CN102329789A - Positive control composition for reverse transcription polymerase chain reaction kit and preparation method thereof - Google Patents
Positive control composition for reverse transcription polymerase chain reaction kit and preparation method thereof Download PDFInfo
- Publication number
- CN102329789A CN102329789A CN201110272456A CN201110272456A CN102329789A CN 102329789 A CN102329789 A CN 102329789A CN 201110272456 A CN201110272456 A CN 201110272456A CN 201110272456 A CN201110272456 A CN 201110272456A CN 102329789 A CN102329789 A CN 102329789A
- Authority
- CN
- China
- Prior art keywords
- virus
- positive control
- preparation
- polymerase chain
- chain reaction
- 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 provides a positive control composition for a reverse transcription polymerase chain reaction kit and a preparation method thereof, and the positive control composition is characterized in that a non-target virus is taken as positive control, a pair of primers are simultaneously designed for amplification, and the length of an amplified fragment is the same with that of a target gene of the reverse transcription polymerase chain reaction kit; and the non-target virus is inactivated by guanidine isothiocyanate. The positive control prepared by the method is safe, thereby avoiding virus expansion caused by direct use of a virus infection material in the prior art, playing a control role during an RNA (ribonucleic acid) extraction process and an RNA reverse transcription process when serving as the reverse transcription positive control of the RNA virus, proving that an RNA extraction reagent and substances used in a reaction system are normal in activity, facilitating the positive control when serving as the commercial kit and having very high practical value.
Description
Technical field
The invention belongs to field of virus detection, relate to a kind of positive control that is used for the inverse transcription polymerase chain reaction test kit, more specifically relate to a kind of positive control compsn that is used for the inverse transcription polymerase chain reaction test kit and preparation method thereof.
Background technology
The effect of the control sample of inverse transcription polymerase chain reaction test kit in experiment is: when each RT-PCR detects; Negative control sample and the positive control sample set up can not lack; When the negative control sample detection is negative, show that the reagent of testing all processes is not polluted; During the positive control sample test positive, show that RNA extraction, RNA reverse transcription, DNA cloning and electrophoresis evaluation work system are normal.Under the prerequisite that negative control sample and positive control sample detected result are all set up, could judge test sample.Therefore, test all should set up RNA extraction, RNA reverse transcription, DNA cloning and electrophoresis to identify contrast at every turn, only sets up the omnidistance contrast of test, could prove the test-results establishment.Therefore, the inverse transcription polymerase chain reaction test kit will be set up positive control, and positive control need satisfy following 3 points in the test kit: 1) good stability, 2) reliable effectively, 3) meet Biosafety.
Biosafety is meant that biological Vector of infection is through direct infection or destroy environment indirectly and cause the true or potential of human, animal or plant dangerous.Therefore, meeting Biosafety is meant and has avoided above-mentioned danger.Said danger for example is highly pathogenic pathogenic micro-organism; The first kind and second type of pathogenic micro-organism are referred to as highly pathogenic pathogenic micro-organism; First kind pathogenic micro-organism; Be meant can cause human or the unusual mikrobe of serious disease of animal, and China mikrobe of finding or announced to eliminate as yet.Second type of pathogenic micro-organism is meant can cause human or the animal serious disease, than being easier to direct or being connected on the mikrobe of propagating between person to person, animals and human beings, animal and animal.Positive control for the inverse transcription polymerase chain reaction test kit of some highly pathogenic pathogenic micro-organisms; At present most of this test kit ways be with the purpose fragment cloning to carrier; Its shortcoming be can not extract as RNA, RNA transcriptive process,reversed contrast, and pollute easily.
Therefore, need badly a kind of good stability, reliable effectively, meet the preparation method of the inverse transcription polymerase chain reaction test kit positive control of Biosafety.
Summary of the invention
Technical problem to be solved by this invention is the deficiency to prior art; A kind of positive compsn contrast of inverse transcription polymerase chain reaction test kit is provided; Positive control compsn and preparation method thereof particularly, the positive control good stability that this method makes, safe and reliable, can significantly reduce the test contamination of heavy.
Technical problem to be solved by this invention realizes through following technical scheme.The invention provides a kind of preparation method of positive control compsn of inverse transcription polymerase chain reaction test kit, it is characterized in that, choose a kind of non-target virus as the positive control template.
One aspect of the present invention; The non-target virus of choosing among the preparation method of the positive control compsn of inverse transcription polymerase chain reaction test kit is RNA viruses; It can be the RNA viruses that meets Biosafety arbitrarily; Include but not limited to newcastle disease virus and porcine reproductive and respiratory syndrome virus, and be selected from comparison safety, inheritance stability, easy virus strain of cultivating.
In embodiment of the present invention, selected non-target virus strain newcastle disease virus lasota strain and porcine reproductive and respiratory syndrome virus MLV strain.
Also aspect of the present invention also comprises the nutrient solution that uses the said non-target virus of deactivation agent treated among the preparation method of the positive control compsn of inverse transcription polymerase chain reaction test kit.Said deactivation reagent is the high density strong denaturant, and it destroys cellularstructure rapidly, and RNA is discharged from cell, also makes intracellular RNA enzyme deactivation, and the RNA that discharges is not degraded.Wherein, the high density strong denaturant includes but not limited to that guanidinium isothiocyanate, guanidine thiocyanate, Guanidinium hydrochloride etc. contain the salt of guanidine radicals.Deactivation reagent can be used for the deactivation of RT-PCR, real-time fluorescence RT-PCR positive control.That is to say, in method of the present invention, non-target virus-culturing fluid is diluted with denaturing agent that the multiple of dilution can easily be confirmed by those skilled in the art, generally should dilute and be not less than 7 times, preferably dilute 50-100 doubly, more preferably dilute 50 times.Extension rate is relevant with viral level, and wherein the requirement of positive control is that viral level can not be too high, can not be too low; Too high, the contamination reagent box is too low easily, makes the preservation period of test kit shorter.Dilute according to the requirement of test kit separately.
Also aspect of the present invention; Also comprise a pair of non-target virus primer of design among the preparation method of the positive control compsn of inverse transcription polymerase chain reaction test kit; The said non-target virus of choosing is increased, and the fragment length of amplification is identical with the length of inverse transcription polymerase chain reaction goal gene that test kit is directed against
The present invention also provides a kind of positive control compsn that is used for the inverse transcription polymerase chain reaction test kit, it is characterized in that comprising that a kind of non-target virus is as the positive control template.
In embodiment of the present invention; The non-target virus of choosing in the positive control compsn is RNA viruses; It can be the RNA viruses that meets Biosafety arbitrarily; Include but not limited to newcastle disease virus and porcine reproductive and respiratory syndrome virus, and be selected from safety, inheritance stability, easy virus strain of cultivating, preferred said virus strain comprises newcastle disease virus lasota strain and porcine reproductive and respiratory syndrome virus MLV strain.
In one aspect of the invention, the positive control compsn also comprises deactivation reagent.Deactivation reagent is the high density strong denaturant.Wherein, the high density strong denaturant includes but not limited to contain the salt of guanidine radicals, preferably guanidinium isothiocyanate, guanidine thiocyanate, Guanidinium hydrochloride.Deactivation reagent can be used for the deactivation of RT-PCR, real-time fluorescence RT-PCR positive control.The nutrient solution of said non-target virus is used according to the said deactivation reagent of viral level and is diluted, and generally should dilute to be not less than 7 times, preferably dilutes 50-100 doubly, more preferably dilutes 50 times.And extension rate can easily be confirmed according to the requirement of test kit separately by those skilled in the art.
In one aspect of the invention, the positive control compsn also comprises a pair of non-target virus primer, and so that said non-target virus is increased, the fragment length of amplification is identical with the length of the goal gene that the inverse transcription polymerase chain reaction test kit is directed against.
The present invention also provides the purposes of positive control compsn mentioned above, and it is as the part of inverse transcription polymerase chain reaction test kit.Positive control compsn of the present invention can place the inverse transcription polymerase chain reaction test kit.
In embodiment of the present invention; The inverse transcription polymerase chain reaction test kit that is suitable for includes but not limited to bird flu virus RT-PCR test kit and foot and mouth disease virus RT-PCR test kit; Wherein bird flu virus RT-PCR test kit can be used for bird, for example chicken, duck, goose, sparrow, pigeon or the like; Foot and mouth disease virus RT-PCR test kit can be used for detecting artiodactyl, for example ox.
Compared with prior art; The positive control safety of the inventive method preparation, the easy preservation; Can play the effect that RNA extracts, the RNA transcriptive process,reversed contrasts; The ThermoScript II isoreactivity that proof RNA extracts in reagent, the reaction system is normal, has avoided directly using in the prior art virus infection material to be prone to cause the shortcoming of virus diffusion.
Embodiment
To combine embodiment that embodiment of the present invention are described in detail below, but it will be understood to those of skill in the art that the following example only is used to explain the present invention, and should not be regarded as limiting scope of the present invention.
Utilizing and detecting target virus is a kind of ideal method as positive control.Because bird flu generation meeting causes serious economy loss to country, and bird flu belongs to zoonosis.Therefore, the avian influenza virus detection kit Biosafety is very important.Particularly bird flu virus may produce some unsafe influences to laboratory environment as positive control.
Newcastle disease virus lasota strain is a strain attenuated vaccine strain, and widespread use China newcastle disease immunization is being brought into play crucial effect aspect the prevention newcastle disease.This virus stain hemagglutinative titer in the chicken embryo culture is very high simultaneously, can reach 2
-13, be comparison safety, inheritance stability, easy virus strain of cultivating.Therefore newcastle disease virus lasota strain is a desirable virus of setting up the reverse transcription positive control.We adopt the reverse transcription positive control of newcastle disease virus lasota strain as RNA viruses, have played the effect that RNA extraction, RNA transcriptive process,reversed contrast, and have proved that the ThermoScript II isoreactivity is normal in RNA extraction reagent, the reaction system.Design a pair of primer to newcastle disease virus simultaneously, this primer requires the clip size of amplification consistent with the bird flu virus expanding fragment length that is detected just.Add the deactivation reagent that contains guanidinium isothiocyanate; With the newcastle disease virus deactivation; Positive control as bird flu virus inverse transcription polymerase chain reaction test kit; Owing to adopted newcastle disease virus lasota strain in the RNA leaching process as extracting contrast, significantly reduced the test contamination of heavy, relatively be fit to carry out for a long time laboratory or the large-scale bird flu monitoring that bird flu detects.
Embodiment
Unreceipted concrete technology or condition person among the embodiment; According to the described technology of the document in this area or condition (for example with reference to works such as J. Sa nurse Brookers; " the molecular cloning experiment guide " that Huang Peitang etc. translate, the third edition, Science Press) or carry out according to product description.The unreceipted person of production firm of agents useful for same or instrument, being can be through the conventional products of commercial acquisition.
The preparation of embodiment 1. bird flu virus RT-PCR test kit positive controls
1, the preparation of deactivation reagent: 4mol/L guanidinium isothiocyanate, 25m mol/L Sodium Citrate, usp, Dihydrate Powder, 0.5% (m/v) sodium lauryl sulphate, 0.1mol/L beta-mercaptoethanol.
2, non-target virus newcastle disease virus lasota strain (deriving from China Veterinery Drug Inspection Office, original seed code name AV100 (L)): the blood clotting valency is higher than 2048 newcastle disease virus lasota strain chick embryo allantoic liquid merges, mixing.
3, the preparation of positive control: with non-target virus newcastle disease virus lasota strain with 100 times of dilutions of sex change liquid after, can be used as the use of bird flu virus RT-PCR test kit positive control.
The application of embodiment 2. bird flu virus RT-PCR test kit positive controls
1, the composition of test kit
0.2mL thin-walled PCR pipe, RNA extract test kit (Rneasy Mini Kit, qiagen company), 50 times of TAE electrophoretic buffers, staining fluid (Goldview), sample-loading buffer (tetrabromophenol sulfonphthalein sucrose solution), negative control (deionized water of promptly sterilizing), positive control (being positive control prepared among the embodiment 1), RT-PCR reaction solution (primer, dNTPs, TaqDNA polysaccharase with reaction buffer, Mg
2+), enzyme mixed solution (AMV ThermoScript II, RNA suppressor factor, TaqDNA polysaccharase), X liquid (being described below).
2, specimen preparation
2.1 sample collecting: larynx tracheae and the lung tissue of the chicken that dies of illness.
2.2 sample preparation: every duplicate samples is handled respectively.
2.2.1 tissue sample is handled: organize respectively for every part and take by weighing the about 1g of sample from three different positions; Shred with operating scissors and to get 0.05g behind the mixing and in mill, grind; Adding 1.5mL saline water continues to grind; Treat to go in the 1.5mL sterilization centrifuge tube after the homogenate, the centrifugal 2min of 8000rpm gets supernatant 100 μ L in 1.5mL sterilization centrifuge tube.
2.2.2 positive control is handled: get positive control 100 μ L, put in the 1.5mL sterilization centrifuge tube.
2.2.3 negative control is handled: get negative control 100 μ L, put in the 1.5mL sterilization centrifuge tube.
3, the extraction of viral RNA (operating) according to the test kit specification sheets
4, RT-PCR schedule of operation
Every part of TV 20 μ L contain 16.8 μ L RT-PCR reaction solutions (using preceding mixing), 1.2 μ L enzyme mixed solutions, 2 μ L template ribonucleic acids.For example: n duplicate samples (n＜10); Preparation n+1 part, 16.8 * (n+1) RT-PCR reaction solutions add 1.2 * (n+1) enzyme mixed solution mixings again; Get 18 μ L and be distributed into n part; Add 2 μ L template ribonucleic acids (the positive control pipe adds 1 μ L positive control RNA and 1 μ L X liquid, and the negative control pipe adds 1 μ L negative control RNA and 1 μ LX liquid) respectively, perform mark.
5, X liquid: be the aqueous solution of the primer sequence in the non-target virus newcastle disease virus lasota pnca gene group, concentration 10pmoL/ μ L.
6, RT-PCR amplification program
On the pcr amplification appearance, carry out following program: 42 ℃ of 45min, 95 ℃ of 3min; Amplification condition is 95 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30s, 35 circulations; 72 ℃ are extended 10min.
7, positive control result
Pcr amplification product is carried out agarose gel electrophoresis, and the 330bp amplified band of expection, result such as Fig. 1 appear in positive control.The fragment length of positive control amplification is identical with the length of the goal gene that the inverse transcription polymerase chain reaction test kit is directed against.
The preparation and the application of embodiment 3. foot and mouth disease virus RT-PCR test kit positive controls
1, non-target virus porcine reproductive and respiratory syndrome virus MLV strain (be purchased and can get): every milliliter of viral level should be not less than 10
7.0TCID
50Virus-culturing fluid.
2, the preparation of positive control: with non-target virus porcine reproductive and respiratory syndrome virus MLV strain cell culture fluid with 50 times of dilutions of deactivation reagent after, can be used as foot and mouth disease virus RT-PCR test kit positive control usefulness.
3, non-target virus primer sequence: be mixed with X liquid with concentration 10pmoL/ μ L
4, specimen preparation
4.1 sample collecting: blister skin or the blister liquid of getting ox place 50% glycerine saline water.
4.2 sample preparation: every duplicate samples is handled respectively.
4.2.1 blister liquid is handled: get 100 μ L, put in the 1.5mL sterilization centrifuge tube.
4.2.2 positive control is handled: get positive control 100 μ L, put in the 1.5mL sterilization centrifuge tube.
4.2.3 negative control is handled: get negative control 100 μ L, put in the 1.5mL sterilization centrifuge tube.
The method of 5, the extraction of viral RNA, RT-PCR schedule of operation is with embodiment 2.
6, RT-PCR amplification program
On the pcr amplification appearance, carry out following program: 42 ℃ of 45min, 95 ℃ of 3min; Amplification condition is 95 ℃ of 15s, 60 ℃ of 30s, 35 circulations; 60 ℃ are extended 3min.
7, positive control result
Pcr amplification product is carried out agarose gel electrophoresis, and the 131bp amplified band of expection, result such as Fig. 2 appear in positive control.The fragment length of positive control amplification is identical with the length of the goal gene that the inverse transcription polymerase chain reaction test kit is directed against.
Description of drawings
Fig. 1. the amplification of positive control among the embodiment 2.Wherein each swimming lane is respectively 1.Marker 2. positive controls 3. samples 4. negative controls.
Fig. 2. the amplification of positive control among the embodiment 3.Wherein each swimming lane is respectively 1.Marker 2. positive controls 3. samples 4.Negative control.
Claims (15)
1. a preparation method who is used for the positive control compsn of inverse transcription polymerase chain reaction test kit is characterized in that, chooses a kind of non-target virus as the positive control template.
2. the preparation method of claim 1; It is characterized in that the non-target virus of choosing is RNA viruses; It can be the RNA viruses that meets Biosafety arbitrarily; Include but not limited to newcastle disease virus and porcine reproductive and respiratory syndrome virus, and be selected from safety, inheritance stability, easy virus strain of cultivating, preferred said virus strain comprises newcastle disease virus lasota strain and porcine reproductive and respiratory syndrome virus MLV strain.
3. claim 1 or 2 preparation method is characterized in that also comprising the nutrient solution that uses the said non-target virus of deactivation agent treated.
4. preparation method according to claim 3, wherein said deactivation reagent is the high density strong denaturant.
5. preparation method according to claim 4, wherein said high density strong denaturant includes but not limited to contain the salt of guanidine radicals, preferably guanidinium isothiocyanate, guanidine thiocyanate, Guanidinium hydrochloride.
6. each described preparation method among the claim 3-5, the nutrient solution of wherein said non-target virus dilutes with said deactivation reagent according to viral level, and dilution is not less than 7 times, preferably dilutes 50-100 times, more preferably dilutes 50 times.
7. each described preparation method among the claim 1-6; It is characterized in that; Design a pair of non-target virus primer, so that said non-target virus is increased, the fragment length of amplification is identical with the length of the goal gene that the inverse transcription polymerase chain reaction test kit is directed against.
8. a positive control compsn that is used for the inverse transcription polymerase chain reaction test kit is characterized in that comprising that a kind of non-target virus is as the positive control template.
9. the described positive control compsn of claim 8; It is characterized in that the non-target virus of choosing is RNA viruses; It can be the RNA viruses that meets Biosafety arbitrarily; Include but not limited to newcastle disease virus and porcine reproductive and respiratory syndrome virus, and be selected from safety, inheritance stability, easy virus strain of cultivating, preferred said virus strain comprises newcastle disease virus lasota strain and porcine reproductive and respiratory syndrome virus MLV strain.
10. claim 8 or 9 described positive control compsns is characterized in that also comprising deactivation reagent.
11. the described positive control compsn of claim 10, wherein deactivation reagent is the high density strong denaturant.
12. the described positive control compsn of claim 11, wherein, the high density strong denaturant includes but not limited to contain the salt of guanidine radicals, preferably guanidinium isothiocyanate, guanidine thiocyanate, Guanidinium hydrochloride.
13. the described positive control compsn of claim 10-12, the nutrient solution of wherein said non-target virus dilutes with said deactivation reagent according to viral level, and dilution is not less than 7 times, preferably dilutes 50-100 doubly, more preferably dilutes 50 times.
14. each described positive control compsn among the claim 8-13; It is characterized in that also comprising a pair of non-target virus primer; So that said non-target virus is increased, the fragment length of amplification is identical with the length of the goal gene that the inverse transcription polymerase chain reaction test kit is directed against.
15. the purposes of each described positive control compsn of claim 8-14, it is as the part of inverse transcription polymerase chain reaction test kit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110272456.XA CN102329789B (en) | 2011-09-15 | 2011-09-15 | Positive control composition for reverse transcription polymerase chain reaction kit and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110272456.XA CN102329789B (en) | 2011-09-15 | 2011-09-15 | Positive control composition for reverse transcription polymerase chain reaction kit and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102329789A true CN102329789A (en) | 2012-01-25 |
| CN102329789B CN102329789B (en) | 2014-02-26 |
Family
ID=45481751
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110272456.XA Active CN102329789B (en) | 2011-09-15 | 2011-09-15 | Positive control composition for reverse transcription polymerase chain reaction kit and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102329789B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030224017A1 (en) * | 2002-03-06 | 2003-12-04 | Samal Siba K. | Recombinant Newcastle disease viruses useful as vaccines or vaccine vectors |
| CN101798602A (en) * | 2010-03-18 | 2010-08-11 | 山东出入境检验检疫局检验检疫技术中心 | Composite kit for detecting avian influenzas and Newcastle disease viruses and detection method |
| CN102154511A (en) * | 2011-03-14 | 2011-08-17 | 秦智锋 | Preparation method and application of reagent for detecting novel type A H1N1 2009 influenza virus with one-step method |
-
2011
- 2011-09-15 CN CN201110272456.XA patent/CN102329789B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030224017A1 (en) * | 2002-03-06 | 2003-12-04 | Samal Siba K. | Recombinant Newcastle disease viruses useful as vaccines or vaccine vectors |
| CN101798602A (en) * | 2010-03-18 | 2010-08-11 | 山东出入境检验检疫局检验检疫技术中心 | Composite kit for detecting avian influenzas and Newcastle disease viruses and detection method |
| CN102154511A (en) * | 2011-03-14 | 2011-08-17 | 秦智锋 | Preparation method and application of reagent for detecting novel type A H1N1 2009 influenza virus with one-step method |
Non-Patent Citations (4)
| Title |
|---|
| 《Journal of Applied Microbiology》 20091231 K.E.Pierce et al. Design and optimization of a novel reverse transcription linear-after-the-exponential PCR for the detection of foot-and-mouth disease virus 1-10 1-15 , * |
| 《Poultry Science》 20091231 S.Rashid et al. Multiplex polymerase chain reaction for the detection and differentiation of avian influenza viruse and other poultry respiratory pathogens 2526-2531 1-15 第88卷, * |
| 《中国兽医学报》 20020131 赵虎等 新城疫单抗酶联免疫试剂盒的完善及其应用 25-28 1-15 第22卷, 第1期 * |
| 《中国预防兽医学报》 20070228 范忠军等 美洲型猪繁殖与呼吸综合征病毒荧光RT-PCR检测方法的建立 134-137 1-15 第29卷, 第2期 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102329789B (en) | 2014-02-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Costa et al. | The effect of age on avian influenza viral shedding in mallards (Anas platyrhynchos) | |
| Intisar et al. | Peste des petits ruminants infection in domestic ruminants in Sudan | |
| He et al. | Lethal infection by a novel reassortant H5N1 avian influenza A virus in a zoo-housed tiger | |
| Costa et al. | Effect of a prior exposure to a low pathogenic avian influenza virus in the outcome of a heterosubtypic low pathogenic avian influenza infection in mallards (Anas platyrhynchos) | |
| Sun et al. | Identification and characterization of avian-origin H3N2 canine influenza viruses in northern China during 2009–2010 | |
| CN108950068B (en) | Kit for identifying and detecting QX-type strains of chicken infectious bronchitis viruses | |
| Abolnik et al. | Susceptibility and status of avian influenza in ostriches | |
| Abolnik et al. | Molecular analysis of the 2011 HPAI H5N2 outbreak in ostriches, South Africa | |
| Yehia et al. | Selenium nanoparticles enhance the efficacy of homologous vaccine against the highly pathogenic avian influenza H5N1 virus in chickens | |
| Mishununa et al. | COMPUTED QUANTUM CHEMICAL MODELING OF THE EFFECT OF NANOSILVER ON CORONAVIRUS COVID-19. | |
| Martín-Valls et al. | Phylogeny of Spanish swine influenza viruses isolated from respiratory disease outbreaks and evolution of swine influenza virus within an endemically infected farm | |
| Hira Akhter et al. | Molecular and serological detection of avian influenza H9N2 virus in asymptomatic commercial layers in Faisalabad district, Punjab. | |
| EP3299459B1 (en) | Staphylococcus lyase and use thereof | |
| CN101260439A (en) | Amplification primer for detecting pigeon source component by PCR-mtDNA, detecting kit and using method thereof | |
| CN102311994A (en) | Fluorescence quantitative PCR (polymerase chain reaction) detection method of aspergillus fumigatus | |
| CN102329789B (en) | Positive control composition for reverse transcription polymerase chain reaction kit and preparation method thereof | |
| Chen et al. | Delayed protein shut down and cytopathic changes lead to high yields of infectious pancreatic necrosis virus cultured in Asian Grouper cells | |
| Prasad et al. | RT-PCR and its detection limit for cell culture grown bluetongue virus 1 using NS1 gene group specific primers | |
| CN106399591B (en) | I type duck hepatitis virus and duck plague virus one-step method PCR detection method | |
| CN108950071A (en) | The qPCR detection method of duck tembusu virus | |
| Marchenko et al. | Characterization of the H5N1 influenza virus isolated during an outbreak among wild birds in Russia (Tuva Republic) in 2010 | |
| CN100567506C (en) | A kind of H5 subtype avian influenza virus detection kit and application thereof | |
| CN105039331A (en) | Peronophythora litchi LAMP primer as well as rapid detection method and application thereof | |
| Anjorin et al. | Detection of Influenza A Virus in Pigs in Lagos, Nigeria | |
| Helal et al. | Avian influenza in live bird markets in the Suez canal region, Egypt |
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 |