CN103160615A - Multiple PCR primer used for simultaneously detecting infectious Bovine Rhinotracheitis virus and akabane virus as well as its design method - Google Patents
Multiple PCR primer used for simultaneously detecting infectious Bovine Rhinotracheitis virus and akabane virus as well as its design method Download PDFInfo
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Abstract
The invention relates to a multiple PCR primer used for simultaneously detecting infectious Bovine Rhinotracheitis virus and akabane virus as well as its design method, and relates to detection of infectious Bovine Rhinotracheitis virus and akabane virus. The size of a PCR amplification fragment corresponded to an infectious Bovine Rhinotracheitis virus primer in the multiple PCR primer is 311bp, the size of the PCR amplification fragment corresponded to akabane virus id 392bp. The method comprises the following steps: 1) designing multiple PCR primer combination; 2) screening the primer in the primer combination, keeping the primer which can not synthesize a primer dimer; 3) determining the competition advantage and disadvantage states of the kept primer, comparing GC% and base number in the kept primer, selecting the kept primer with high GC content and determining as the primer with excellent competition state, performing a step 5); otherwise, performing a step 4); 4) selecting the primer with poor competition state again, wherein the concrete step repeats the step 2) and determining the kept primer according to the step 3) again; and 5) performing amplification on the primer with the excellent competition state.
Description
Technical field
The present invention relates to the detection of infectious bovine rhinotrachetis virus and Akabane virus, especially relate to a kind of multiple PCR primer and method of design thereof for detecting simultaneously infectious bovine rhinotrachetis virus and Akabane virus.
Background technology
Infectious bovine rhinotrachetis (Infectious bovine rhinotracheitis, IBR) is the febris acuta respiratory infectious disease of ox.At first nineteen fifty is found in the beef cattle group of Western United States, comes across afterwards the milk cows, isolates virus in 1956, through being accredited as the simplexvirus of ox, easily obscures mutually with Niu Liuhang heat, ox influenza, maligant catarrh fever and bovine mucosal disease syndromes clinically.The IBR Clinical symptoms is heating, upper respiratory tract mucosa inflammation, oedema, hemorrhage, necrotic spot even occurs.Infectivity vaginal orifice vaginitis (IVP) after infecting, cow occurs.Also can induce out ox infectivity balanoposthitis, eye conjunctivitis, keratoconjunctivitis, miscarriage, mastitis etc.This disease pathogen is to contain huge double-stranded DNA, can ox kidney monolayer cell, newborn rabbit kidney horse kidney and Hela Growth of Cells, and make its pathology.It is very fast that this disease is propagated, and by air (spittle) contact infection, mortality ratio reaches 10%, the encephalitis type is up to 50%.IBR all has generation all over the world, the many areas of China also have report that detecting of the positive ox of IBR arranged, and eradicates in case this sick being difficult to occurs cows.
Akabane Disease virus (Akabane disease virus) is distributed in the ground such as Australia, Japan, Israel, Africa; Take mosquito, storehouse grasshopper as communication media; Main infection ox, sheep, goat.Conceived cow infects rear without clinical symptom; Fetus shows as encephalomyelitis, polymyositis by after placental infection, and severe patient is dead, miscarriage, and congenital archrogryposis, hydranencephaly (cerebellum forms large plaque) occur anti-mistake person.The ox in pregnancy period, sheep, goat are to the susceptible of AKAV, and horse, buffalo, camel also can infect, and the susceptibility of people and pig is lower.Nineteen ninety, confirm that this disease exists in China.Its equality of Lee of Animal Quarantine Station, Agricultural Ministry in 1998 is separated to 3 strain akabane viruses at District of Shanghai for the first time.AK has caused serious financial loss to livestock industry, is the emphasis Quarantine Objects in China's Animal diseases prevention and control and international animal trade.
Along with improving constantly and the development of China's large-scale cultivation industry of China's national life level, the demand of beef cattle and milk cow just is being exponential growth, be badly in need of a large amount of high-qualitys, high yield beef cattle and milk cow, impel the ox of import kind in recent years quantity suddenly to increase thereupon.Yet, infected cattle is recovered, and recessive ox rear, band poison throughout one's life and wildlife such as goat, antelope, pronghorn, gnu, mink and ferret are all the viral places of this disease of storage, and they do not show clinical symptom, but scatter virus, be the cattle disease long-term existence, be difficult to the basic reason of eradicating.And some diseases is as the characteristic of the latent infection of infectious bovine rhinotrachetis etc. with do not have at present effective vaccine to promote the use of, except by forbidding inoculation, slaughtering the seropositivity ox and also do not have better method to eradicate this type of transmissible disease.In the urgent need to setting up in early days cattle disease diagnostic method fast and accurately, to prevent and to remove such transmissible disease, protect the healthy and rapid development of China's livestock industry.The laboratory diagnosis of two-strain comprises viral separation, Protein Detection and detection of nucleic acids, and characteristics such as detection of nucleic acids, high specificity short with the cycle and become the prior development direction that virus detects wherein.
The application of multiple PCR technique can greatly reduce testing cost, can greatly reduce workload, is adapted to the needs of port rapid detection, be a kind of accurately, the molecular biology method of reliable, science.In view of this, develop as early as possible a kind of imperative for the multiplex PCR that detects simultaneously infectious bovine rhinotrachetis and Akabane Disease.
Polymerase chain reaction (Polymerase Chain Reaction, be called for short PCR) be the method for external rapid amplifying DNA a kind of, be used for amplifying specific DNA fragmentation, can make the goal gene fragment amplification to the Protocols in Molecular Biology of millions of copies in a few hours.A reaction often has 25～35 circulations, and a circulation comprises 3 steps, and at first making the template DNA two strands is strand 94～95 ℃ of sex change, then at a lower temperature, primer is combined, then under the effect of the guiding of primer and Taq enzyme, in 72 ℃ of synthetic template DNA complementary strands with template.The special DNA replication dna that can regard in vitro as.General PCR only uses pair of primers, produces a nucleic acid fragment by pcr amplification.Multiplex PCR claims again Multiplex PCR or composite PCR, and it is to add primer more than two pairs in same PCR reaction system, amplifies simultaneously the PCR reaction of a plurality of nucleic acid fragments, its reaction principle, and the reaction kit operating process is identical with general PCR.Multiplex PCR is transformed on the basis of regular-PCR, adds Auele Specific Primer in a PCR reaction system, for the round pcr of a plurality of purpose fragments of the different zones of a plurality of DNA profilings or same template amplification.Just can increase simultaneously in primary first-order equation a plurality of target sequences of a gene of multiplex PCR.Multiplex PCR is just by Chamberlain(Chamberlain JS.Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification[J at first in 1988] .Nucleic Acids Research, 1988,16:11141-11156) propose, within these short 22 days, the application that it has been succeeded in a plurality of fields of DNA detection.Comprise that specifically detection transgenation, disappearance etc. are polymorphic, quantitative PCR, reverse transcription PCR.Henegariu has designed multiplex PCR in 1997 and has optimized agreement step by step, how to instruct the reaction system of multiplex PCR.But the problem of multiplex PCR maximum still is present in the design of primer.Compare with general PCR, multiplex PCR amplifies the gene fragment of a plurality of cause of diseases simultaneously in same PCR reaction tubes, once complete the amplification of a plurality of templates.The systematicness of multiplex PCR is mainly reflected in it and can be according to demand disposablely amplifies all genes involveds, will greatly save time, and saves reagent, and the reduction of expenditure spending provides more diagnostic messages more accurately for clinical.
Just can increase simultaneously in the primary first-order equation round pcr of a plurality of purpose fragments of different zones amplification of a template of multiplex PCR detection technique.With it fast, efficiently, the characteristics that specificity is good, highly sensitive have vital role in food pathogenic micro-organism, non-pathogenic microorganism and environmental microorganism detect; But multiplex PCR improves hygienic standard and strengthens still having on technician's level many problems to need to solve in the interference that improves Microbiological detection of foods sensitivity, removes the food supressor.Multiplex PCR improvement technology has been simplified the Microbiological detection of foods process, has shortened detection time, has been reduced the sensitivity that testing cost has improved Microbiological detection of foods simultaneously; But also there is some problems in the improvement technology of multiplex PCR.following research mainly concentrates on the improvement Sample Pretreatment Technique, removing the food supressor disturbs, next is the integration application of multiplex PCR and other technology, as multiplex PCR and denaturing gradient gel electrophoresis, gene chip, the fluorescent probe quantitative technique, the technology such as immunomagnetic beads absorption are in conjunction with application, further improve the sensitivity of Microbiological detection of foods, repeatable, realize food inspection in enormous quantities, effective detection of the multiple-microorganism in complex sample matrix, the stdn of Microbiological detection of foods, extraordinary application prospect will be arranged in following Microbiological detection of foods.
The characteristics such as PCR is the technology of DNA replication dna process in a kind of analogue body, and is quick owing to having, special and responsive are used widely in the swine disease diagnosis in recent years.Multiplex PCR is a kind of special PCR form,, more saving more faster than single PCR reaction, the characteristics that it is the most outstanding, i.e. a PCR reaction, can detect simultaneously, identify multiple pathogens, have its unique advantage and very high use value in the differential diagnosis of clinical polyinfection.
The application of multiple PCR technique can greatly reduce testing cost, can greatly reduce workload, is adapted to the needs of port rapid detection, be a kind of accurately, the molecular biology method of reliable, science.In view of this, develop as early as possible a kind of imperative for the multiplex PCR that detects simultaneously infectious bovine rhinotrachetis virus and Akabane virus.This has very important significance for these 2 kinds of diseases of early diagnosis, for strengthening port quarantine, control and these two kinds of diseases of elimination and all bringing into play huge effect.
Summary of the invention
The object of the present invention is to provide a kind of multiple PCR primer and method of design thereof for detecting simultaneously infectious bovine rhinotrachetis virus and Akabane virus, but the present invention's rapid detection is imported and exported infectious bovine rhinotrachetis and Akabane Disease in kind of ox.
Described multiple PCR primer for detecting simultaneously infectious bovine rhinotrachetis virus and Akabane virus, wherein, the pcr amplified fragment size that the infectious bovine rhinotrachetis virus primer pair is answered is 311bp, and the pcr amplified fragment size that the Akabane virus primer pair is answered is 392bp, and concrete primer sequence is as follows:
IBRV?F1：-TGACGGTAGCCTGGGACTGG-3’
IBRV?R1：5’-CCGGAAGGCCACGACAAA-3’
AKAV?F1：5’-AGGGTATGTGGCATTTATCA-3’
AKAV?R1：5’-CCAGAAACATCTCAGCACC-3’
Wherein, IBRV is the english abbreviation of infectious bovine rhinotrachetis virus, and AKAV is the english abbreviation of Akabane virus;
IBRV F1 and IBRV R1 are according to the upstream primer F1 of infectious bovine rhinotrachetis virus gB gene design and downstream primer R1; AKAV F1 and AKAV R1 are according to the upstream primer F1 of Akabane virus S gene design and downstream primer R1.
The method of design of described multiple PCR primer for detecting simultaneously infectious bovine rhinotrachetis virus and Akabane virus comprises the following steps:
In step 1, described primer-design software is Primer Premier5.0, and the melting temperature (Tm) Tm value of described multiple PCR primer combination is 52～62 ℃.
In step 2, the described concrete grammar that primer in combination of primers is screened can be: primer used is carried out pcr amplification, the best combination of primers of screening expanding effect; The described base number that can not form the primer of primer dimer is 20～24.
In step 5, the denaturation temperature of described PCR method can be 94 ℃, and annealing temperature can be 60 ℃, and elongating temperature can be 72 ℃.
Beneficial effect of the present invention is as follows:
The multiple PCR technique that adopts the present invention to set up detect simultaneously infectious bovine rhinotrachetis virus and Akabane virus method, have easy and simple to handle, quick, high specificity, the advantages such as sensitivity height, only need a PCR reaction just can detect simultaneously infectious bovine rhinotrachetis virus and Akabane virus, by using the method to the detection of 25 parts of nose of an ox swabs, bovine serum 25 duplicate samples, compare with the ordinary method detection simultaneously.Multiple PCR method detects 3 parts of infectious bovine rhinotrachetis virus positives, fits like a glove with the Virus Isolation result.The present invention can accelerate the detection of infectious bovine rhinotrachetis virus and Akabane virus greatly.
Adopt multiple PCR technique of the present invention to detect simultaneously the method for infectious bovine rhinotrachetis virus and Akabane virus, have the advantages such as simple, sensitive, quick, special, all significant in food sanitation safe, public health security and port health quarantine.Can be applicable to the to enter and leave the border rapid detection of import ox two-strain, with prevention with remove such transmissible disease, the healthy and rapid development of protection China livestock industry.
The present invention detect at the same time have advantages of in the application of infectious bovine rhinotrachetis and Akabane Disease simple, sensitive, quick, special.
Description of drawings
Fig. 1 is the optimization of multi-PRC reaction annealing temperature.In Fig. 1, M:100bp λ DNA Marker; 1～6:53 ℃, 55 ℃, 57 ℃, 59 ℃, 61 ℃, 63 ℃.
Fig. 2 is the optimization of multiplex PCR magnesium ion.In Fig. 2, M:100bp λ DNA Marker; 1～5:0.5,, 1.0,1.5,2.0,2.5 μ L.
Fig. 3 is the optimization of multi-PRC reaction primer concentration.In Fig. 3, M:100bp λ DNA Marker; 1～9 is different primers concentration combination of primers.
Fig. 4 multiplex PCR system sensitivity test.In Fig. 4, M:100bp λ DNA Marker, 1～6 for being respectively dilution plasmid concentration 10
-2-10
-7, 7 negative contrasts; In figure, the 392bp band is AKAV, and the 311bp band is IBRV.
Fig. 5 multiple PCR primer specific test.In Fig. 5, M:100bp λ DNA Marker; 1, infectious bovine rhinotrachetis virus and Akabane virus; 2, infectious bovine rhinotrachetis virus; 3, Akabane virus; 4, adenovirus; 5, pseudorabies virus; 6, pseudocowpox virus; 7, bovine viral diarrhea virus; 8, the ox vesicular stomatitis virus; 9, foot and mouth disease virus; 10, bovine leukemia virus.
Embodiment
The present invention is further illustrated below in conjunction with the drawings and specific embodiments, but the drawings and specific embodiments should not be understood as the restriction that the present invention is carried out.
The method of design of described multiple PCR primer for detecting simultaneously infectious bovine rhinotrachetis virus and Akabane virus comprises the following steps:
A) utilize increase the simultaneously multi-primers combination of infectious bovine rhinotrachetis virus and Akabane virus of Primer Premier5.0 primer-design software design.
B) primer of PCR detection infectious bovine rhinotrachetis virus in the combination of screening multi-primers.
C) primer of PCR detection Akabane virus in the combination of screening multi-primers.
D) the screening multiple PCR technique detects the combination of primers of infectious bovine rhinotrachetis virus and Akabane virus simultaneously.
E) multiple PCR technique detects the mensuration of infectious bovine rhinotrachetis virus and Akabane virus method optimum reaction condition simultaneously.
In step a), related primer is for (IBRV gB is GenBank AJ004801.1 with reference to delivering on GenBank, AKAV S is GenBank AB426280.1) infectious bovine rhinotrachetis virus and the gene order of Akabane virus, detect the Auele Specific Primer combination of infectious bovine rhinotrachetis virus and Akabane virus when using Primer Premier5.0 design.
At step b), c) and d) in, through screening, draw the pcr amplified fragment size that the infectious bovine rhinotrachetis virus primer pair answers and be 311bp, the pcr amplified fragment size that the Akabane virus primer pair is answered is 392bp; Concrete primer sequence is as follows:
IBRV?F1：-TGACGGTAGCCTGGGACTGG-3’；
IBRV?R1：5’-CCGGAAGGCCACGACAAA-3’；
AKAV?F1：5’-AGGGTATGTGGCATTTATCA-3’；
AKAV?R1：5’-CCAGAAACATCTCAGCACC-3’。
In step e), multiple PCR technique detects the mensuration of infectious bovine rhinotrachetis virus and Akabane virus method optimum reaction condition simultaneously, utilizes respectively 2 pairs of primers to carry out 2 kinds of virus multiple PCR method optimum annealing temperatures, primer concentration, Mg
2+The mensuration of concentration, template concentrations etc.; The reaction system that multiple PCR technique detects infectious bovine rhinotrachetis virus and Akabane virus method simultaneously is 25 μ L, and is specific as follows: 10 * PCR Buffer2.5 μ L, 25mmol/L MgCl
21.5 μ L, 10mmol/L dNTP2 μ L, the upstream and downstream primer mixture amount of the two-strain of 20 μ mol/L is followed successively by: infectious bovine rhinotrachetis virus 1.4 μ L; Akabane virus 1.4 μ L; 5u/ μ L Taq enzyme 0.125 μ L, ddH
2O6.075 μ L, each 5 μ L of the DNA profiling of two all viruses or cDNA template.Amplification condition is 94 ℃ of 5min; 95 ℃ of 45s, 59 ℃ of 45s, 72 ℃ of 45s35 circulations; 72 ℃ of 10min.
The structure of 1, viral template extraction, reverse transcription method and plasmid
Build the plasmid of infectious bovine rhinotrachetis virus and Akabane virus, be used as the optimization that template is carried out reaction conditions.The virus genom DNA that the extraction of template is produced with reference to sky root company/RNA extracts test kit specification sheets (Cat.#DP315) to carry out, with reference to precious biological reverse transcription test kit (TaKaRa Code:D2680A), use AKAV R1 that the RNA reverse transcription of AKAV is cDNA.
2, the multiplex PCR one trip technique detects the foundation of infectious bovine rhinotrachetis virus and Akabane virus method simultaneously
With the plasmid template of infectious bovine rhinotrachetis virus and the plasmid template of Akabane virus, carry out pcr amplification.Reaction system is 25 μ L, and is specific as follows: 10 * PCR Buffer2.5 μ L, 25mmol/L MgCl
21.5 μ L, 10mmol/L dNTP2 μ L, the upstream and downstream primer mixture amount of the two-strain of 20 μ mol/L is followed successively by: infectious bovine rhinotrachetis virus 1.4 μ L; Akabane virus 1.4 μ L; 5u/ μ L Taq enzyme 0.125 μ L, ddH
2O6.075 μ L, each 5 μ L of the DNA profiling of two all viruses or cDNA template.Amplification condition is 94 ℃ of 5min; 95 ℃ of 45s, 59 ℃ of 45s, 72 ℃ of 45s35 circulations; 72 ℃ of 10min.Get pcr amplification product 7 μ L and carry out agarose (2%) electrophoresis detection amplification.
3, multiplex PCR single stage method optimum reaction condition gropes
Utilize respectively 2 pairs of primers to carry out 2 kinds of virus multiple PCR method optimum annealing temperatures, primer concentration, Mg
2+The mensuration of concentration.
Result records the multiplex PCR single stage method, and annealing temperature is seen Fig. 1 with 59 ℃ of the bests;
Multiplex PCR single stage method magnesium ion concentration is in the reaction system of 25 μ L, and design 25mmol/L Mg2+ gradient is that 0.5,1.0,1.5,2.0,2.5 μ L pass through relatively, and finally selecting 1.5 μ L is optimum addition, sees Fig. 2;
The addition optimization of combination of primers sees Table 1, and determine that finally each primer concentration of component is as follows: infectious bovine rhinotrachetis virus upstream and downstream primer addition is respectively 0.7 μ L; Akabane virus upstream and downstream primer addition is respectively 0.7 μ L,, see Fig. 3;
Table 1 multiple PCR primer concentration combination
4, the sensitivity of multiplex PCR single stage method detects
With 2 kinds of virus recombinant plasmid stostes extracting respectively quantitatively and mix, 10 times of gradient dilutions then, every individual system is added the template of 10 μ L, carries out the mensuration of susceptibility with reference to the multiplex PCR system after having optimized.As can be known, the detection sensitivity of system is from result: the sensitivity of IBRV is 0.13pg/25 μ L, and the sensitivity of AKAV is 1.04pg/25 μ L.See Fig. 4.
5, multiplex PCR single stage method specific assay
According to the multiplex PCR condition of optimizing, respectively 2 kinds of viruses and adenovirus (BAV), pseudorabies virus (PRV), pseudocowpox virus, bovine viral diarrhea virus (BVDV), ox vesicular stomatitis virus (VSV), foot and mouth disease virus (FMDV), bovine leukemia virus (BLV) are increased, detect the specificity of the method.Result shows can the increase band of correspondence of single or 2 kinds of combinations, respectively at 311bp, 392bp, two amplified bands is arranged, and illustrate that the method specificity of the present invention's foundation is good.See Fig. 5.
6, multiplex PCR single stage method Stability Determination
Packing after according to the above optimal conditions of groping, the PCR reaction system beyond removing template being mixed is put in-20 ℃ frozenly, regularly takes out detection according to optimum reaction condition.The PCR mixture that will be pre-mixed between detection period is placed to preserve under-20 ℃ of conditions and still can be amplified clearly band the same as the PCR mixture of now joining in 1,2,3,4,5,6,7,8,9,10,11,12 month, the stability that can find out this reagent by the detection of a year is better, can keep more than 1 year at least.
7, multiplex PCR single stage method clinical detection
By using the method, 25 parts of nose of an ox swabs, bovine serum 25 duplicate samples are detected, compare with the ordinary method detection simultaneously.Multiple PCR method detects 3 parts of infectious bovine rhinotrachetis virus positives, fits like a glove with the Virus Isolation result.
The variation that is appreciated that a lot of details is possible, but therefore this do not run counter to scope and spirit of the present invention, and any person of an ordinary skill in the technical field all should be considered as not breaking away from category of the present invention to its suitable variation of doing.
Claims (7)
1. be used for detecting simultaneously the multiple PCR primer of infectious bovine rhinotrachetis virus and Akabane virus, it is characterized in that wherein, the pcr amplified fragment size that the infectious bovine rhinotrachetis virus primer pair is answered is 311bp, the pcr amplified fragment size that the Akabane virus primer pair is answered is 392bp, and concrete primer sequence is as follows:
IBRV?F1：-TGACGGTAGCCTGGGACTGG-3’
IBRV?R1：5’-CCGGAAGGCCACGACAAA-3’
AKAV?F1：5’-AGGGTATGTGGCATTTATCA-3’
AKAV?R1：5’-CCAGAAACATCTCAGCACC-3’
Wherein, IBRV is the english abbreviation of infectious bovine rhinotrachetis virus, and AKAV is the english abbreviation of Akabane virus;
IBRV F1 and IBRV R1 are according to the upstream primer F1 of infectious bovine rhinotrachetis virus gB gene design and downstream primer R1; AKAV F1 and AKAV R1 are according to the upstream primer F1 of Akabane virus S gene design and downstream primer R1.
2. be used for detecting the method for design of the multiple PCR primer of infectious bovine rhinotrachetis virus and Akabane virus simultaneously, it is characterized in that comprising the following steps:
Step 1, utilize the primer-design software design to detect simultaneously the multiple PCR primer combination of infectious bovine rhinotrachetis virus and Akabane virus, the primer base number that formed combination of primers comprises is 20～24, the melting temperature (Tm) Tm value of combination of primers is 52～62 ℃, and the GC% of combination of primers is 40%～60%;
Step 2 is screened the primer in combination of primers, and reservation can not the dimeric primer of synthetic primer;
Step 3, the good and bad state of the competition of primer that judgement keeps compares above-mentioned GC% and the base number that keeps primer, chooses the high race condition advantage that the is judged as primer of GC content, carry out step 5; Otherwise be judged as race condition inferior position primer, carry out step 4;
Step 4 is screened the bad primer of race condition again, and concrete steps are: repeating step 2 judges according to step 3 again to the primer that keeps;
Step 5 utilizes PCR method that the excellent primer of race condition is increased.
3. be used for as claimed in claim 2 detecting the method for design of the multiple PCR primer of infectious bovine rhinotrachetis virus and Akabane virus simultaneously, it is characterized in that in step 1, described primer-design software is Primer Premier5.0.
4. be used for as claimed in claim 2 detecting the method for design of the multiple PCR primer of infectious bovine rhinotrachetis virus and Akabane virus simultaneously, it is characterized in that in step 1, the melting temperature (Tm) Tm value of described multiple PCR primer combination is 52～62 ℃.
5. be used for as claimed in claim 2 detecting simultaneously the method for design of the multiple PCR primer of infectious bovine rhinotrachetis virus and Akabane virus, it is characterized in that in step 2, the described concrete grammar that primer in combination of primers is screened is: primer used is carried out pcr amplification, the best combination of primers of screening expanding effect.
6. be used for as claimed in claim 2 detecting the method for design of the multiple PCR primer of infectious bovine rhinotrachetis virus and Akabane virus simultaneously, it is characterized in that in step 2, the described base number that can not form the primer of primer dimer is 20～24.
7. be used for as claimed in claim 2 detecting simultaneously the method for design of the multiple PCR primer of infectious bovine rhinotrachetis virus and Akabane virus, it is characterized in that in step 5, the denaturation temperature of described PCR method is 94 ℃, and annealing temperature is 60 ℃, and elongating temperature is 72 ℃.
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| CN103498009A (en) * | 2013-10-10 | 2014-01-08 | 中国农业科学院特产研究所 | Multiplex-PCR (polymerase chain reaction) detection kit for bovine respiratory disease complex and preparation method thereof |
| CN103789455A (en) * | 2014-02-28 | 2014-05-14 | 广东出入境检验检疫局检验检疫技术中心 | Group of real-time fluorescent QRT-PCR primers capable of detecting cattle, sheep akabane virus by one-step method, probe and usage method thereof |
| CN103789455B (en) * | 2014-02-28 | 2015-04-22 | 广东出入境检验检疫局检验检疫技术中心 | Group of real-time fluorescent QRT-PCR primers capable of detecting cattle, sheep akabane virus by one-step method, probe and usage method thereof |
| CN104726614A (en) * | 2015-03-31 | 2015-06-24 | 河南牧业经济学院 | Detection primer, kit and detection method of infectious bovine rhinotracheitis virus |
| CN105132590A (en) * | 2015-10-15 | 2015-12-09 | 上海市农业科学院 | LAMP visual detection method of infectious bovine rhinotracheitis viruses |
| CN105132590B (en) * | 2015-10-15 | 2022-02-08 | 上海市农业科学院 | LAMP visual detection method of infectious bovine rhinotracheitis virus |
| CN105567869A (en) * | 2015-11-17 | 2016-05-11 | 天津出入境检验检疫局动植物与食品检测中心 | GeXP multi-primers and method for detecting akabane viruses, bovine viral diarrhea viruses and foot and mouth disease viruses |
| CN105567869B (en) * | 2015-11-17 | 2021-07-27 | 天津海关动植物与食品检测中心 | GeXP multiple primers and method for detecting akabane virus, bovine viral diarrhea virus and foot-and-mouth disease virus |
| CN107012260A (en) * | 2017-05-08 | 2017-08-04 | 河南科技大学 | Neospora, Brucella and infectious bovine rhinotrachetis virus multiplex-nested PCR detection primer, kit and detection method |
| CN111560471A (en) * | 2020-04-26 | 2020-08-21 | 北京市动物疫病预防控制中心 | Nucleic acid, kit and micro-drop digital PCR method for detecting infectious bovine rhinotracheitis virus |
| CN113862397A (en) * | 2021-10-18 | 2021-12-31 | 广西壮族自治区兽医研究所 | Fluorescent quantitative RT-PCR primer for detecting novel acarbovirus S gene and kit thereof |
| CN113862397B (en) * | 2021-10-18 | 2024-07-12 | 广西壮族自治区兽医研究所 | Fluorescent quantitative RT-PCR primer for detecting novel acarbose virus S gene and kit thereof |
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