Fig. 2 is universal primer amplification figure, wherein, and M.DL2000DNA marker, 1-2.814,3-4.CVI988,5.HVT (Fc-126), 6.SB1,7. strong malicious GA.
Fig. 3 diagnostic primers specific test result, wherein, M is DL2000; 1 negative contrast; 2 is MDV814 strain positive control; 3 is CVI988 strain; 4 is NDV; 5 is IBV; 6 is AIV H5; 7 is AIV H9; 8 is IBDV; 9 is REOV; 10 is ILTV; 11 is IBHV; 12 is CAV.
Fig. 4 is diagnostic primers sensitivity test result, and wherein, M is DL2000; 1-8 is universal primer uMDv-1/2 detected result, and wherein 1-8 is respectively MD814 strain 10
7~ 1PFU extent of dilution; 9-16 is diagnostic primers 814-3 and 814-4 primer PCR detected result, and wherein 9-16 is respectively MD814 strain 10
71PFU.
Fig. 5 is diagnostic primers replica test result, and wherein M is DL2000; 1-6 is 6 different batches of MD814 strain; 7-12 is 6 different batches of CVI988 strain.
Fig. 6 is universal primer specific test result, and wherein, M is DL2000; 1MD814 strain positive control; 2 is NDV; 3 is IBV; 4 is AIV H5; 5 is AIV H9; 6 is IBDV; 7 is REOV; 8 is ILTV; 9 is IBHV.
Fig. 7 is universal primer replica test result, and wherein M is DL2000; 1-3 is 3 batches of 814 strains; 4-6 is CVI9883 different batches.
Fig. 8 is clinical sample diagnostic primers detected result, and wherein, M is DL2000; 1: sample 1,2: sample 2; 3: sample 3.
Fig. 9 is clinical sample universal primer detected result, and wherein M is DL2000; 1: sample 1,2: sample 2; 3: sample 3.
Embodiment
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the modification that the inventive method, step or condition are done or replacement, all belong to scope of the present invention.
If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art.If do not specialize, in embodiment, reagent used is commercially available.
The design of embodiment 1 primer
By at present disclosed MDV complete sequence carry out in detail relatively and analyze, reference sequences of the present invention number of logging in NCBI is respectively: CVI988-DQ530348, GA-AF147806, LMS-JQ314003, MD5-AF243438, MD11-AY510475, RB-1B-EF523390, MD814-JF742597, the special primer that goes out to differentiate marek's disease virus serum 1 type vaccine virus 814 and CVI988 for the sequences Design of Marek 814 vaccine virus.
The nucleotide sequence of the primer of the discriminating marek's disease virus serum 1 type vaccine virus of design is as follows:
Upstream primer 814-3:GGAAGAAACAGCGTACTTGTACCGG-3 ' (SEQID NO.1)
Downstream primer 814-4:5 '-CCCCGGAGTTCACTGTATCGTACGT-3 ' (SEQID NO.2)
The present invention has also designed a pair of universal primer, and this primer can be identified marek's disease virus by PCR method.The sequence of this universal primer is as follows:
uMDv-1:5’-CGCCCACCACGATTACTACCT-3’(SEQ?ID?NO.3)
uMDv-2:5’-AGACCACGCGGCCCTGAGAC-3’(SEQ?ID?NO.4)
The object fragment of pcr amplification is 277bp, and sample DNA is from marek's disease virus.
The foundation of embodiment 2PCR discrimination method
1, the processing of strain
From liquid nitrogen, take out frozen strain: 814 strains of marek's disease virus serum 1 type living vaccine and CVI988, the 2 type living vaccine SB1 strains of marek's disease virus serum, marek's disease virus Serotype-3 living vaccine HVT(Fc-126) (814 strains of serum I type are from Beijing Hua Doushi China biological products company limited, other strains are purchased from Intervet Internat B. V., put into rapidly 37 ℃ of water-baths and make its thawing, after sucking-off, join containing in the centrifuge tube of 10 milliliters of serum-free cell culture mediums (DMEM), the centrifugal 5min of room temperature 1500r/min, supernatant discarded, with 5mL, contain after the DMEM Eddy diffusion of 10%FBS, be transferred to and in T25 Tissue Culture Flask, carry out virus recovery and cultivation.After 24 hours, carry out cytopathy observation every day.To within 36-96 hour, there is the sample of pathology, carry out the extraction of cell total genomic dna.
2, the extraction of DNA adopts commercially available reagent box
Adopt commercially available reagent box: TIANamp Virus DNA/RNA Kit(virus genom DNA/RNA extracts test kit, catalog number (Cat.No.): DP315).Preparation work before test kit is used: the configuration of Carrier RNA solution: to the RNase-free ddH2O that adds 310 μ l in the pipe of Carrier RNA lyophilized powder of 310 μ g is housed, after Carrier RNA thoroughly dissolves, use EP without the 0.5ml of RNase to be distributed into 10 pipes and mark is frozen in-30 ℃ of refrigerators.To the dehydrated alcohol that adds 60ml in the RW washing lotion reagent bottle of 15ml.
The experimental procedure of DNA extraction:
A. with pipettor, 20 μ l Proteinase Ks are added in a clean 1.5ml centrifuge tube;
B. in centrifuge tube, add 200 μ l cell suspensions (noting: sample needs balance to room temperature).
If sample volume is less than 200 μ l, can add 0.9%NaCl solution to supplement; Preparation Carrier RNA working fluid: damping fluid GB mixes with Carrier RNA solution, formula: the Carrier RNA solution of the GB+6.2 μ l of 220 μ l;
C. add 200 μ l Carrier RNA working fluids, lid upper tube cap, vortex vibration mixes for 15 seconds.
D. sample is placed on 56 ℃ and hatches after 15 minutes, and wink is centrifugal to collect the liquid that is attached to tube wall and pipe lid;
E. add 250 μ l dehydrated alcohols (now may occur flocks), lid upper tube cap vortex vibration 15 seconds;
F. in room temperature (15 25 ℃ of –), place 5 minutes.Attention: if environment is higher than 25 ℃, ethanol needs to add after ice bath again;
G. centrifugal rear collection of wink is attached to the liquid of tube wall and pipe lid;
H. the solution in centrifuge tube and flocks being all transferred to RNase-free adsorption column CR2(adsorption column is placed in collection tube), lid upper tube cap, centrifugal 1 minute of 8000rpm (~ 6000 * g), abandons waste liquid, and adsorption column is put back in collection tube; Attention: fail all centrifugally to collection tube as the liquid on adsorption column, please strengthen rotating speed, extend centrifugation time to liquid and transfer in collection tube completely;
I. carefully open adsorption column lid, please first check whether added dehydrated alcohol before adding 500 μ l solution RW(to use), lid upper tube cap, standing 2 minutes, centrifugal 1 minute of 8000rpm (6000 * g), abandoned waste liquid, and adsorption column is put back to collection tube;
J. repeating 9) step is once;
K. carefully open adsorption column lid, add 500 μ l dehydrated alcohols, lid upper tube cap, centrifugal 1 minute of 8000rpm (6000 * g), abandons waste liquid; Attention: the residual of ethanol may impact subsequent experimental;
L. adsorption column is put back in collection tube, and centrifugal 3 minutes of 12,000rpm (~ 13,400 * g), becomes dry adsorption film completely, abandons waste liquid;
M. adsorption column is put into a RNase-free centrifuge tube (1.5ml), carefully opened the lid of adsorption column, to film central authorities, add 60 μ l RNase-free ddH2O, cover lid, room temperature is placed 5 minutes.Centrifugal 1 minute of 12,000rpm.
N. extracting the liquid obtaining is DNA liquid, can carry out the detection test of MDV.
Attention: for guaranteeing elutriant (RNase-free ddH
2o) after equilibrium at room temperature, re-use, for the DNA on film is fully eluted, should note elutriant to be added to the middle position of film; When wintry weather is too cold or hot, room conditioning need be opened so that classes of agents temperature is held in 20-25 ℃, to guarantee the stable of nucleic acid extraction; The DNA obtaining should carry out PCR immediately, or it is standby to put into immediately-30 ℃ of Refrigerator stores.
3, PCR reaction system
Adopt the reaction system of 50 μ L: under condition of ice bath, by following reagent dosage, in 14 PCR reaction tubess, add respectively each composition: 2 * Taq Mix12.5 μ L(is containing loading dye, Tian Gen company produces), PCR reaction water (sterilizing distilled water) 8.5 μ L, each 1.0 μ L(primer working concentration 10mM of upstream and downstream primer), each reaction tubes adds respectively the total DNA of cellular genome of the extraction of 2.0 μ L.
When 4, the 814-3 of employing embodiment 1 and 814-4 primer carry out PCR reaction, PCR reaction conditions: 95 ℃ of denaturation 3min; 94 ℃ of sex change 30sec, 60 ℃ of annealing 30sec, 72 ℃ of extensions 1min, totally 40 circulations; 72 ℃ are extended 10min.When MDV universal primer (uMDV-1+uMDV-2) primer of employing embodiment 1 carries out PCR reaction, PCR reaction conditions: 94 ℃ of denaturation 3min; 94 ℃ of sex change 30sec, 55 ℃ of annealing 30sec, 72 ℃ of extensions 30sec, totally 35 circulations; 72 ℃ are extended 10min.
5, the detection of PCR product: get 8 μ LPCR reaction product, in 1% sepharose hole of the ethidium bromide containing 0.5 μ g/mL concentration, carry out electrophoresis, under the condition of 80V voltage, electrophoresis is 40 minutes, under ultraviolet transilluminator, observe PCR product, check quantity and the size of PCR product, under gel imaging system, take pictures and record result.
6, the results are shown in Figure 1, Fig. 2.Under ultraviolet lamp, observe and contrast in standard molecular weight, using the MDV universal primer (uMDV-1+uMDV-2) of embodiment 1, all MDV serum 1 type virus can amplify the band (comprising HVT Fc-126 strain) of 1 band 277bp, sees Fig. 2.Use 814 special primers (814-3+814-4) sequence of embodiment 1 as shown in SEQID NO.1 ~ 2, while increasing, MD vaccine strain 814 can amplify 250 and 750bp two bands, and MD vaccine strain CVI988 can only amplify the band of 1 250bp, the wild poison of other MDV serum 2 types and MDV Serotype-3 cannot amplify any band, see Fig. 1.
7, sequence verification and further judgement:
A. with QIAGEN gel, reclaiming test kit reclaims;
The clone of B.PCR product: PCR product is connected with carrier pMD18-T Vector, according to a conventional method by converted product difference transformed competence colibacillus cell, the single colony inoculation of picking white is to containing in ampicillin medium, and 37 ℃ of isothermal vibrations increase bacterium and cultivate 16h;
C. to step 8) bacterium liquid PCR evaluation: the preparation same 4 of a. reaction system) identical, result is judged and step 6) identical;
D. the sequencing of amplified production and comparative analysis: the positive colony screening transfers to associated nucleic acid sequences mensuration company to carry out determined dna sequence, and measured sequence is carried out to Blast analysis at GenBank, thereby determine that the nucleotide sequence of pcr amplification gained belongs to the genome of which kind of marek's disease virus.Sequencing result demonstration, 750bp fragment is MD814 gene order, 250bp gene fragment is that MD814 strain and the common fragment of CVI988 strain can be distinguished 814 strains and CVI988 strain with this.
The optimization of embodiment 3 experiment parameters
814 special primers in test kit (814-3 and 814-4) reaction conditions is optimized to exploration, first, primer concentration in reaction is optimized, by the primer (concentration 0.4mM-0.05mM) of twice dilution is detected and finds best primer concentration, result shows that the susceptibility of test is best when primer concentration is 0.1mM.Secondly, reaction conditions is optimized in the test that annealing temperature by the PCR in test kit is 50 ℃ to 65 ℃, and when the annealing temperature of finally finding each reaction is 60 ℃, it is best that test kit detects effect.
The present invention is to the MDV universal primer (uMDV-1+uMDV-2) in test kit, and reaction conditions is optimized exploration.First, the primer concentration in reaction is optimized, by the primer (concentration 0.4mM-0.05mM) of twice dilution is detected and finds best primer concentration, result shows that the susceptibility of test is best when primer concentration is 0.2mM.Secondly, reaction conditions is optimized in the test that annealing temperature by the PCR in test kit is 50 ℃ to 65 ℃, and when the annealing temperature of finally finding each reaction is 55 ℃, it is best that test kit detects effect.
Composition (1) and sensitivity, specific degree, detection limit and the repeatability of embodiment 4 test kits
Evaluate
The test kit of the present embodiment is used for differentiating 814 strains of Marek serum 1 type virus vaccine strain and CVI988 strain, contains 814-3 and 814-4 pair of primers (nucleotide sequence is as shown in SEQ ID NO.1 ~ 2), and PCR reaction reagent and enzyme.
1, specific test
With test kit increase other viral cDNA or RNA, carry out primer specificity test. subjects comprises MDV814 strain, MDV CVI988 strain and RNA viruses, RNA viruses: NDV, IBV, AIV H5, AIV H9, IBDV and REOV, and DNA virus: ILTV, IBHV and CAV, the above-mentioned common bird virus that is, from Beijing Hua Doushi China biological products company limited.Adopt the method for embodiment 2 to carry out pcr amplification, through agarose gel electrophoresis, detect, only have MDV814 strain can amplify two bands (250bp and 750bp), MDV CVI988 strain can only amplify the band of a 250bp, and the amplification of other virus strain is all negative (test-results is shown in Fig. 3).Test-results shows that test kit specificity of the present invention is 100%, has stronger specificity.
2, replica test
Apply this test kit, carry out the replica test of this test kit.Get (201210 batches-201215 batches of 6 batch products of MDV814 strain, by Beijing Hua Doushi China biological products company limited, produced) and the MDV CVI988 vaccine of 6 different batches of market purchase, adopt the method for embodiment 2 to carry out repeatability detection, 6 batches of two bands that 750bp and 250bp can be detected finding MDV814 strain, CVI988 strain can only detect a band (test-results is shown in Fig. 4) of 250bp.It is stronger that test-results illustrates that this test kit repeats.
3, sensitivity test
In test kit detects, 1 ~ 1 * 10
6the MDV814 strain virus of PFU/mL dilution detects, and the detection limit of MDV814 strain is that 10PFU(test-results is shown in Fig. 5).Result shows to use Auele Specific Primer of the present invention to differentiate that Marek serum 1 type 814 strain virus have good susceptibility.
Composition (2) and sensitivity, specific degree, detection limit and the repeatability of embodiment 5 test kits
Evaluate
The test kit of the present embodiment is used for differentiating Mareks disease virus, and further differentiate 814 strains of Marek serum 1 type virus vaccine strain and CVI988 strain, contain 814-3 and 814-4 primer (nucleotide sequence is as shown in SEQ ID NO.1 ~ 2) and MDV universal primer (uMDV-1+uMDV-2), and PCR reaction reagent and enzyme.
1, specific test
With test kit increase other viral cDNA or RNA, carry out primer specificity test.Comprise MDV814 strain, MDV CVI988 strain and RNA viruses, RNA viruses: NDV, IBV, AIV H5, AIV H9, IBD and REO, and DNA virus: ILTV, IBHV and CAV.Pcr amplification, detects through agarose gel electrophoresis, only have MDV814 strain CVI988 strain can amplify the band about a 277bp, and the amplification of other virus strain is all negative (test-results is shown in Fig. 6).Test-results shows that the specificity of this test kit detection Mareks disease virus is 100%.
The cDNA or the RNA that with reference to the method for embodiment 4, further apply 814-3 and the above-mentioned virus of 814-4 primer pair carry out PCR detection, result is consistent with embodiment 4 results, can detect specifically the vaccine strain of Mareks disease virus serum 1 type, and vaccine strain 814 strains and CVI988 strain have been realized to accurate discriminating.
2, replica test
Apply this test kit, carry out the replica test result of this test kit.Get 3 batch products (201210 batches-201215 batches) of MDV814 strain and the MDV CVI988 vaccine of 3 different batches that market is bought, utilize test kit to carry out repeatability and detect, find 3 batches and 3 batches of bands (test-results is shown in Fig. 7) that a 277bp left and right can be detected of CVI988 strain of MDV814 strain.It is stronger that test-results illustrates that this test kit repeats.
The cDNA or the RNA that with reference to the method for embodiment 4, further apply 814-3 and the above-mentioned virus of 814-4 primer pair carry out PCR detection, result is consistent with embodiment 4 results, vaccine virus 814 strains and CVI988 strain to each batch have realized accurate discriminating, detect repeatability good.
3, test kit sensitivity test
In test kit detects, get 1 ~ 1 * 10
6the MDV814 strain virus of PFU/mL dilution is that template detects, and found that application MDV universal primer (uMDV-1+uMDV-2) is 1PFU to the detection limit of MDV814 strain.Result shows that the susceptibility of this test kit is high.
Guangxi raiser has just reached to obtain 3 batches of chick from different areas, for detecting Immune Profile In Chicks Marek serum I type vaccine situation, extract respectively 1 part of serum sample from every batch of chick, gets altogether 3 parts of serum samples, and the method for recording according to embodiment 2 detects.
Test-results is shown in Fig. 8 and Fig. 9, and 814 strains of serum I type vaccine are crossed in interpret sample 1 immunity, the not immune serum I type of sample 2 vaccine, and serum I type vaccine CVI988 strain is crossed in sample 3 immunity.Through the Immune Profile In Chicks situation in 3 parts of serum sources is reviewed, the Immune Profile In Chicks of confirmatory sample 1 is crossed 814 strains of serum I type vaccine and not immune CVI988 strain and other MD vaccines, the not immune MDV serum I of the chick of sample 2 type vaccine, but immunity HVT(FC126 strain) vaccine; The Immune Profile In Chicks of sample 3 is crossed CVI988 strain and 814 strains of not immune serum I type vaccine and other MD vaccines.Clinical detection presentation of results, test kit of the present invention can be differentiated vaccine virus 814 and the CVI988 of Marek serum 1 type virus accurately and rapidly, and highly sensitive, high specificity, can occur for investigation chicken group reason, investigation chicken group early infection MDV epidemiology, the detection of environment virus and the prevention of MD and the control of poison, disease by force of MDV immuning failure thering is wide market using value.
Although, used general explanation, embodiment and test above, the present invention is described in detail, on basis of the present invention, can make some modifications or improvement to it, and this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (9)
1. for differentiating a primer pair for marek's disease virus serum 1 type vaccine virus, it is characterized in that, it is the nucleotide sequence shown in SEQ ID NO.1～2.
2. the test kit that contains the discriminating marek's disease virus serum 1 type vaccine virus of primer pair described in claim 1.
3. test kit as claimed in claim 2, is characterized in that, adopts primer pair described in claim 1 to carry out after pcr amplification, and the sample that has the amplification of object band is that marek's disease virus serum 1 type vaccine virus is positive, otherwise negative.
4. test kit as claimed in claim 2 or claim 3, it is characterized in that, described in employing claim 1, primer pair carries out after pcr amplification, the sample that only has length and be the amplified production band of 250bp is the strain of marek's disease virus serum 1 type vaccine strain CVI988, and the sample that has length and be 250bp and two special product bands of 750bp is 814 strains of marek's disease virus serum 1 type vaccine virus.
5. test kit as claimed in claim 2 or claim 3, is characterized in that, PCR reaction conditions is: 95 ℃ of 3min; 94 ℃ of sex change 30sec, 60 ℃ of annealing 30sec, 72 ℃ of 1min, circulate 40 times; 72 ℃ of 10min.
6. a diagnostic reagent of differentiating Marek serum 1 type vaccine strain, is characterized in that, comprises primer pair claimed in claim 1.
7. test kit as claimed in claim 2, is characterized in that, also comprises the primer of a pair of nucleotide sequence as shown in SEQ ID NO.3～4.
8. test kit as claimed in claim 7, is characterized in that, its working routine is:
1) primer pair shown in sample DNA application SEQ ID NO.3～4 is carried out to PCR reaction, the sample that has length and be the amplified production band of 277bp is that marek's disease virus is positive, otherwise negative;
2) to the sample DNA application rights of the step 1) positive, require the primer pair described in 1 again to carry out PCR reaction, the sample that only has length and be the amplified production band of 250bp is the strain of marek's disease virus serum 1 type vaccine strain CVI988, and the sample that has length and be 250bp and two special product bands of 750bp is 814 strains of marek's disease virus serum 1 type vaccine virus.
9. test kit as claimed in claim 8, is characterized in that, in step 1), PCR reaction conditions is: 94 ℃ of 3min; 94 ℃ of sex change 30sec, 55 ℃ of annealing 30sec, 72 ℃ of 30s, circulate 35 times; 72 ℃ of 10min.
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| CN111733296A (en) * | 2020-08-10 | 2020-10-02 | 中国兽医药品监察所 | Marek's disease virus serum 1 type fluorescent quantitative PCR kit and application thereof |
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| WO2012092226A1 (en) * | 2010-12-27 | 2012-07-05 | Biomune Company | Veterinary vaccine composition against infections caused by salmonella |
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