CN101671748A - Primer for detecting infectious bursal disease viruses and method and kit thereof - Google Patents
Primer for detecting infectious bursal disease viruses and method and kit thereof Download PDFInfo
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Abstract
The invention provides a primer for detecting infectious bursal disease viruses (IBDV). The primer comprises sequences in VP2 genes of the IBDV or complementary sequences thereof, such as oligonucleotide or complementary strands as shown in SEQ ID NO.1-4. The invention further provides a method and a kit for detecting IBDV, particularly a method and a kit for amplifying the nucleotide sequences ofthe IBDV on the basis of loop-mediated isothermal amplification (LAMP) method, wherein, the primer comprises sequences in VP2 genes of the IBDV or complementary sequences thereof. The primer of the invention has the advantages of high specificity and sensitivity. The method and the kit of the invention have the advantages of high sensitivity and specificity, convenient operation and short reaction time, and dispense with temperature-variable amplification equipment. Therefore, the invention is applicable to detection in simply equipped laboratories and fields.
Description
Technical field
The present invention relates to the virus detection techniques field, particularly be used to detect primer and the detection method and the test kit of infectious bursal disease virus.
Background technology
Infectious bursal disease (Infectious bursal disease, IBD) be by infectious bursal disease virus (Infectious bursal disease virus, IBDV) a kind of acute, the height contagious disease that cause, and be considered to one of disease of bringing very big loss to aviculture.IBDV mainly encroaches on the central immune organ fabricius bursa of 3-12 chick in age in week.It is dead that this disease not only can make infected chicken cause a disease, and production performance descends, but also can cause immunosuppression and immune deficiency, causes other vaccine immunity failures.Late 1980s U.S. variation strain and European highly virulent strain (very virulent IBDV, appearance vvIBDV) makes the anti-system of this disease become more severe.Because breaking through the protection that causes weak vaccine strain or classical strains inactivated vaccine by classical strains, variant and vvIBDV cause the chick morbidity.The particularly appearance of highly virulent strain can make the mortality ratio of infected chicken reach more than 60%, has brought serious economy loss for world's aviculture.
Because the use of vaccine is incorrect and the factors such as appearance of variation strain, causes this disease generally popular, set up some high specificities, diagnostic method that susceptibility is high seems very urgent and necessary.Originally people mainly make tentative diagnosis according to clinical pathology of chicken group and disease progression process to IBDV.Mainly comprise viral isolation identification and agar diffusion test (AGP), virus neutralization tests (VN), fluorescent antibody test (IFA), enzyme-linked immunosorbent assay (ELISA) etc. when making a definite diagnosis, but, do not reach the requirement of quick, accurate and early diagnosis because the isolation identification required time of virus is oversize.In recent years, the investigator transfers to adopt molecular biology method such as RT-PCR that different I BDV strain is diagnosed.In order to distinguish the different IBDV strain of antigenicity, RT-PCR technology and multiple restriction enzyme combine.But because the limitation of this method is replaced with RFLP bonded RT-PCR soon, this technology also can be used to IBDV is carried out somatotype simultaneously.In addition, real-time RT-PCR also is applied to viral diagnosis.Though these methods are simple fast, and are highly sensitive, reaction times method than before is short, but still needs the long period, and needs to use expensive instrument such as PCR instrument, and is therefore restricted to use range.Therefore, need the detection primer of a kind of new chicken infectivity bursa of Fabricius virus of invention and application method thereof to address the above problem.
Summary of the invention
One of main purpose of the present invention is to provide based on the deficiencies in the prior art a kind of primer that is used to detect infectious bursal disease virus of highly sensitive, high specificity.
One group of primer that is used for detecting infectious bursal disease virus provided by the invention is sequence or its complementary sequence of the VP2 gene of infectious bursal disease virus.
As the preferred embodiment for the present invention, the primer that the present invention is used to detect infectious bursal disease virus is the oligonucleotide shown in SEQ ID NO:1～4, the perhaps complementary strand of the oligonucleotide shown in SEQ ID NO:1～4.
Oligonucleotide sequence shown in SEQ ID NO:1～4 is as follows:
5′-GGCATCTTGCAGGTTTGT-3′；
5′-TCAGGAGCATCTGATCGAA-3′；
5′-GTGATTCTCTAGGGTGTTTTTCCATACGGAGCCTTCTGA-3′；
5′-CTCAATTGTGGGTGCTTTTTGTTCCATTGCTCGTCAGTGT-3′；
The perhaps complementary strand of the oligonucleotide shown in SEQ ID NO:1～4.
Another object of the present invention provides the method for short, the simple to operate detection infectious bursal disease virus of a kind of highly sensitive, high specificity, reaction times.
The method that the present invention detects infectious bursal disease virus is that the isothermal amplification method by the ring mediation increases to the nucleotide sequence of infectious bursal disease virus, and wherein primer is sequence or its complementary sequence in the VP2 gene of infectious bursal disease virus.
Preferably, the oligonucleotide sequence of described primer shown in SEQ ID NO:1～4:
F3：5′-GGCATCTTGCAGGTTTGT-3′；
B3：5′-TCAGGAGCATCTGATCGAA-3′；
FIP：5′-GTGATTCTCTAGGGTGTTTTTCCATACGGAGCCTTCTGA-3′；
BIP：5′-CTCAATTGTGGGTGCTTTTTGTTCCATTGCTCGTCAGTGT-3′；
The perhaps complementary strand of the oligonucleotide shown in SEQ ID NO:1～4.
As the preferred embodiment for the present invention, the detection method of infectious bursal disease virus of the present invention comprises the steps: to extract total RNA of sample; RNA is carried out reverse transcription reaction, obtain cDNA; CDNA is encircled the isothermal nucleic acid amplification reaction of mediation, and temperature of reaction is 61～65 ℃, and the reaction times is 10～60 minutes; Termination reaction detects amplified production at last again.
Perhaps extract total RNA of sample; The reverse transcription reaction of RNA and the isothermal nucleic acid amplification reaction of ring mediation are carried out under identical conditions simultaneously, and temperature of reaction is 61～65 ℃, and the reaction times is 10～60 minutes; Termination reaction detects amplified production at last again.
The 3rd purpose of the present invention is to provide based on the deficiencies in the prior art the detection kit of short, the simple to operate infectious bursal disease virus of a kind of highly sensitive, high specificity, reaction times.Described test kit comprises the primer that detects infectious bursal disease virus, and this primer is sequence or its complementary sequence in the VP2 gene of infectious bursal disease virus.
Preferably, the oligonucleotide of described primer shown in SEQ ID NO:1～4:
F3：5′-GGCATCTTGCAGGTTTGT-3′；
B3：5′-TCAGGAGCATCTGATCGAA-3′；
FIP：5′-GTGATTCTCTAGGGTGTTTTTCCATACGGAGCCTTCTGA-3′；
BIP：5′-CTCAATTGTGGGTGCTTTTTGTTCCATTGCTCGTCAGTGT-3′；
The perhaps complementary strand of the oligonucleotide shown in SEQ ID NO:1～4.
This test kit also comprises dNTPs mixture, trimethyl-glycine, MgSO
4, the big fragment of Bst archaeal dna polymerase, Bst dna polymerase buffer liquid and AMV reversed transcriptive enzyme.
Highly sensitive, the high specificity of detection infectious bursal disease virus primer of the present invention.According to VP2 gene conservative sequences Design primer, specificity between both having guaranteed to plant, can not increase he plant virus (IBV for example, NDV, H3N2AIV); Guarantee the kind internal specific again, can detect the strain of IBDV as much as possible.Article 4, primer fully guarantees its specificity, and wherein F3 and B3 are outer primers, works when initial amplified reaction; FIP and BIP are called inner primer, all work at whole amplification procedure, are a pair of primers of most critical, cause producing the cyclic loop-stem structure and unlimited amplification.
The method of detection infectious bursal disease virus provided by the invention has following advantage: highly sensitive, high specificity, amplification template can reach 10 the copy or still less; Easy and simple to handle, the loop-mediated isothermal amplification of employing can directly use water-bath to react, and does not need alternating temperature amplification instrument; Reaction times is short, and whole amplification can be finished less than 1h, can accelerate the LAMP reaction by 2 ring-type primers, make the reaction times shorten near half; The RT-LAMP single stage method does not need independent reverse transcription process; Identify easy, visual inspection by product white precipitate and colorimetry.The RT-LAMP technology sensitivity of detection of the present invention IBDV, special, easy is applicable to that simple and easy laboratory and field detect, for the detection of virus provides a kind of new selection.
The test kit of detection infectious bursal disease virus provided by the invention, the detection primer of employing is highly sensitive, high specificity, reaction times short, simple to operate, can satisfy the demand in market.
Description of drawings
Fig. 1 is the electrophorogram of a resulting product of preferred embodiment increasing under the differential responses temperature of the loop-mediated isothermal amplification of detection infectious bursal disease virus of the present invention;
Fig. 2 is the electrophorogram of a resulting product of preferred embodiment increasing under the differential responses time of the loop-mediated isothermal amplification of detection infectious bursal disease virus of the present invention;
Fig. 3 is the result who detects the preferred embodiment that the susceptibility of infectious bursal disease virus contrasts with RT-PCR and RT-LAMP method;
Fig. 4 is the result of a preferred embodiment of specificity test of the RT-LAMP of detection infectious bursal disease virus of the present invention.
Embodiment
For making the present invention easier to understand, will further set forth specific embodiments of the invention below.
The fabricius bursa sample that infects infectious bursal disease virus picks up from ground such as domestic Guangxi, Guangdong.Infectious bronchitis virus (IBV) vaccine strain H52 is a Cimmeria animal health company product, infectious bursal disease virus (IBDV) is provided by Guangdong Wen Shi group, and Lowly Pathogenic Avian Influenza Virus (LPAIV) H9N2 is provided by animal science institute of Agricultural University Of South China poultry research department.All samples comprises the fabricius bursa sample of 25 parts of clinical doubtful infection IBDV.Before handling, be stored in the PBS damping fluid of sterilization, frozen in-80 ℃.
Embodiment one: the isothermal amplification method by the ring mediation detects infectious bursal disease virus
Sample preparation: sample preparation is with reference to the standard method of OIE's regulation (OIE regulation).
The Qu Fashi of institute lens capsule tissue sample is cut into small pieces with different scissors places different vessels, and mill grinds, and adds the PBS that contains penicillin 2000IU/ml, Streptomycin sulphate 1000IU/ml and makes 20% (W/V) suspension.The centrifugal 10min of 1000g gets supernatant liquor and filters after allantoic cavity is inoculated SPF chicken embryo (available from Cimmeria animal health company) with disposable bacterial filter.
5 piece of 10 age in days SPF chicken embryo of supernatant liquor inoculation of each sample, every piece of inoculation 0.2ml.Every 12h observes chicken embryonic development situation.Discard chicken embryo dead in the 24h, dead germ and the dying embryo of collecting 36-72h place 4 ℃ of refrigerators earlier; The embryo of will living behind the 72h also places 4 ℃ of refrigerators, and the aseptic extraction allantoic fluid in back spends the night.The fresh allantoic fluid of a part is used for RT-LAMP test, and the rest part packing is also put-80 ℃ of preservations and is used for other experiment.Can prepare with reference to the experimental technique of " the molecular cloning experiment guide " of Cold Spring Harbor Laboratory press the preparation of sample.
Total RNA extracting:
Supernatant liquor after the allantoic fluid of 200 μ l or sample homogenization are centrifugal is used for the extraction of total RNA.Extraction step is undertaken by the process specifications of RNAiso reagent (Takara company), and chloroform, primary isoamyl alcohol, dehydrated alcohol are available from Guangzhou Chemical Reagent Factory; 75% ethanol and DEPC handle sterilized water (RNase-free water) and prepare voluntarily; High-speed refrigerated centrifuge is available from sigma company; Bechtop is available from net plant company limited of Soviet Union.
Total extractive step of RNA is summarized as follows:
(1) add 200ul virus liquid in the 1.5ml centrifuge tube, add 800 μ l TRIZOL again, jolting is thick until becoming sticky.Room temperature was placed 5 minutes, and nucleic acid and albumen are fully dissociated.
(2) add 200 μ l chloroforms, thermal agitation 15s mixes.Room temperature was placed 15 minutes.
(3) 4 ℃, 13,000r/min, centrifugal 15min gets the upper strata water in another new 1.5ml centrifuge tube, and adds the equal-volume primary isoamyl alcohol, and room temperature was placed 10～15 minutes behind the mixing.
(4) 4 ℃, 13, the centrifugal 10～15min of 000rpm, precipitated rna.Carefully abandon most supernatant, last with 4 ℃ 75% ethanol 1ml washing precipitation, 13, the centrifugal 10min of 000r/min.
(5) carefully abandon clean supernatant, air-dry RNA precipitation in the Bechtop.
(6) with 20 μ l RNase-free water dissolution precipitation, 2 μ l are used for RT-LAMP (reaction system is 25 μ l) 4 μ l and are used for RT-PCR (reaction system is 50 μ l).
Design of primers and synthetic:
Download the sequence of the VP2 gene of the IBDV on the GeneBank, find conservative zone, be used for designing the LAMP primer, comprise 4 primers altogether, be complementary to 8 fragments of template by software aligned sequences such as DNAStar, ClustalX.Primer sequence sees Table 1, and table 1 is the primer tabulation of RT-LAMP and RT-PCR.
Table 1
It is synthetic that primer sequence is delivered to Shanghai living worker company limited, is diluted to 10 μ M after synthesizing, and is placed in-20 ℃ of preservations after packing.
The RT-LAMP reaction:
Reaction system is by each 2M of inner primer, outer primer 0.2 μ M, dNTPs mix (TaKaRa company) 1.4mM, trimethyl-glycine betaine (Sigma company) 0.6M, MgSO
45mM, Bst archaeal dna polymerase (large fragment; New England Biolabs company) Bst dna polymerase buffer liquid, AMV reversed transcriptive enzyme (TaKaRa) 0.125U of 16U, 1 * provide, the RNA template of 2 μ l is formed, and reaction system is totally 25 μ l.In addition, pollute, on system, add the mineral oil (Sigma) of 20 μ l, play the effect of fluid-tight for preventing aerosol particles.
React last 80 ℃ of lasting 5min and come termination reaction with deactivation Bst archaeal dna polymerase.After having reacted, get product electrophoresis on 2% concentration, usefulness EB stained gel of 5 μ l.The sterilized water that uses DEPC to handle is done negative template.The characteristic electrophoretic band of the product of RT-LAMP is the scalariform band.
Embodiment two: the susceptibility of RT-LAMP and specificity experiment
In sensitivity test, get the positive allantoic fluid extracted total RNA of the infection IBDV of 200 μ l, RNA handles the sterilized water dissolving with the DEPC of 10 μ l, dilutes by 10 times multiple proportions then, each RNA concentration gradient is all got 2 μ l and is used for RT-LAMP, so as to judging the dilution limit that detects.
The specificity experiment is the RNA that detects the IBDV strain isolated with RT-LAMP, and with the RNA of NDV (Avian pneumo-encephalitis virus), IBV (infectious bronchitis virus), SIV (simian immunodeficiency virus), the sterilized water that DEPC handles is done negative control.
For the susceptibility and the specificity of relatively RT-LAMP amplification, (Promega, Madison WI) carry out synchronously with the RT-LAMP method to use AccessQuick RT-PCR single stage method test kit.
According to the test kit specification sheets, 50 μ l reaction systems comprise UP and the DN primer of 0.4 μ M, certain TflDNA polysaccharase, the RNA template of 5U AMV ThermoScript II and 4 μ l.
The program of RT-PCR reaction is:
50℃，30min
94℃，2min
72℃，10min
React last 80 ℃ of lasting 5min and come termination reaction with deactivation Bst archaeal dna polymerase.According to the demonstration of PCR instrument (Biometra company), the RT-PCR reaction probably needs 100min.
After having reacted, get use EB stained gel on the electrophoresis of the product of 5 μ l in 2% concentration, the amplified fragments of expection is 1226bp.
Embodiment three: the isothermal duplication method by the ring mediation detects the optimal reaction temperature of infectious bursal disease virus and groping of time conditions
Temperature of reaction is a most important parameter of RT-LAMP amplification, and four primers come initial sum continuity entire reaction in proper order according to the priority annealed.In order to determine best temperature of reaction and time, designed the thermograde and the time gradient of reaction respectively.RT-LAMP is reflected at 61 ℃, and 62 ℃, 63 ℃, 64 ℃ and 65 ℃ of reaction 60min (thermograde); Perhaps at 65 ℃ of reaction 10min, 20min, 30min, 40min, 50min and 60min (time gradient).Fig. 1 is the product electrophorogram of a preferred embodiment increasing under the differential responses temperature of the loop-mediated isothermal amplification of detection infectious bursal disease virus of the present invention; The M swimming lane is the Marker of 2000bp, the negative contrast of N swimming lane; The swimming lane of mark 61～65 is represented the different temperature of reaction between 61～65 ℃.Fig. 1 shows, increase in 61～65 ℃ of scopes and can both carry out, but the band of 61 ℃ of 65 ℃ of ratios and 62 ℃ becomes clear, and selects 65 ℃ as optimal reaction temperature.Fig. 2 is the product electrophorogram of a preferred embodiment increasing under the differential responses time of the loop-mediated isothermal amplification of detection infectious bursal disease virus of the present invention, wherein, the M swimming lane is represented the Marker of 2000bp, the different reaction times (min) of 60～20 expressions.In the reaction times, as shown in Figure 2,30min just can see distinctive scalariform band, produces bright band at 50min, and is faster than RT-PCR (needing 90min).
Embodiment four: RT-PCR and RT-LAMP method detect the susceptibility contrast experiment of infectious bursal disease virus
By extracting the RNA that infects the IBDV allantoic fluid, behind 10 times of doubling dilutions, the RNA that gets with isoconcentration is used for RT-LAMP and RT-PCR reaction.Fig. 3 is the result who detects the preferred embodiment that the susceptibility of infectious bursal disease virus contrasts with RT-PCR and RT-LAMP method; M represents the DNA Marker of 2000bp, 1～10
-5The swimming lane of sign is respectively the amplification of template after by 10 times of dilutions; Fig. 3 shows that the limit of detection of RT-PCR is RNA dilution 10
3Times, and the limit of detection of RT-LAMP is dilution 10
5Doubly.Therefore, RT-LAMP is than RT-PCR about responsive 100 times.From the reaction times, RT-LAMP is than the fast nearly 50min of RT-PCR.In the sensitivity, the RT-LAMP susceptibility is higher.
Embodiment five: RT-LAMP detects the specificity experiment of infectious bursal disease virus
Fig. 4 is the result of a preferred embodiment of specificity test of the RT-LAMP of detection infectious bursal disease virus of the present invention.Wherein, the M swimming lane is represented 2000bp DNA Marker, and the 1st, 3,5 swimming lanes are IBDV positive detected results, and the 2nd swimming lane is the detected result of NDV, and the 4th swimming lane is the detected result of IV (avian influenza virus), and the 6th swimming lane is the detected result of IBV.Table 2 is the result that 20 strain IBDV strains and 3 strains contrast strain detect with different methods.As table 2 and shown in Figure 4, all bursal disease virus poison strains all show the positive (+), and the strain that detects other three strains bird viruses shows feminine gender (-), both there be not omission bursal disease virus poison strain, do not have to occur and other viral cross reactions yet, illustrate that the RT-LAMP technology of being set up has very strong specificity.
Table 2
Embodiment six: RT-LAMP is applied to the detection of the clinical sample of infectious bursal disease virus
All detected results see Table 3.Table 3 is that different methods is to the doubtful test result of samples in field.25 all duplicate samples are all carried out traditional virus separation earlier and are identified, wherein 23 parts is positive the infection, and 2 parts negative.All negative samples are detected negative by RT-PCR and RT-LAMP, in all 23 parts of positive, 1 part by the RT-LAMP omission, and 3 parts by the RT-PCR omission.May be because viral level is lower than limit of detection in the sample, or because viral genome is undergone mutation at primer location, another may be that the improper or RNA extracting of sample preparation causes the degraded of RNA template.The presentation of results that 25 all duplicate samples detect, RT-LAMP shows 95.65% susceptibility, and RT-PCR is 86.96%.
Table 3
The LAMP amplification has mainly utilized the big segmental two big characteristics of Bst archaeal dna polymerase: the one, and archaeal dna polymerase has 5 ' → 3 ' dna polymerase activity, can be at DNA amplification nucleic acid under the temperature constant state; The 2nd, the Bst archaeal dna polymerase does not have 5 ' → 3 ' exonuclease activity, new synthetic nucleic acid strand can be got off from newly-generated dna double chain displacement (replacement), and independently the nucleic acid strand is synthetic with the DNA nucleic acid of opening next round to form two; And then removed DNA sex change link before each amplification from.On this basis, design can be discerned the LAMP primer sets (4 primer sets or 6 primer sets) of 6 (or 8) independent dna sequence dnas dexterously, makes two big characteristics of Bst archaeal dna polymerase give full play to.Wherein, the design of inner primer (BIP, FIP) is bright spot, the difficult point of LAMP design of primers.
The big fragment of Bst archaeal dna polymerase is the part of bacstearothermophilus archaeal dna polymerase, derives from the E.coli bacterial strain, and this bacterial strain contains the gene from the bacstearothermophilus archaeal dna polymerase, and this gene does not have 5 ' → 3 ' exonuclease activity.The applying gene recombinant technology with maltose binding protein (MBP) gene and this gene fusion expression, is removed maltose binding protein again behind the purifying.
Fs: the thing formation of starting of increasing
1 step: when double-stranded DNA was in the running balance about 65 ℃, the BIP primer in the primer sets just can be attached on the target dna two strands by complementation, under the effect of Bst archaeal dna polymerase, started DNA and synthesized.
2 steps: begin the complementary strand of synthetic DNA template to F3C from the B2 zone 3 ' end of BIP;
3 steps: the B3 primer annealing is attached to the complementary region B3C of template DNA, and it is synthetic to start the chain replacement, discharges simultaneously by BIP primer guiding synthetic complementary strand.
4 steps: the B1C zone of the DNA of release forms loop-stem structure in conjunction with the B1 zone; 1.3 the DNA chain that the step discharges as the template of FIP primer and F3 primer, is pressed, and the identical pattern of BIP end starts synthesizing of DNA and chain replaces, the DNA chain of release is at the same formation of FIP end loop-stem structure.And then, form the dumbbell-shaped structure amplification thing that starts.
Subordinate phase: extend
5 steps: the dumbbell structure is by the synthetic loop-stem structure DNA that converts to rapidly of bootstrap DNA;
6 steps: the BIP primer combines and begins new DNA with the cyclic single strand zone of loop-stem structure and synthesizes, because the complementary relationship of F1 and F1C forms loop-stem structure once more, previous synthetic DNA chain was released;
7 steps: the cyclic single strand zone combination of the loop-stem structure that the FIP primer is same and corresponding, because the complementary relationship of B1 and B1C forms loop-stem structure once more, previous synthetic DNA chain was released;
8 steps: the amplification that so goes round and begins again finally forms the inverted repeats of different lengths (the natural several times of purpose fragment length) Cauliflower cauliflower shape.
The LAMP primer design is more than conventional PCR complexity.A reaction comprises 4 primers at least, comprises a pair of inner primer (FIP and BIP, each about 40bp) and a pair of outer primer (F3 and B3, each about 25bp); If add a pair of ring primer (loop primer), just reaction can be quickened, the reaction times reduces by half, and increases the efficient of augmentation detection greatly.Primer Explorer version 3 softwares can the various special LAMP primers of specialized designs, and the user only need submit purpose segment sequence on request, and correlation parameter is set, and system just can produce multiple combination of primers and select for the user.In addition, the LAMP primer can manually design, the composition of each primer (asking for an interview table 4, the explanation that table 4 is formed for the LAMP primer), and the other technologies details of design of primers and precaution can be with reference to pertinent literature (Tsugunori Notomi etc., 2000; K.Nagamine etc., 2002).
Table 4
Because the LAMP reaction need not temperature cycling, so only need water-bath or other constent temperature heaters just can.
Amplification has three kinds of decision methods: the 1) agarose gel electrophoresis of nucleic acid, dyestuff can be that EB also can be SYBR Green I, and the concentration of sepharose is advisable with 2%, and damping fluid adopts tbe buffer liquid.2) naked eyes are directly observed by product magnesium pyrophosphate precipitation.The reaction that product is positive, the liquid in pipe muddiness, centrifugal or by white precipitate combine in the pipe end, negative reaction does not then have this phenomenon.3) SYBR Green I fluorescence dye directly dyes to product, also can pass through the naked eyes direct viewing.The reaction that product is positive adds and is green behind the dyestuff, and it is orange red that negative reaction then is.
Embodiment seven
One group of primer that is used to detect infectious bursal disease virus, this primer are sequence or its complementary sequence in the VP2 gene of infectious bursal disease virus.In the present embodiment, shown in the oligonucleotide of the sequence of this primer shown in SEQ IDNO:1～4, the perhaps complementary strand of the oligonucleotide shown in SEQ ID NO:1～4.
Oligonucleotide sequence shown in SEQ ID NO:1～4 is as follows:
5′-GGCATCTTGCAGGTTTGT-3′；
5′-TCAGGAGCATCTGATCGAA-3′；
5′-GTGATTCTCTAGGGTGTTTTTCCATACGGAGCCTTCTGA-3′；
5′-CTCAATTGTGGGTGCTTTTTGTTCCATTGCTCGTCAGTGT-3′；
The perhaps complementary strand of the oligonucleotide shown in SEQ ID NO:1～4.
Here used " oligonucleotide " is meant the purine-containing of any length and the polymkeric substance of pyrimidine, can be polyribonucleotide, can be polydeoxyribonucleotide, or blended multinuclear sugar-polydeoxyribonucleotide.It comprises strand and duplex molecule, DNA-DNA for example, and DNA-RNA and RNA-RNA heterozygote, and by " the protein nucleic acid " that forms with amino acid backbone conjugation base (PNA).It also comprises the nucleic acid that contains modified base.
Here used " primer " is that length is about 5 oligonucleotide to 50 Nucleotide, preferred length is about 6 to 25 Nucleotide, special preferred length is about 6 to 18 Nucleotide, it and relevant duplex of single-chain nucleic acid sequence formation, and can make the complementary strand polymerization reaction take place with for example reversed transcriptive enzyme or archaeal dna polymerase.
" complementary strand " of used here nucleotide sequence is meant the antisense sequences that participates in the original series Watson-Crick base pairing.
Embodiment eight
The test kit of infectious bursal disease virus in a kind of detection of biological sample, this test kit includes the primer that is used to detect infectious bursal disease virus, and described primer is sequence or its complementary sequence in the VP2 gene of infectious bursal disease virus.In the present embodiment, shown in the oligonucleotide of the sequence of this primer shown in SEQ ID NO:1～4, the oligonucleotide sequence shown in SEQ ID NO:1～4 is as follows:
5′-GGCATCTTGCAGGTTTGT-3′；
5′-TCAGGAGCATCTGATCGAA-3′；
5′-GTGATTCTCTAGGGTGTTTTTCCATACGGAGCCTTCTGA-3′；
5′-CTCAATTGTGGGTGCTTTTTGTTCCATTGCTCGTCAGTGT-3′；
The perhaps complementary strand of the oligonucleotide shown in SEQ ID NO:1～4.
Last institute should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention but not limiting the scope of the invention; although the present invention has been done detailed description with reference to preferred embodiment; those of ordinary skill in the art is to be understood that; can make amendment or be equal to replacement technical scheme of the present invention, and not break away from the essence and the scope of technical solution of the present invention.
Sequence table
＜110〉Zhongshan University
＜120〉be used to detect primer and the detection method and the test kit of infectious bursal disease virus
<160>6
<170>PatentIn?version?3.3
<210>1
<211>18
<212>DNA
＜213〉artificial sequence F3
<400>1
ggcatcttgc?aggtttgt 18
<210>2
<211>19
<212>DNA
＜213〉artificial sequence B3
<400>2
tcaggagcat?ctgatcgaa 19
<210>3
<211>39
<212>DNA
＜213〉artificial sequence FIP
<400>3
gtgattctct?agggtgtttt?tccatacgga?gccttctga 39
<210>4
<211>40
<212>DNA
＜213〉artificial sequence BIP
<400>4
ctcaattgtg?ggtgcttttt?gttccattgc?tcgtcagtgt 40
<210>5
<211>21
<212>DNA
＜213〉artificial sequence UP
<400>5
attgttccgt?tcatacggag?c 21
<210>6
<211>21
<212>DNA
＜213〉artificial sequence DN
<400>6
gctccgtatg?aacggaacaa?t 21
Claims (9)
1. be used to detect the primer of infectious bursal disease virus, it is characterized in that, described primer is sequence or its complementary sequence in the VP2 gene of described infectious bursal disease virus.
2. primer according to claim 1 is characterized in that, described primer is the oligonucleotide shown in SEQ ID NO:1～4:
5′-GGCATCTTGCAGGTTTGT-3′；
5′-TCAGGAGCATCTGATCGAA-3′；
5′-GTGATTCTCTAGGGTGTTTTTCCATACGGAGCCTTCTGA-3′；
5 '-CTCAATTGTGGGTGCTTTTTGTTCCATTGCTCGTCAGTGT-3 '; The perhaps complementary strand of the oligonucleotide shown in SEQ ID NO:1～4.
3. method that detects infectious bursal disease virus, it is characterized in that, isothermal duplication method by ring mediation increases to the nucleotide sequence of described infectious bursal disease virus, and wherein primer is sequence or its complementary sequence in the VP2 gene of described infectious bursal disease virus.
4. the method for detection infectious bursal disease virus according to claim 3 is characterized in that, described primer is the oligonucleotide shown in SEQ ID NO:1～4:
5′-GGCATCTTGCAGGTTTGT-3′；
5′-TCAGGAGCATCTGATCGAA-3′；
5′-GTGATTCTCTAGGGTGTTTTTCCATACGGAGCCTTCTGA-3′；
5 '-CTCAATTGTGGGTGCTTTTTGTTCCATTGCTCGTCAGTGT-3 '; The perhaps complementary strand of the oligonucleotide shown in SEQ ID NO:1～4.
5. the method for detection infectious bursal disease virus according to claim 3 is characterized in that, comprises the steps: to extract total RNA of sample; RNA is carried out reverse transcription reaction, obtain cDNA; CDNA is encircled the isothermal nucleic acid amplification reaction of mediation, and temperature of reaction is 61～65 ℃, and the reaction times is 10～60 minutes; Termination reaction detects amplified production at last again.
6. the method for detection infectious bursal disease virus according to claim 3 is characterized in that, comprises the steps: to extract total RNA of sample; The reverse transcription reaction of RNA and the isothermal nucleic acid amplification reaction of ring mediation are carried out under identical conditions simultaneously, and temperature of reaction is 61～65 ℃, and the reaction times is 10～60 minutes; Termination reaction detects amplified production at last again.
7. a test kit that detects infectious bursal disease virus is characterized in that, described test kit comprises the primer that detects infectious bursal disease virus, and described primer is sequence or its complementary sequence in the VP2 gene of described infectious bursal disease virus.
8. the test kit of detection infectious bursal disease virus according to claim 7 is characterized in that, described primer is the oligonucleotide shown in SEQ ID NO:1～4:
5′-GGCATCTTGCAGGTTTGT-3′；
5′-TCAGGAGCATCTGATCGAA-3′；
5′-GTGATTCTCTAGGGTGTTTTTCCATACGGAGCCTTCTGA-3′；
5 '-CTCAATTGTGGGTGCTTTTTGTTCCATTGCTCGTCAGTGT-3 '; The perhaps complementary strand of the oligonucleotide shown in SEQ ID NO:1～4.
9. the test kit of detection infectious bursal disease virus according to claim 7 is characterized in that, described test kit also comprises dNTPs mixture, trimethyl-glycine, MgSO
4, the big fragment of Bst archaeal dna polymerase, Bst dna polymerase buffer liquid and AMV reversed transcriptive enzyme.
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| CN200910193197A CN101671748A (en) | 2009-10-21 | 2009-10-21 | Primer for detecting infectious bursal disease viruses and method and kit thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102296064A (en) * | 2010-06-22 | 2011-12-28 | 中国人民解放军军事医学科学院微生物流行病研究所 | Specific primer for detecting enterovirus 71 (EV71) and application thereof |
| CN111019909A (en) * | 2019-12-27 | 2020-04-17 | 中国水产科学研究院黄海水产研究所 | Crustacean flavivirus and detection method and application thereof |
| CN112760424A (en) * | 2021-03-04 | 2021-05-07 | 山东信达基因科技有限公司 | Primer probe combination and kit for detecting chicken infectious bursal disease virus based on RT-RAA technology |
-
2009
- 2009-10-21 CN CN200910193197A patent/CN101671748A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102296064A (en) * | 2010-06-22 | 2011-12-28 | 中国人民解放军军事医学科学院微生物流行病研究所 | Specific primer for detecting enterovirus 71 (EV71) and application thereof |
| CN102296064B (en) * | 2010-06-22 | 2012-12-26 | 中国人民解放军军事医学科学院微生物流行病研究所 | Specific primer for detecting enterovirus 71 (EV71) and application thereof |
| CN111019909A (en) * | 2019-12-27 | 2020-04-17 | 中国水产科学研究院黄海水产研究所 | Crustacean flavivirus and detection method and application thereof |
| CN112760424A (en) * | 2021-03-04 | 2021-05-07 | 山东信达基因科技有限公司 | Primer probe combination and kit for detecting chicken infectious bursal disease virus based on RT-RAA technology |
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Open date: 20100317 |