CN108546780A - PCR primer that is a kind of while detecting RHDV and RHDVa and its kit and non-diagnostic purpose detection method - Google Patents
PCR primer that is a kind of while detecting RHDV and RHDVa and its kit and non-diagnostic purpose detection method Download PDFInfo
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Abstract
The present invention relates to detection technique field, more particularly to PCR primer that is a kind of while detecting RHDV and RHDVa and its kit and non-diagnostic purpose detection method.The PCR primer is made of RHDV detection primers and RHDVa detection primers；RHDV detection primers are by SEQ ID NO：Sense primer shown in 1 and SEQ ID NO：Downstream primer shown in 2 forms；RHDVa detection primers are by SEQ ID NO：Sense primer shown in 3 and SEQ ID NO：Downstream primer shown in 4 forms.The present invention have many advantages, such as specificity it is good, sensitive and accurate, without large-scale instrument and equipment or follow-up sequence verification, easy to operate and quick.
Description
Technical field
The present invention relates to detection technique field, more particularly to PCR primer that is a kind of while detecting RHDV and RHDVa and its examination
Agent box and non-diagnostic purpose detection method.
Background technology
The production and trade of rabbit product are the important components of animal husbandry.China rabbit delivers for sale, livestock on hand and rabbit meat yield
40%, 25% and the 41.09% of the whole world is accounted for respectively.Rabbit haemorrhagic disease (being commonly called as rabbit pest) is the common disease for threatening rabbit group's life and health
Disease seriously affects the production composition of rabbit product.Under natural conditions, 2 monthly age of the disease main harm above adult rabbits.It is to exhale
Desorption system bleeding, organa parenchymatosum's extravasated blood, enlargement, bleeding and hepatonecrosis are main lesion characteristics.Its break out and spread has latent
The volt phase is short, morbidity is anxious, the course of disease is short (rabbit 48~72h usually after infection is dead), propagation is fast, the death rate is high (80%~
100%).Rabbit haemorrhagic disease is after 1984 are reported for the first time by China, and in the world, morbidity and popular report occur in many countries, sternly
It threatens the sound development of world's rabbit keeping again, two class animal epidemic of inspection and quarantining for import/export is classified as by China.
The pathogen of rabbit haemorrhagic disease be rabbit hemorrhagic disease virus (Rabbit hemorrhagic disease virus,
RHDV), it is single-stranded underlying stock RNA virus, Caliciviridae (Caliciviridae) Lagovirus should be belonged in classification
(Lagovirus).In addition to classical strains, there are anomaly a (Antigenic variant of rabbit for rabbit hemorrhagic disease virus
Hemorrhagic disease virus, RHDVa).Classical RHDV and RHDVa genome structures having the same, overall length are about
7400 bases, contain 2 open reading frame (Open reading frame, ORF), and wherein ORF1 originates in 5 ' ' end, accounts for overall length
94%, ORF1 encode 1 albumen, which is subsequently formed ripe non-structural protein and 1 main structural proteins-
VP60 capsid proteins, non-structural protein are arranged in order in viral genome as p16, p23/p26,2C-like, p29, p13/p14,
3C-like, p58 (RNA polymerase that RNA is relied on, RdRP).VP60 protein variants are the foundations of virogene type classification,
It is virus immunity protective antigens.ORF2 is located at 3 ' ' end, encodes VP10 albumen, is the secondary structure albumen of virion.
In rabbit breeding process, can effectively control rabbit, including rabbit bleeding heat symptom-complex, depend greatly on and
When, accurate monitoring rabbit fashion trend.Classical RHDV and anomaly RHDVa has prodigious difference in epidemiology characteristic.
Compared to classical RHDV, anomaly RHDVa is easier to propagate in the rabbit group of high-density rearing.But China rabbit owner wants
Diversified feature, intensive large-scale production pattern, cooperative association's production model and peasant household front yard is presented in production management planning
Institute's production model is simultaneously deposited.Although industrial upgrading, scale standardization cultivation become trend, dispersion it is small-scale due to it
It has the advantage that, such as small investment, quick, can also get rapid development.The above-mentioned mode of production is without the propagation suspected of different RHDV
Advantageous external environment is provided, challenge also is proposed to the outburst of timely, Accurate Diagnosis and prevention and control rabbit haemorrhagic disease.
Currently, for the molecular detecting method of rabbit hemorrhagic disease virus, RT-PCR technology and fluorescence RT-PCR are related generally to
Technology：
(1) round pcr specificity is dependent on the Oligonucleolide primers with the complementation of target sequence both ends, in the ginseng of archaeal dna polymerase
With under, the replication in vitro to specific gene is completed.
(2) as the development of round pcr, quantitative fluorescent PCR by the probe of the specificity of fluorescent dye or fluorescent marker,
Tracking is marked to PCR product, the intensity of real time on-line monitoring fluorescence signal divides product in conjunction with corresponding software
Analysis, it is quantitative to achieve the purpose that carry out product.
The molecule method of inspection for a variety of rabbit hemorrhagic disease virus established at present has played weight in the diagnosis and prevention and control of epidemic disease
It acts on.But these examine classical rabbit hemorrhagic disease virus with rabbit hemorrhagic disease virus anomaly a as simple target
It surveys.Moreover, the specificity of PCR reactions depends on the specific binding of primer and target sequence, due to the viral heredity of itself
Variation features, the above method face the risk of false negative or false positive.For the inspection result of PCR, if you need to further confirm that, then
The nucleic acid sequence for needing measurement product, leads to the increase of round of visits and expense；Although quantitative fluorescent PCR can be by using spy
Specific probes improve the reliability examined to the comparison of PCR amplification curve, and still, result still depends on primer and target
The specific binding of sequence can not still avoid false negative or the risk of false positive results, and this method is complicated for operation, reagent expense
Costliness needs Special experimental equipment.For the small-scale cultivation mould for the dispersion peasant household that China is current and will be widely present from now on
Formula, the above method can all increase the financial burden of peasant household.
Invention content
In view of this, the present invention provides a kind of while detecting the PCR primer of RHDV and RHDVa and its kit and non-examine
Disconnected purpose detection method.The PCR primer and its kit specificity are good, sensitive and accurate, without large-scale instrument and equipment or subsequently
Sequence verification saves money；It is easy to operate and quick.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme：
Classical rabbit hemorrhagic disease virus (RHDV) and rabbit hemorrhagic disease virus anomaly a are detected the present invention provides a kind of simultaneously
(RHDVa) PCR primer is made of classical rabbit hemorrhagic disease virus detection primer and rabbit hemorrhagic disease virus anomaly a detection primers；
Classical rabbit hemorrhagic disease virus detection primer is by SEQ ID NO：Sense primer shown in 1 and SEQ ID NO：Shown in 2
Downstream primer composition；
Rabbit hemorrhagic disease virus anomaly a detection primers are by SEQ ID NO：Sense primer shown in 3 and SEQ ID NO：4 institutes
The downstream primer composition shown.
The purpose of patent of the present invention is to provide the high detection of high specificity, reliability and differentiates classical rabbit mass formed by blood stasis virus
(RHDV) with the PCR primer of rabbit hemorrhagic disease virus anomaly a (RHDVa).Classical rabbit hemorrhagic disease virus (Rabbit
Hemorrhagic disease virus, RHDV) and rabbit hemorrhagic disease virus anomaly a (Antigenic variant of
Rabbit hemorrhagic disease virus, RHDVa) there is higher genetic similarity, but in terms of epidemiology
A degree of difference is presented.The technical problem to be solved by the present invention is to establish one kind to detect and differentiate that classical rabbit goes out classics
The molecular diagnosis method and its kit of mass formed by blood stasis virus and rabbit hemorrhagic disease virus anomaly a.This method should have specificity height, take
With cheap and quick simple operation feature.In terms of production application, this method can by once sampling, once analyze
It obtains as a result, low-cost and simple operation is quick.
The present invention also provides a kind of while detecting the RT- of classical rabbit hemorrhagic disease virus and rabbit hemorrhagic disease virus anomaly a
PCR kit, including reverse transcription system, PCR system, PCR system include PCR primer as claimed in claim 1.
Another purpose of patent of the present invention is to provide high specificity, the low-cost classical rabbit mass formed by blood stasis of detectable and discriminating
The detection kit of virus and rabbit hemorrhagic disease virus anomaly RT-PCR.
Preferably, reverse transcription system includes RNA PCR buffer solutions, dNTP, RNase-free ddH2O、MgCl2、RNase
Inhibitor, AMV reverse transcriptases, random primer and viral RNA.
Preferably, in terms of 10 μ L, reverse transcription system is：
Preferably, PCR system further includes Taq DNA polymerase buffers liquid, dNTP, MgCl2, Taq archaeal dna polymerases and
Reverse transcription product.
Preferably, in terms of 50 μ L, PCR system is：
Preferably, the kit further includes negative control and positive control.
Classical rabbit hemorrhagic disease virus is detected simultaneously the present invention also provides a kind of non-diagnostic purpose and rabbit hemorrhagic disease virus becomes
The method of special-shaped a, includes the following steps：
Extract sample RNA；
Reverse transcription system is prepared, reverse transcription is carried out, obtains reverse transcription product；Reverse transcription system include RNA PCR buffer solutions,
dNTP、RNase-free ddH2O、MgCl2, RNase inhibitor, AMV reverse transcriptases, random primer and RNA；
PCR system is prepared, carries out PCR amplification, PCR system includes primer provided by the invention (classical rabbit hemorrhagic disease virus
Detection primer is by SEQ ID NO：Sense primer shown in 1 and SEQ ID NO：Downstream primer shown in 2 forms；Rabbit haemorrhagic disease disease
Malicious anomaly a detection primers are by SEQ ID NO：Sense primer shown in 3 and SEQ ID NO：Downstream primer shown in 4 forms),
Amplified production is subjected to electrophoresis, classical rabbit hemorrhagic disease virus is detected and rabbit hemorrhagic disease virus anomaly a whether there is；PCR system
Further include Taq DNA polymerase buffers liquid, dNTP, MgCl2, Taq archaeal dna polymerases and reverse transcription product.
Preferably, the response procedures of reverse transcription are：
Preferably, the response procedures of PCR amplification are：
Classical rabbit hemorrhagic disease virus detection primer：
Rabbit hemorrhagic disease virus anomaly a detection primers：
Preferably, the classical rabbit hemorrhagic disease virus of detection and rabbit hemorrhagic disease virus anomaly a are with the presence or absence of specially：
(1) size of classical rabbit hemorrhagic disease virus pcr amplification product is 170bp, and rabbit hemorrhagic disease virus anomaly a PCR expand
The size for increasing production object is 167bp；
(2) on the basis of (1), by two groups of PCR reaction products of comprehensive analysis with/without excluding the false negative of result
And false positive, i.e.,
The present invention provides it is a kind of and meanwhile detect RHDV and RHDVa PCR primer and its kit and non-diagnostic purpose inspection
Survey method.The PCR primer is made of classical rabbit hemorrhagic disease virus detection primer and rabbit hemorrhagic disease virus anomaly a detection primers；
Classical rabbit hemorrhagic disease virus detection primer is by SEQ ID NO：Sense primer shown in 1 and SEQ ID NO：Draw in downstream shown in 2
Object forms；Rabbit hemorrhagic disease virus anomaly a detection primers are by SEQ ID NO：Sense primer shown in 3 and SEQ ID NO：4 institutes
The downstream primer composition shown.The technique effect that the present invention has is：
(1) specific：By the feature of genetic sequence, the primer of specificity is designed, can detect and is clearly differentiated classical
Rabbit mass formed by blood stasis virus and rabbit hemorrhagic disease virus anomaly a, are conducive to the pathogen type of Accurate Diagnosis epidemic disease；
(2) sensitive and accurate：Reacted by PCR, can large amplification target fragment, and two groups of PCR of comprehensive analysis can be passed through
Reaction as a result, eliminating the false negative and false positive of diagnostic result；
(3) it is not necessarily to large-scale instrument and equipment or follow-up sequence verification, is saved money；
(4) easy to operate and quick：This method by once sampling, once analysis can be obtained reliable results, and used time
It is short.
Description of the drawings
Fig. 1 shows has good amplification efficiency for primer sets RH-FP+RH-RP to classical Rabbit pest virus, and same
Under the conditions of, the sample of rabbit hemorrhagic disease virus anomaly a is then generated without product；
Fig. 2 shows has good amplification efficiency for primer sets RHa-FP+RHa-RP to rabbit hemorrhagic disease virus anomaly a,
And under identical condition, the sample of classical Rabbit pest virus is then generated without product.
Specific implementation mode
The invention discloses it is a kind of and meanwhile detect RHDV and RHDVa PCR primer and its kit and non-diagnostic purpose inspection
Survey method, those skilled in the art can use for reference present disclosure, be suitably modified technological parameter realization.In particular, it should be pointed out that
All similar substitutions and modifications are apparent to those skilled in the art, they are considered as being included in this hair
It is bright.The method of the present invention and application are described by preferred embodiment, and related personnel can obviously not depart from this hair
Method described herein and application are modified or are suitably changed and combined in bright content, spirit and scope, to realize and answer
Use the technology of the present invention.
Term is explained：
RHD：Rabbit hemorrhagic disease, rabbit haemorrhagic disease, rabbit pest；
RHDV：Rabbit hemorrhagic disease virus, rabbit hemorrhagic disease virus；
RHDVa：Antigenic variant of rabbit hemorrhagic disease virus, rabbit haemorrhagic disease
Virus variation type；
PCR：Polymerase Chain Reaction, PCR；
RT-PCR：Reverse transcription PCR, reverse transcriptase chain reaction.
The target sequence of the downstream primer of every group of primer of the present invention is in classical rabbit mass formed by blood stasis virus and rabbit hemorrhagic disease virus anomaly a
Inside, is presented more conservative feature, and hereditary variation is smaller；But between amphitypy virus, which then has apparent point
Change.Primer specificity of the present invention is good, easy to operate and low-cost.Based on the experimental result of two groups of RT-PCR, by simple
Compare, you can accurate detection and the classical rabbit mass formed by blood stasis virus of discriminating and rabbit hemorrhagic disease virus anomaly a can effectively avoid to eliminate and examine
The false negative and false positive of disconnected result, and take short, low-cost.
PCR primer that is provided by the invention while detecting RHDV and RHDVa and its kit and non-diagnostic purpose detection side
Agents useful for same or instrument are available on the market in method.
With reference to embodiment, the present invention is further explained：
Embodiment 1
Downstream primer is designed using target sequence (the VP60 1099-1103nt of M67473 and DQ280493).Make above-mentioned sequence
Positioned at 3 ' ends of primer, it is made to generate apparent influence for the specificity of product.Corresponding to the features described above of target sequence, primer
The difference of sequence will cause the result of the PCR of different type rabbit hemorrhagic disease virus that apparent differentiation is presented.Primer is as shown in table 1：
The classical rabbit mass formed by blood stasis of table 1 viral (RHDV) and rabbit hemorrhagic disease virus anomaly a (RHDVa) detections, identification primer
Note：A, adenine；C, cytimidine；G, guanine；T, thymidine.Degeneracy base R, A or G；W, A or T；Y, C or
T。
The present embodiment additionally provides PCR that is detectable and differentiating classical rabbit mass formed by blood stasis virus and rabbit hemorrhagic disease virus anomaly a
Detection kit.
Except above-mentioned primer, this detection kit further includes：Reverse transcriptase, polymerase and reaction buffer solution.
It can detect simultaneously and differentiate that classical rabbit mass formed by blood stasis virus and the PCR detection method of rabbit hemorrhagic disease virus anomaly a include
Following steps：
(1) RNA of sample to be tested is extracted；
(2) RT-PCR reaction solutions are prepared；
(3) RNA obtained using step (1) as template, with above-mentioned 2 groups of primers be respectively adopted One step RT-PCR method into
Row PCR reactions；
(4) result judgement standard：Referring to table 2.
Table 2RT-PCR one-step method detects and differentiates classical rabbit mass formed by blood stasis viral (RHDV) and rabbit hemorrhagic disease virus anomaly a
(RHDVa) result judgement standard
It is as follows：
(1) extraction of RHDV RNA is reacted with RT
Operating extraction RHDV artificial challenges according to RNAiso Plus reagent specifications organizes total serum IgE in pathological material of disease (to preserve
In -70 DEG C) it is template, according to RNA LA PCRTMKit (AMV) Ver.1.1 (DRR012A) specification carries out reverse transcription (RT).
Reaction system (10 μ L)：
Response procedures are：
Collect RT products be stored in -70 DEG C it is spare.
(2) optimization of PCR method of the present invention
To establish the PCR method of a sensitivity and stabilization, the PCR parameters reacted are optimized in the present embodiment, including
The temperature of annealing.
PCR reaction systems (50 μ L) are：
Reaction condition is：
5 μ L products are taken to carry out Ago-Gel (10g/L) electrophoretic analysis after reaction.
(3) experimental result
PCR testing results show, primer sets RH-FP+RH-RP and primer sets RHa-FP+RHa-RP show good
Expanding effect and specificity.
There is good amplification efficiency to classical Rabbit pest virus for primer sets RH-FP+RH-RP, and in same condition
Under, the sample of rabbit hemorrhagic disease virus anomaly a then generates (Fig. 1) without product.
There is good amplification efficiency to rabbit hemorrhagic disease virus anomaly a for primer sets RHa-FP+RHa-RP, and same
Under conditions of sample, the sample of classical Rabbit pest virus then generates (Fig. 2) without product.
(4) identification of amplified production
Recycling positive products band, which is sent to Hua Da-six directions sequencing company, carries out sequencing.Sequencing result has with NCBI
Rabbit pest virus sequence alignment determines that it is respectively classical Rabbit pest virus and rabbit hemorrhagic disease virus anomaly a.
Examples detailed above result illustrates the characteristic that PCR detection method of the present invention has specificity good, easy to operate, only logical
Spend simple compare, you can accurate detection and the classical rabbit mass formed by blood stasis virus of discriminating and rabbit hemorrhagic disease virus anomaly a can effectively avoid
The false negative and false positive of diagnostic result are eliminated, and is taken short, low-cost.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Sichuan Agricultural University
<120>PCR primer that is a kind of while detecting RHDV and RHDVa and its kit and non-diagnostic purpose detection method
<130> MO180147
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 1
gccaatgctg ggtctgca 18
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 2
tgcacagtcg twacgttg 18
<210> 3
<211> 18
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 3
atgctgggtc tgcaattg 18
<210> 4
<211> 18
<212> DNA
<213>Artificial sequence (Artificial sequence)
<400> 4
ctgcacagty gtrgcagc 18
Claims (10)
1. PCR primer that is a kind of while detecting classical rabbit hemorrhagic disease virus and rabbit hemorrhagic disease virus anomaly a, which is characterized in that
It is made of classical rabbit hemorrhagic disease virus detection primer and rabbit hemorrhagic disease virus anomaly a detection primers；
The classics rabbit hemorrhagic disease virus detection primer is by SEQ ID NO：Sense primer shown in 1 and SEQ ID NO：Shown in 2
Downstream primer composition；
The rabbit hemorrhagic disease virus anomaly a detection primers are by SEQ ID NO：Sense primer shown in 3 and SEQ ID NO：4 institutes
The downstream primer composition shown.
2. RT-PCR kit that is a kind of while detecting classical rabbit hemorrhagic disease virus and rabbit hemorrhagic disease virus anomaly a, feature
It is, including reverse transcription system, PCR system, the PCR system includes PCR primer as described in claim 1.
3. RT-PCR kit according to claim 2, which is characterized in that the reverse transcription system includes RNA PCR slow
Fliud flushing, dNTP, RNase-free ddH2O、MgCl2, RNase inhibitor, AMV reverse transcriptases, random primer and viral RNA.
4. RT-PCR kit according to claim 3, which is characterized in that in terms of 10 μ L, the reverse transcription system is：
5. RT-PCR kit according to claim 2, which is characterized in that the PCR system further includes Taq DNA polymerizations
Enzyme buffer liquid, dNTP, MgCl2, Taq archaeal dna polymerases and reverse transcription product.
6. RT-PCR kit according to claim 5, which is characterized in that in terms of 50 μ L, the PCR system is：
7. a kind of method that non-diagnostic purpose detects classical rabbit hemorrhagic disease virus and rabbit hemorrhagic disease virus anomaly a simultaneously, special
Sign is, includes the following steps：
Extract sample RNA；
Reverse transcription system is prepared, reverse transcription is carried out, obtains reverse transcription product；The reverse transcription system include RNA PCR buffer solutions,
dNTP、RNase-free ddH2O、MgCl2, RNase inhibitor, AMV reverse transcriptases, random primer and RNA；
PCR system is prepared, PCR amplification is carried out, amplified production is subjected to electrophoresis, detects classical rabbit hemorrhagic disease virus and rabbit haemorrhagic disease
Virus variation type a whether there is；The PCR system further includes Taq DNA polymerase buffers liquid, dNTP, MgCl2, Taq DNA it is poly-
Synthase and reverse transcription product.
8. the method according to the description of claim 7 is characterized in that the response procedures of the reverse transcription are：
9. the method according to the description of claim 7 is characterized in that the response procedures of the PCR amplification are：
Classical rabbit hemorrhagic disease virus detection primer：
Rabbit hemorrhagic disease virus anomaly a detection primers：
10. the method according to the description of claim 7 is characterized in that detection classics rabbit hemorrhagic disease virus and rabbit haemorrhagic disease
Virus variation type a is with the presence or absence of specially：
(1) size of classical rabbit hemorrhagic disease virus pcr amplification product is 170bp, the aPCR amplification productions of rabbit hemorrhagic disease virus anomaly
The size of object is 167bp；
(2) on the basis of (1), by two groups of PCR reaction products of comprehensive analysis with/without excluding false negative and the vacation of result
The positive, i.e.,
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112280898A (en) * | 2020-10-26 | 2021-01-29 | 宁波爱基因科技有限公司 | Efficient duplex primer and kit for detecting rabbit plague and rabbit plague type 2 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2034012A1 (en) * | 2007-09-05 | 2009-03-11 | Laboratorios Ovejero, S.A. | Vaccine against viral rabbit hemorrhagic disease |
| CN103146842A (en) * | 2011-03-15 | 2013-06-12 | 中国检验检疫科学研究院 | Rabbit hemorrhagic disease virus rt-pcr detection method |
| CN106702027A (en) * | 2017-01-16 | 2017-05-24 | 山东省农业科学院家禽研究所 | Isothermal amplification detection method for RHDV recombinant polymerase |
-
2018
- 2018-04-26 CN CN201810383767.5A patent/CN108546780A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2034012A1 (en) * | 2007-09-05 | 2009-03-11 | Laboratorios Ovejero, S.A. | Vaccine against viral rabbit hemorrhagic disease |
| CN103146842A (en) * | 2011-03-15 | 2013-06-12 | 中国检验检疫科学研究院 | Rabbit hemorrhagic disease virus rt-pcr detection method |
| CN106702027A (en) * | 2017-01-16 | 2017-05-24 | 山东省农业科学院家禽研究所 | Isothermal amplification detection method for RHDV recombinant polymerase |
Non-Patent Citations (4)
| Title |
|---|
| CAPUCCI L ET AL.: "A further step in the evolution of rabbit hemorrhagic disease virus: the appearance of the first consistent antigenic variant", 《VIRUS RESEARCH》 * |
| WANG X ET AL.: "Phylogenetic analysis of rabbit hemorrhagic disease virus in China and the antigenic variation of new strains", 《ARCHVIROL》 * |
| 宋艳华等: "兔出血症病毒经典毒株和变异毒株的RT-PCR鉴定", 《江苏农业学报》 * |
| 谭永贵等: "新型兔出血症病毒研究进展", 《中国动物传染病学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112280898A (en) * | 2020-10-26 | 2021-01-29 | 宁波爱基因科技有限公司 | Efficient duplex primer and kit for detecting rabbit plague and rabbit plague type 2 |
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