CN102329889A - Primer and probe and method for detecting poxvirus muris - Google Patents
Primer and probe and method for detecting poxvirus muris Download PDFInfo
- Publication number
- CN102329889A CN102329889A CN201110233951A CN201110233951A CN102329889A CN 102329889 A CN102329889 A CN 102329889A CN 201110233951 A CN201110233951 A CN 201110233951A CN 201110233951 A CN201110233951 A CN 201110233951A CN 102329889 A CN102329889 A CN 102329889A
- Authority
- CN
- China
- Prior art keywords
- primer
- probe
- real
- seq
- pcr
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
The invention discloses a primer and a probe and a method for detecting poxvirus muris. The primer is a pair of oligonucleotides consisting of a forward primer with a SEQ ID NO:1 in a sequence table and a reverse primer with a SEQ ID NO:2 in the sequence table, and the probe matched with the primer has a nucleotide sequence of SEQ ID NO:3 in the sequence table. The method and a kit for detecting the poxvirus muris have the advantages of rapidness, simpleness, high sensitivity, strong specificity and high yield, solve the defects of complex operation, time waste, long period, and a certain requirement on an operated laboratory of the traditional detection means, and have better application prospect.
Description
Technical field
The present invention relates to field of biological detection, particularly relate to a kind of primer, probe and method thereof that is used to detect infectious ectromelia virus.
Background technology
The virus of natural infection laboratory animal is a lot, according to the hazardness to the mankind, can it be divided three classes; One type is Amphixenosis virus, can infected person and primate; Two types are not still had sign at present and show infected person, but can in people, ape and the monkey source sexual cell of vitro culture, duplicate, to being potentially dangerous property of the mankind; Three viroids are infection animal itself only under field conditions (factors), and not having still at present that sign shows can infected person, therefore little to threat of human.
Now, mouse has the potential virus pollution as a main source of biological products such as monoclonal antibody, protein medicaments.In " Pharmacopoeia of People's Republic of China (version in 2010) " three ones; Stipulated that mouse source property biological products need 8 kinds of mouse source viruses of quality inspection; Wherein infectious ectromelia virus belongs to the II class; Still do not have sign at present and show infected person, but can in people, ape and the monkey source sexual cell of vitro culture, duplicate, being potentially dangerous property of the mankind.The formulation of mouse borne virus examination criteria guarantees that to objective evaluation biological products quality people ' s health will play a positive role.
Infectious ectromelia virus also is mouse pox virus, and (poxvirus of mice MPV), can cause mousepox.It is explosive popular that mousepox is more, and lethality rate is higher, often causes the full crowd of mouse to eliminate, very harmful.Clinical manifestation with four limbs, tail and head swelling, fester, necrosis even toe come off is characteristic, so mouse pox virus claims to take off pedopathy virus (ectromelis virus, below abbreviate this virus as EV) again.Be widely current among the laboratory mouse crowd of this disease all over the world, in China mouse crowd, be sporadic popular.
The mouse source method for detecting virus of pharmacopeia regulation has: test cell line, animal's antibody produce experiment and chicken embryo infection experiment.These methods detect the potentially contaminated of mouse source virus from the biological effect angle, and the detection means complicacy is time-consuming, and the cycle is long, and there is certain requirement in the laboratory of operation, should not be as a kind of Quality Control means of guidance production of routine.
And the very sophisticated real-time fluorescence quantitative PCR technology of development at present (Real-time Fluorence Quantitative Polymerase Chain Reaction is called for short Real Time PCR) detection sensitivity is high; Simple and convenient, and also very low to Laboratory Request.Real Time PCR was released by U.S. Applied biosystems company in 1996; Be in the PCR reaction system, to add fluorophor; Utilize the fluorescent signal accumulation whole PCR process of monitoring in real time; Each circulation is become " visible ", the method for through Ct value and typical curve the initial concentration of the DNA in the sample (orcDNA) being carried out quantitative analysis at last.This method since producing, constantly develop perfect, particularly along with the widespread use of Taqman fluorescent probe, up to the present should technology very ripe.The PCR of Taqman fluorescent probe detects and is meant when carrying out pcr amplification; Also add a specific fluorescent probe when in reaction system, adding a pair of primer; This probe is an oligonucleotide, and two ends are mark report fluorogene and a cancellation fluorophor respectively.When probe is complete; Reporter gene institute fluorescent signal emitted is absorbed by the cancellation gene, and along with the Taq enzyme runs in the chain extension process and template bonded probe, its 5 '-3 ' exonuclease activity is cut degraded with probe enzyme during amplification; The report fluorophor separates with the cancellation fluorophor; Thereby fluorescence detecting system can monitor fluorescent signal, and template is whenever duplicated once, just has a probe to be cut off the release of following a fluorescent signal.Because d/d fluorophor number and PCR product quantity are one-one relationships, so signal accumulation and PCR product are synchronous fully.After entire reaction finishes, just can obtain an amplification curve, can obtain a typical curve, can carry out quantitative analysis to sample according to the amplification curve in this typical curve and the sample by the amplification curve of concentration known standard model.
The real-time fluorescence quantitative PCR technology not only realized to the DNA/RNA template quantitatively; And have sensitivity and specificity high, can realize multiple reaction, level of automation is high, pollution-free, real-time and characteristics such as accurate, this has been widely used in a plurality of fields such as immunoassay, bacterium, viral detection.Up to now, real-time fluorescence quantitative PCR technology for detection infectious ectromelia virus aspect does not see that also report is arranged, and very wide development space is arranged undoubtedly.
Summary of the invention
In order to solve the deficiency of existing infectious ectromelia virus detection method, the invention provides primer, probe and method thereof that a kind of real-time fluorescence quantitative PCR detects infectious ectromelia virus, this method is simple and convenient, highly sensitive and detection time short.
An object of the present invention is to provide a pair of primer that is used to detect infectious ectromelia virus.
Primer provided by the invention, a pair of oligonucleotide of forming by upstream primer with the SEQ ID NO:1 in the sequence table and the downstream primer with the SEQ ID NO:2 in the sequence table.
SEQ ID NO:1 in the sequence table is by 31 based compositions; SEQ IDNO:2 in the sequence table is by 22 based compositions.
The inventor has designed many to the PCR primer, and it is right that the long-term a large amount of numerous primer centerings of experiment of process have filtered out the primer of being made up of above-mentioned upstream primer and downstream primer.Its amplification segment size is 119bp.This primer has the conservative property of height to infectious ectromelia virus, and does not have significant homology with the mouse genome, uses this primer to carry out pcr amplification, can realize accurate qualitative and quantitative analysis.
Another object of the present invention provides a kind of probe that is used to detect infectious ectromelia virus; Said probe has the nucleotide sequence of the SEQ ID NO:3 in the sequence table; Said probe 5 ' end is connected with the fluorescence report group, and 3 ' end is connected with the fluorescent quenching group.
The fluorescence report group can be selected from any one among FAM, JOE, the HEX in the said probe, is preferably FAM; Said fluorescent quenching group is selected from TAMRA or Eclipse, is preferably TAMRA.
SEQ ID NO:3 in the said sequence table is by 29 based compositions.
A further object of the present invention provides a kind of method that detects infectious ectromelia virus, and said method is template with the sample DNA, uses the above-mentioned real-time fluorescence quantitative PCR detection method of mentioning that primer and probe carry out.
Preferably, the reaction system of said real-time fluorescence quantitative PCR also comprises and takes off pedopathy viral nucleic acid pcr amplification reagent, positive quantitative criterion article, negative quality control product, blank.
Preferably, the annealing temperature of said real-time fluorescence quantitative PCR is 62 ℃.
Preferably, the reaction conditions of said real-time fluorescence quantitative PCR is: 50 ℃, and 2min; 95 ℃, 10min; 95 ℃, 15s, 62 ℃, 1min, totally 40 circulations.
Preferably; Said method also comprises the foundation of typical curve; Method is: use the pMA-T recombinant plasmid that contains the base homologous sequence shown in the SEQ ID NO:4 in the sequence table as positive quantitative criterion article; With its ten times positive quantitative criterion article that are diluted to different concns, carry out real-time fluorescence quantitative PCR with said positive quantitative criterion article as template and detect, obtain detecting the residual typical curve of living infectious ectromelia virus.
Another purpose of the present invention provides a kind of test kit that detects infectious ectromelia virus, and said test kit contains above-mentioned primer of mentioning and probe.
Preferably, the reaction system of said test kit also comprises and takes off pedopathy viral nucleic acid pcr amplification reagent, positive quantitative criterion article, negative quality control product, blank.
Preferably, said pcr amplification reagent is 2 * Taqman PCR Mix.
Preferably, said positive quantitative criterion article are for containing the pMA-T recombinant plasmid of the base homologous sequence shown in the SEQ ID NO:4 in the sequence table.
Also purpose of the present invention provides the above-mentioned primer of mentioning or probe and in detecting mouse source biological products, takes off the application of pedopathy virus when residual.
Compare with existing detection method, fluorescence quantifying PCR method provided by the invention detects infectious ectromelia virus and has the following advantages:
1, fast simple: existing pharmacopeia infectious ectromelia virus detection method is: test cell line, animal's antibody produce experiment and chicken embryo infection experiment; The latent infection of Ectromelia virus from biological effect angular detection biological products; Take and grow (1-4 time-of-week), and the present invention detects taking off pedopathy virus from the nucleic acid angle, only needs can go out the result in about 6 hours; And it is not high to the operation laboratory environmental requirement; Evaded the issuable leak of former sampling observation product, can further improve biological products Quality Control quality in enterprises implement batch detection.
2, highly sensitive: existing detection method is the latent infection of Ectromelia virus from biological effect angular detection biological products; Wherein in the preparation process, passed through the processing of a plurality of process steps because of preparation; The possibility that the live virus antigen of residual mouse source virus and antiviral antibody exist is minimum; And the present invention detects from the viral nucleic acid angle, and aforementioned relatively pharmacopeia prescriptive procedure has improved the sensitivity that detects.Utilize when Ectromelia is viral in the real-time fluorescence quantitative PCR detection of biological goods among the present invention, can be accurately quantitative, target dna is 10
2-10
7In the concentration range, good linear relationship is arranged all, the highly sensitive 100 copy/μ l that reach.
3, repeatability, accuracy and specificity: through the homology analysis to the conservative gene EVM170 of ECTV-Mos, the primer among the present invention and probe design according to its homology sequence, and do not have homology with the mouse genome, and detection specificity is strong, good reproducibility;
4, the recovery is high: the universal standard requires the same yield of detection method will reach more than 50% in the industry, and the real time fluorescence quantifying PCR method recovery of the present invention has all reached more than 80%, is the detection method of the alternative existing pharmacopeia regulation of a kind of ideal.
Description of drawings
The structural representation of the positive quantitative criterion article of Fig. 1 plasmid;
Fig. 2 is the quantitative curve of real-time fluorescence quantitative PCR;
Fig. 3 is the real-time fluorescence quantitative PCR typical curve.
Embodiment
The following embodiment that lifts only is used to explain the present invention, is not to be used to limit protection scope of the present invention.
Reagent raw material described in the following embodiment is commercially available common raw material except that indicating the source especially, and ordinary method is adopted in the preparation of reagent.The method that does not detail among the embodiment is this area routine operation, sees " molecular cloning " third edition for details.
The design of embodiment 1 infectious ectromelia virus primer special and probe
According to document (David J.Esteban et al.; 2005), the genome of ECTV-Mos is analyzed, and combined document (Gloria Ribas et al.; 2003), select the conservative gene EVM170 (being the crmD gene) of ECTV-Mos to carry out the ECTV homology analysis.This gene order and GenBank+EMBL+DDBJ+PDB DB are carried out sequence alignment, and carry out sequence alignment, confirm that homologous sequence is following: i.e. nucleotide sequence shown in the SEQ ID NO:4 with the mouse genome database.
Conservative homologous sequence zone design real-time fluorescence quantitative PCR to finding out detects primer special and probe sequence, and the purpose clip size that amplifies is 119 bases.Wherein preferred primer, probe sequence are:
Upstream primer: 5 '-TGACTCATTCCTGTAATACCACTTCTAATAC-3 ', i.e. nucleotide sequence shown in the SEQID NO:1;
Downstream primer: 5 '-ACTGCTACATTTGCCTCGACAA-3 ', i.e. nucleotide sequence shown in the SEQ ID NO:2;
Said probe: 5 '-TCCATTCCTAATCATAGTCCCGCGTGTCT-3 ', i.e. nucleotide sequence shown in the SEQID NO:3;
5 ' end of said probe is with fluorescence report group FAM mark, and 3 ' end is with fluorescent quenching group TAMRA mark.
Carry out homology comparison back with designed primer, probe and find that it has the high conservative type to taking off pedopathy virus, with mouse genome/EST, and other nearly edge viruses homology not, its specificity is higher.
The preparation of embodiment 2 positive quantitative criterion article
Nucleotide sequence shown in the synthetic SEQ ID NO:4, and be inserted on the pMA-T carrier and be prepared from (synthetic by Invitrogen company), called after pMA-T-EV, collection of illustrative plates is as shown in Figure 1.PMA-T-EV plasmid total length is 2854b altogether, molecular weight 1.88 * 10
6Da, calculating can get, 1 μ g plasmid=3.2 * 10
11Copies.
The glycerol stock (Invitrogen company provides) that will contain the pMA-T-EV plasmid is after 37 ℃ of concussions of spending the night are cultivated in LB substratum (containing 100ng/ml Amp); Extract the pMA-T-EV plasmid with the little extraction reagent kit of plasmid (QIAgen company provides); The ultraviolet light absorption standard measure also calculates copy number, is diluted to 10
8Copies/ μ l is as positive quantitative criterion article, packing be stored in-80 ℃ subsequent use.
The foundation of embodiment 3 real-time fluorescence quantitative PCR amplification methods
1, real-time fluorescence quantitative PCR template preparation---total DNA extraction:
According to operation instruction, use Trizol/Trizol LS test kit (Invitrogen company provides) to extract sample DNA respectively, concrete grammar is following:
1) in sample (as need to grind homogenate for tissue), add an amount of TRIzol//Trizol LS, mixing, room temperature leaves standstill 5min;
2) add chloroform: primary isoamyl alcohol (24: 1) solution 200ul, with the violent mixing 10s of vortex.Room temperature is placed 5min;
3) 4 ℃ of centrifugal 15min of 15000prm.Exhaust supernatant as far as possible, abandon supernatant;
4) middle layer and organic layer add 0.5mlDNA recovery liquid, mixing, and room temperature leaves standstill 10min;
5) 4 ℃ of centrifugal 15min of 15000prm;
6) shift supernatant, add equal-volume phenol: chloroform: primary isoamyl alcohol (25: 24: 1), concussion mixing;
7) shift supernatant to new pipe, add 0.8 times of volume Virahol, room temperature leaves standstill 5min;
8) 4 ℃ of centrifugal 15min of 15000prm abandon supernatant, and the 75% ethanol 1ml that adds precooling washes the DNA deposition;
9) 4 ℃ of centrifugal 10min of 15000prm;
10) with liquid-transfering gun supernatant is removed totally as far as possible vacuum-drying 5～10min;
11) use the 100ulTE dissolution precipitation.
2, real-time fluorescence quantitative PCR reaction system
The inventor is a template with positive quantitative criterion article pMA-T-EV, and the various combination of probe, primer is groped, and has finally confirmed the best of breed of probe and primer: 10 μ M probes, 0.4 μ l, each 0.6 μ l of 10 μ M upstream and downstream primers in the 20 μ l reaction systems.
Particularly, when carrying out sample determination, be template with total DNA, carry out real-time fluorescence quantitative PCR, wherein reaction system is:
Reaction system (20 μ l):
3, real-time fluorescence quantitative PCR reaction conditions
Carry out real-time fluorescence quantitative PCR with above-mentioned reaction system, the annealing temperature in the response procedures is groped, confirm that optimum annealing temperature is 62 ℃.
Concrete reaction conditions is:
50 ℃, 2min; 95 ℃, 10min; 95 ℃, 15s, 62 ℃, 1min, totally 40 circulations.Image data after each loop ends, reaction finish the back according to the amplification curve result of determination.
The checking of embodiment 4 methodologies
1, the sensitivity of the foundation of typical curve and real-time fluorescence quantitative PCR
Be diluted to 10 with pMA-T-EV
1, 10
2, 10
3, 10
4, 10
5, 10
6, 10
7Copies/ μ l is a template; Carry out the real-time fluorescence quantitative PCR reaction according to reaction system among the embodiment 3 and reaction conditions; Measure light absorption value after each loop ends along with the PCR system; Just having obtained with the cycle number is X-coordinate, and light absorption value is the quantitative curve of ordinate zou and is X-coordinate with the logarithmic value of standard substance concentration that Ct is the typical curve of ordinate zou.Wherein, the implication of Ct value is: the cycle number that the fluorescent signal in each reaction tubes is experienced when arriving the thresholding of setting.
The detection lower bound of real-time fluorescence quantitative PCR of the present invention: the dilution lower bound that credible Ct value in 40 circulations, occurs; Differ from 10 times of template amounts when every; The Ct value differs 3.3 Ct value and is considered to believable Ct value; Quantitative curve by shown in Figure 2 can draw, and the sensitivity of this real-time fluorescence quantitative PCR can reach 100copies/ μ l.
Research shows that there is linear relationship in the logarithm of the Ct value of each template and the initial copy number of this template, and initial copy number is many more, and the Ct value is more little.Utilize the positive quantitative criterion article of known initial copy number to make typical curve, wherein X-coordinate is represented the logarithm of initial copy number, and ordinate zou is for the Ct value.Therefore, as long as obtain the Ct value of unknown sample, can calculate the initial copy number of this sample from typical curve.
Typical curve is as shown in Figure 3, typical curve R
2=0.995, linear dependence is good.
2, the repeatability of real-time fluorescence quantitative PCR, accuracy and specificity
It is 10 that positive quantitative criterion article pMA-T-EV is diluted to concentration respectively
6, 10
4, 10
2Copies/ μ l with the negative contrast of anosis mouse DNA (negative quality control product), is a blank with ddH2O as being senior middle school's low value Quality Control, carries out real-time fluorescence quantitative PCR, measuring result such as following table 1:
Table 1
Can know by The above results,
1) blank is set up, and experimental result is effective; Anosis mouse DNA detected result is negative;
It is all positive that positive quantitative criterion article detect the result, so this detection method of explanation has very strong specificity;
2) 3 experimental standard deviations are explained good reproducibility of the present invention in 15%, and precision meets the requirements;
3) accuracy meets the requirements.
3, recovery experiment
Get 4 parts of mouse liver tissues (numbering 1-4) and 4 parts of commercially available Soviet Union peptides and give birth to (numbering 4-8, the injection liquid after the dissolving), positive quantitative criterion article pMA-T-EV is diluted to concentration 10
8, 10
6, 10
4Copies/ μ l, the preparation of according to the form below 2 proportionings supplies test agent (simulation positive).
Table 2 supplies the test agent preparation
The process for extracting of pressing among the embodiment 3 extracts DNA, as the template of PCR reaction, carries out the real-time fluorescence quantitative PCR reaction by reaction system and the condition of embodiment 3 then, and wherein blank is with ddH
2O is a template, and three times reproducible results is as shown in table 3, calculates the recovery of this method.
Table 3
The real-time fluorescence detection kit of embodiment 5 infectious ectromelia virus
Above-mentioned being used for carried out the upstream and downstream primer probe mixed solution 500 μ l that real-time fluorescence quantitative PCR detects, Taqman Gene Expression Master MiX 10ml, positive quantitative criterion article (1 * 10 to infectious ectromelia virus
7Copies/ μ l) 50 μ l and diluent 10ml, negative control article 25 μ l, aseptic ddH
2O 10ml packs jointly, obtains the real-time fluorescence detection kit of infectious ectromelia virus.
This test kit method of use:
With by the sample total DNA of extracting among the embodiment 3-1 as template, positive quantitative criterion article 1 * 10
7Copies/ μ l uses negative control, ddH simultaneously as the positive control template
2The O blank; Carry out real-time fluorescence quantitative PCR according to the reaction system of embodiment 3-2 and the reaction conditions of embodiment 3-3, with reference to the method for embodiment 4-1 positive quantitative criterion article dilution is carried out real-time fluorescence quantitative PCR production standard curve for different concns simultaneously.
Interpretation of result, when positive control, negative control, blank all just often, as long as obtain the Ct value of unknown sample, can calculate the initial copy number of this sample from typical curve.
The above is merely preferred embodiment of the present invention, and is in order to restriction the present invention, not all within spirit of the present invention and principle, any modification of being done, is equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (15)
1. be used to detect the primer of infectious ectromelia virus, it is characterized in that, a pair of oligonucleotide that said primer is made up of upstream primer with the SEQ ID NO:1 in the sequence table and the downstream primer with the SEQ ID NO:2 in the sequence table.
2. a probe that is used with the said primer of claim 1 is characterized in that, said probe has the nucleotide sequence of the SEQ ID NO:3 in the sequence table.
3. probe according to claim 2 is characterized in that, said probe 5 ' end is connected with the fluorescence report group, and 3 ' end is connected with the fluorescent quenching group.
4. according to claim 2 or 3 described probes, it is characterized in that said fluorescence report group is selected from any one among FAM, JOE, the HEX, said fluorescent quenching group is selected from TAMRA or Eclipse.
5. according to the arbitrary described probe of claim 2-4, it is characterized in that said fluorescence report group is FAM, said fluorescent quenching group is TAMRA.
6. a method that detects infectious ectromelia virus is characterized in that, said method is template with the sample DNA, the real-time fluorescence quantitative PCR detection method that uses primer described in the claim 1 and the arbitrary described probe of claim 2-4 to carry out.
7. method according to claim 6 is characterized in that, the reaction system of said real-time fluorescence quantitative PCR also comprises takes off pedopathy viral nucleic acid pcr amplification reagent, positive quantitative criterion article, negative quality control product, blank.
8. according to claim 6 or 7 described methods, it is characterized in that the annealing temperature of said real-time fluorescence quantitative PCR is 62 ℃.
9. according to the arbitrary described method of claim 6-8, it is characterized in that the reaction conditions of said real-time fluorescence quantitative PCR is: 50 ℃, 2min; 95 ℃, 10min; 95 ℃, 15s, 62 ℃, 1min, totally 40 circulations.
10. according to the arbitrary described method of claim 6-9; It is characterized in that; Said method also comprises the foundation of typical curve, and method is: use the pMA-T recombinant plasmid that contains the base homologous sequence shown in the SEQ ID NO:4 in the sequence table as positive quantitative criterion article, with its ten times positive quantitative criterion article that are diluted to different concns; Carry out real-time fluorescence quantitative PCR with said positive quantitative criterion article as template and detect, obtain detecting the typical curve of infectious ectromelia virus.
11. a test kit that detects infectious ectromelia virus is characterized in that, said test kit contains primer described in the claim 1 and the arbitrary described probe of claim 2-4.
12. test kit according to claim 11 is characterized in that, the reaction system of said test kit also comprises takes off pedopathy viral nucleic acid pcr amplification reagent, positive quantitative criterion article, negative quality control product, blank.
13., it is characterized in that said pcr amplification reagent is 2 * Taqman PCR Mix according to claim 11 or 12 described test kits.
14., it is characterized in that said positive quantitative criterion article are for containing the pMA-T recombinant plasmid of the base homologous sequence shown in the SEQ ID NO:4 in the sequence table according to the arbitrary described test kit of claim 11-13.
15. the arbitrary described probe of primer described in the claim 1 or claim 2-4 takes off the application of pedopathy virus when residual in detecting mouse source biological products.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110233951 CN102329889B (en) | 2011-08-16 | 2011-08-16 | Primer and probe and method for detecting poxvirus muris |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110233951 CN102329889B (en) | 2011-08-16 | 2011-08-16 | Primer and probe and method for detecting poxvirus muris |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102329889A true CN102329889A (en) | 2012-01-25 |
| CN102329889B CN102329889B (en) | 2013-05-29 |
Family
ID=45481849
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110233951 Active CN102329889B (en) | 2011-08-16 | 2011-08-16 | Primer and probe and method for detecting poxvirus muris |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102329889B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103215382A (en) * | 2013-04-16 | 2013-07-24 | 武汉珈创生物技术有限公司 | Fluorescent quantitative PCR (Polymerase Chain Reaction) kit for detecting ectromelia virus and application |
| CN104450964A (en) * | 2014-12-05 | 2015-03-25 | 浙江省医学科学院 | Fluorescent quantitative polymerase chain reaction (PCR) method for detecting mouse poxvirus as well as special primer pair, probe and kit of method |
| CN104894291A (en) * | 2014-03-09 | 2015-09-09 | 中华人民共和国上海出入境检验检疫局 | Method for real-time fluorescent PCR detection of poxvirus of mice |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101044158A (en) * | 2004-09-02 | 2007-09-26 | 安东尼奥·阿尔卡米佩特霍 | Chemokine binding activity of viral TNF receptors and related proteins |
-
2011
- 2011-08-16 CN CN 201110233951 patent/CN102329889B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101044158A (en) * | 2004-09-02 | 2007-09-26 | 安东尼奥·阿尔卡米佩特霍 | Chemokine binding activity of viral TNF receptors and related proteins |
Non-Patent Citations (2)
| Title |
|---|
| 肖杭: "《实验动物科学与应用》", 31 December 2008 * |
| 蒋健敏: "《实用医学实验动物学》", 31 December 2009 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103215382A (en) * | 2013-04-16 | 2013-07-24 | 武汉珈创生物技术有限公司 | Fluorescent quantitative PCR (Polymerase Chain Reaction) kit for detecting ectromelia virus and application |
| CN104894291A (en) * | 2014-03-09 | 2015-09-09 | 中华人民共和国上海出入境检验检疫局 | Method for real-time fluorescent PCR detection of poxvirus of mice |
| CN104450964A (en) * | 2014-12-05 | 2015-03-25 | 浙江省医学科学院 | Fluorescent quantitative polymerase chain reaction (PCR) method for detecting mouse poxvirus as well as special primer pair, probe and kit of method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102329889B (en) | 2013-05-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103382507B (en) | 1 type and 3 type duck hepatitis A virus (HAV) single stage method dual RT-PCR detection kit, primer pair and method | |
| CN102352416A (en) | Primer, probe and kit for detecting mouse hantaviruses | |
| CN103820573A (en) | Fluorescent quantitation PCR (polymerase chain reaction) detection kit for four conventional herpesvirus hominises | |
| CN108676920A (en) | It is a kind of quickly to detect mouse norovirus primer, kit and its RT-RPA methods | |
| CN105861641A (en) | Primer, kit and method for detecting CHO cell DNA residues | |
| CN102337359A (en) | Primers and probe for detecting mouse leukemia virus and method thereof | |
| CN103773898A (en) | Triple detection kit for human herpes viruses HSV-1, HSV-2 and HCMV | |
| CN102329890B (en) | Primer, probe and method for detecting mouse adenovirus | |
| CN102329889B (en) | Primer and probe and method for detecting poxvirus muris | |
| CN110343784A (en) | The composition and kit of quadruple influenza nucleic acids detection based on melting curve | |
| CN110305988A (en) | Pigeon with newcastle disease LAMP-TaqMan detection kit | |
| CN102634605A (en) | Method for detecting egg drop syndrome viruses and kit for method | |
| CN107686865A (en) | Detect primer pair, probe, kit and the method for I type bovine herpes virus | |
| CN102367494B (en) | Primer, probe and kit with primer and probe for multiple real-time fluorescence PCR (polymerase chain reaction) detection of mouse-derived RNA (ribonucleic acid) viruses | |
| CN102676697A (en) | Primers and probe for detecting peste des petits ruminants virus and kit | |
| CN105525038A (en) | Newcastle disease virus strong/weak virulent one-step real-time fluorescence RT-PCR detection kit | |
| CN102399907B (en) | Multiplex real-time fluorescent polymerase chain reaction (PCR) primer for simultaneously detecting mouse adenovirus and poxvirus muris mouse pox virus, probe as well as reagent kit thereof | |
| CN109536625A (en) | A kind of the iiPCR detection method and detection kit of Mycoplasma bovis | |
| CN102337357A (en) | Primers and probe for detecting mouse reovirus type III and method thereof | |
| CN102952897A (en) | RT-PCR (Reverse Transcription-Polymerase Chain Reaction) fluorescence detection kit for rubella virus and detection method thereof | |
| CN102337358B (en) | Primers and probe for detecting mouse Sendai virus and method thereof | |
| WO2016078215A1 (en) | Primers, probes and kit for detecting and typing five ebola virus subtypes by one-step method reverse transcription pcr | |
| CN102816865A (en) | H1N1, PRRSV and CSFV quadruple detection kit | |
| CN105861751A (en) | Primer pair and fluorescent quantitative PCR kit for detecting mouse adenovirus and application thereof | |
| CN110616200A (en) | Porcine reproductive and respiratory syndrome virus European strain nucleic acid standard substance and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |