CN104894245A - Detection primer, kit and method for identifying components of cashmere and components of sheep wool at the same time - Google Patents
Detection primer, kit and method for identifying components of cashmere and components of sheep wool at the same time Download PDFInfo
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- CN104894245A CN104894245A CN201510240326.6A CN201510240326A CN104894245A CN 104894245 A CN104894245 A CN 104894245A CN 201510240326 A CN201510240326 A CN 201510240326A CN 104894245 A CN104894245 A CN 104894245A
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Abstract
The invention discloses a detection primer, kit and method for identifying the components of cashmere and the components of sheep wool at the same time and particularly relates to a PCR-DHPLC detection primer, kit and method for identifying the components of cashmere and the components of sheep wool at the same time. The method comprises the steps that a detection primer pair SEQ ID NO.1-2 and a detection primer pair SEQ ID NO.3-4 are designed respectively for the mitochondrial DNA sequence of cashmere and the mitochondrial DNA sequence of sheep wool firstly, and the components of cashmere and the components of sheep wool are identified at the same time through the PCR-DHPLC method. The kit contains 2.5 mM of PCR reaction liquid, 2.5 mM of dNTP, 5 U/[mu]l of Taq DNA polymerase, 10 [mu] M of the detection primer pair SEQ ID NO.1-2 and 10 [mu] M of the detection primer pair SEQ ID NO.3-4. The primer is high in specificity, and the detection kit and the method are simple and easy to use; whether a textile contains cashmere and sheep wool can be identified rapidly, and the result is accurate; the primer has high specificity and high sensitivity.
Description
Technical field
The invention belongs to biology field, relate to the detection kit and detection method thereof that detect cashmere and sheep's wool composition in textiles simultaneously, particularly relate to and utilize the detection method that PCR-DHPLC technology carries out cashmere and sheep's wool detection and the reagent used.
Background technology
Existing examination of fibers method accurately can be examined and determine by instrument unlike other elements of inspection, even if in the world, be also adopt traditional organoleptic test method, tester identifies its attribute by vision, sense of touch.The maximum test item of generally acknowledged technical difficulty to the discriminating of wool and cashmere fiber.A large amount of fiber check and measure workers is devoted to the research of the authentication technique of wool and cashmere fiber for a long time always.At present, differentiating cashmere, the universal method of wool fiber is the physical methods such as optical projection microscopy, scanning electron microscope method and solution method, is all distinguish from the mode of appearance of fiber and microtexture.But along with the application of modern technique such as wool stripping squama and stretching etc., even if veteran senior reviewer is also difficult to accurate discrimination cashmere and wool sometimes.Employing chemical process differentiates cashmere by the analytical results difference of amino acid, polypeptide and fat in animal fibre, the research of wool fiber also has more bibliographical information, but these methods also respectively have feature, can not entirely accurate identify, because the content of amino acid, polypeptide and fat is different with the change of processing treatment process, growth of animal environment and feed for nursing in cashmere, wool fiber.。The shortcoming of microscopy is that assay affects larger by reviewer's specialist, traditional discrimination method based on experience can not meet the needs of Scientific control, for feeler mechanism, set up science, accurately textiles and other light industrial goods method for quick identification extremely urgent.
Summary of the invention
In order to solve above-mentioned practical problems, namely the present invention is intended to the characteristic utilizing the hereditary material DNA of different animals fiber there are differences, adopt polymerase chain reaction PCR and denaturing high-performance chromatography DHPLC technology, Auele Specific Primer pair is set up for cashmere in textiles and sheep's wool, and sensitive detection method easily, and set up the quick detection kit being applied to the method.Relate to and detect cashmere and sheep's wool two kinds of fibers simultaneously, apply test kit of the present invention, two kinds of fibre compositions just can detect through PCR reaction, a PCR system.In single job process, detect wool and cashmere two kinds of fibers simultaneously, make detection time, amount detection and detection level all there occurs very large leap.The present invention uses denaturing high-performance chromatography (DHPLC) to adopt the principle of Ion-pairing RP-HPLC, by the mode using special high temperature resistant liquid chromatography separation column to adopt temperature adjusting simultaneously, analytical separation is carried out to nucleotide fragments molecule.Its fast high-flux, full-automatic operation, the advantage that accuracy is high, reproducible and susceptibility is high make it in foranalysis of nucleic acids, have the advantage that can not be substituted.Compared with traditional microscopy, the method that the present invention sets up and test kit, greatly shorten the time of operation, and greatly reduce the impact of artificial sense datum, makes result more accurately, reliably.
The technical solution used in the present invention is: extract sample DNA, take DNA as template, adopt the specific detection primer of cashmere and sheep's wool to carry out pcr amplification simultaneously, the profile information of goal gene is gathered again by DHPLC technology, directly read detected result from profile information, on this basis, the present invention has also carried out specificity and sensitivity technique to this method; Its concrete operation step is as follows:
An aspect of of the present present invention is: provide a kind of detection primer differentiating cashmere, and a kind of detection primer differentiating sheep's wool, is specially following base sequence:
An aspect of of the present present invention is: provide a kind of detection kit simultaneously differentiating cashmere and sheep's wool composition, it comprises right primer SEQ ID NO.1 ~ 4 mentioned above.
For the detection kit differentiating cashmere and sheep's wool composition while described in technique scheme, comprise Taq archaeal dna polymerase and the PCR reaction solution of 5U/ μ l; Independently pack; Wherein, described PCR reaction solution is mixed solution, comprises outside each 10 μMs of SEQ ID NO.1 ~ 4 mentioned above, also and comprises 10mM TrisCl, 50mM KCl, each 2.5mM of 25mM MgCl2, dNTP.
Another aspect of the present invention is: provide the detection method simultaneously differentiating cashmere and sheep's wool composition in a kind of textiles, and it utilizes the detection kit described in technique scheme, carries out PCR method detection to textiles DNA.
For the detection method differentiating cashmere and sheep's wool composition in the textiles described in technique scheme simultaneously, it comprises following operation steps: carry out PCR detection as follows to sample respectively:
1. PCR reaction system is 50 μ l, wherein: Taq archaeal dna polymerase 0.4 μ l, PCR reaction solution 24 μ l of sample DNA 4 μ l, 5U/ μ l, sterilizing ultrapure water 21.6 μ l.
2. PCR reaction conditions: denaturation: 94 DEG C, 3min; Enter circulation: 94 DEG C of sex change 30s, 56 DEG C of annealing 30s, 72 DEG C extend 30s, 35 circulations; Stop extending: 72 DEG C, 7min.
For the detection method differentiating cashmere and sheep's wool composition in the textiles described in technique scheme simultaneously, in its operation steps, can also comprise the steps:
DHPLC analysis is carried out to the pcr amplification product that cashmere mentioned above and sheep's wool composition PCR detection method obtain.
DHPLC principle of work: DHPLC adopts high-pressure closing liquid-phase flow path, DNA sample is automatically injected and under damping fluid carries, flows through DNA sample separator column to be measured, by the different graded of damping fluid, under different separator column temperature condition, realize the analysis different to DNA sample to be measured; By ultraviolet detection or the separated DNA sample of fluoroscopic examination.
DHPLC analytical procedure:
Chromatographic column: PS-DVB & C18DNASep chromatographic column (4.6mm × 50mm, granularity 3 μm);
Column temperature: 50 DEG C;
Moving phase (volume ratio): 0.0min, 48.0% buffered soln A, 52.0% buffered soln B;
0.5min, 42.9% buffered soln A, 57.1% buffered soln B;
3.6min, 38.8% buffered soln A, 61.2% buffered soln B;
6.8min, 36.6% buffered soln A, 63.4% buffered soln B
9.9min, 35.2% buffered soln A, 64.8% buffered soln B;
13min, 34.3% buffered soln A, 65.7% buffered soln B;
Wherein, buffered soln A is 50ml TEAA and the mixing of 250 μ l acetonitriles, adds sterilizing ultrapure water and is settled to 1000ml gained solution; Buffered soln B is 50ml TEAA and the mixing of 250ml acetonitrile, adds sterilizing ultrapure water and is settled to 1000ml gained solution;
Flow velocity: 0.9mL/min;
Detector: fluorimetric detector (light source: 150W Xenon lamp; Excitation spectrum bandwidth: 15nm; Emission spectrum bandwidth: 15.3nm; Detection sensitivity: at wavelength 350nm integration 2s;
Applied sample amount: PCR primer 5 μ L.
After detection terminates, detect the peak shape of Characteristic chromatographic peak in collection of illustrative plates according to DHPLC, retention time and with the contrasting of positive control collection of illustrative plates, whether determine in sample containing cashmere and sheep's wool.
Positive control and negative control can be established in testing process.Positive control is cashmere DNA and sheep's wool DNA, and negative control is the DNA of non-targeted fiber.
Negative control: under above-mentioned DHPLC condition, occur without absorption peak;
Positive control: under above-mentioned DHPLC analysis condition, sheep's wool occurs PCR primer absorption peak at about 4.0min; PCR absorption peak is there is in cashmere at about 6.7min.And above typical PCR primer absorption peak is greater than 2mV;
Under above-mentioned DHPLC analysis condition, there is PCR primer absorption peak in about 4.0min, for containing sheep's wool composition in sample; There is PCR absorption peak in about 6.7min, for containing cashmere composition in sample.
Beneficial effect: use detection kit of the present invention and detection method, can differentiate the cashmere in textiles and sheep's wool composition in high sensitivity, detects consuming time short, simple to operation, can save a large amount of labour's material resources, is applicable to the requirement of rapid detection.Identification efficiency and the sensitivity succeeded in developing and apply improving animal fibre of the method, improve inspection technology and be with a wide range of applications.
Accompanying drawing explanation
Fig. 1 is the specific test result figure of cashmere.
Fig. 2 is the specific test result figure of sheep's wool;
Fig. 3 is the specific test result figure simultaneously containing cashmere and sheep's wool in sample.
Embodiment
Following non-limiting example, it is further described the foundation of present method and application thereof, can make the present invention of those of ordinary skill in the art's comprehend, but not limit the present invention in any way.
If without specified otherwise, the main agents that this part uses, instrument obtain by commercial sources, and part merchandise resources is: DNA extraction kit (tissue and hair extract test kit, Promega company); The reagent such as Taq enzyme and PCR reaction solution are all purchased from precious biotechnology (Dalian) company limited; Triethylamine acetyl salt (TEAA, chromatographically pure) is purchased from Transgenomic company; Acetonitrile (chromatographically pure) is purchased from Fisher company; Regular-PCR instrument PE24000 (PerkinElmer company, the U.S.); Denaturing high-performance chromatography instrument NAV-99-4500 (Transgenomic company, the U.S.).
Embodiment 1
1. the preparation of template
Following template DNA extracting method, is the routine operation of those skilled in the art, and all reagent and solution are conventional route preparation or are obtained by commercial sources.
1. in the 1.5mL centrifuge tube that sample is housed, add 400 μ L DNA extraction buffers and 75 μ L Proteinase Ks, after mixing, at 56 DEG C, temperature bath is spent the night;
2. add 350 μ L lysates, centrifugal 2min, supernatant liquor moves in new 1.5mL centrifuge tube;
3. the DNA IQ that 7 μ L suspend completely is added
tMresin, vibration, room temperature places 10min;
4. by centrifuge tube high speed vortex oscillation 2s, be placed on Magneto separate frame, suck solution, add 100 μ L lysates, again vibrate, Magneto separate; Suck lysate, washings washs three times, air-dry;
5. add 70 μ L elutriants, at a high speed vibration, 65 DEG C of temperature bath 5min, be placed on Magneto separate frame, careful draw solution, be the template solution containing DNA ,-20 DEG C save backup.
2.PCR-DHPLC detects
1. cashmere and sheep's wool composition amplification primers and PCR detection method:
Cashmere and sheep's wool Auele Specific Primer SEQ ID NO.1 ~ 4, synthesize by precious biotechnology (Dalian) company limited; Amplified fragments is respectively 293bp and 174bp.
2. the test kit that PCR-DHPLC detects is designed on this basis.This test kit comprises: concentration is Taq archaeal dna polymerase and the PCR reaction solution of 5U/ μ L; Primer pair (SEQ ID NO.1 ~ 4) each 10 μMs are detected containing 10mM TrisHCl, 50mM KCl, 25mM MgCl2, dNTP (each 2.5mM of dATP, dGTP, dCTP and dTTP) and two kinds of fiber-specifics in described PCR reaction solution.During use, extract testing sample components D NA respectively, add reactant in following ratio and to go forward side by side performing PCR reaction:
Contain in PCR reaction system: 4 μ l testing sample DNA solutions, add 24 μ l PCR reaction solutions, the Taq archaeal dna polymerase of 0.4 μ l and sterilizing ultrapure water 21.6 μ l, cumulative volume 50 μ l; The centrifugal 10s of 5000r/min, then carries out pcr amplification by following parameters:
3. PCR reaction parameter: denaturation: 94 DEG C, 3min; Enter circulation: 94 DEG C of sex change 30s, 56 DEG C of annealing 30s, 72 DEG C extend 30s, 35 circulations; Stop extending: 72 DEG C, 7min;
PCR primer 4 DEG C preservation;
4. DHPLC analyzes.
PCR primer is carried out DHPLC analysis, and DHPLC analysis condition is as follows:
Chromatographic column: PS-DVB & C18DNASep chromatographic column (4.6mm × 50mm, granularity 3 μm);
Column temperature: 50 DEG C;
Moving phase (volume ratio): 0.0min, 48.0% buffered soln A, 52.0% buffered soln B;
0.5min, 42.9% buffered soln A, 57.1% buffered soln B;
3.6min, 38.8% buffered soln A, 61.2% buffered soln B;
6.8min, 36.6% buffered soln A, 63.4% buffered soln B
9.9min, 35.2% buffered soln A, 64.8% buffered soln B;
13min, 34.3% buffered soln A, 65.7% buffered soln B;
Wherein, buffered soln A is 50ml TEAA and the mixing of 250 μ l acetonitriles, adds sterilizing ultrapure water and is settled to 1000ml gained solution; Buffered soln B is 50ml TEAA and the mixing of 250ml acetonitrile, adds sterilizing ultrapure water and is settled to 1000ml gained solution;
Flow velocity: 0.9mL/min;
Detector: fluorimetric detector (light source: 150W Xenon lamp; Excitation spectrum bandwidth: 15nm; Emission spectrum bandwidth: 15.3nm; Detection sensitivity: at wavelength 350nm integration 2s;
Applied sample amount: PCR primer 5 μ L.
5. with accompanying drawing, experimental result illustrates that middle collection of illustrative plates form exports, and the criterion of PCR-DHPLC result is:
After detection terminates, detect sample and occur without amplification absorption peak, can judge in sample not containing cashmere and sheep's wool composition; Detection sample is about 4.0min and occurs PCR primer absorption peak, and when absorption peak is greater than 2mV, can judge in sample containing sheep's wool composition; Detection sample is about 6.7min and occurs PCR primer absorption peak, and when absorption peak is greater than 2mV, can be judged to be in sample containing cashmere composition; Not only detect sample and all occur PCR primer absorption peak at about 4.0min and 6.7min place, and when absorption peak is greater than 2mV, can judge in sample containing cashmere composition but also containing sheep's wool composition.
6. the PCR-DHPLC detected result of the present embodiment as shown in Figure 1, can be judged to be in sample containing cashmere composition; As shown in Figure 2, can judge in sample containing sheep's wool composition; Not only as shown in Figure 3, can judge in sample containing cashmere composition but also containing sheep's wool composition.
The above; be only the present invention's preferably embodiment; but protection scope of the present invention is not limited thereto; anyly be familiar with those skilled in the art in the technical scope that the present invention discloses; be equal to according to technical scheme of the present invention and inventive concept thereof and replace or change, all should be encompassed within protection scope of the present invention.
Claims (7)
1. differentiate that cashmere PCR-DHPLC detects a primer, is characterized in that, comprises base sequence: SEQ ID NO.1 ~ 2.
2. differentiate that sheep's wool PCR-DHPLC detects a primer, is characterized in that, comprises base sequence: SEQ ID NO.3 ~ 4.
3. differentiate a PCR-DHPLC detection kit for cashmere and sheep's wool composition simultaneously, it is characterized in that: comprise primer SEQ ID NO.1 ~ 4 described in claim 1 and 2.
4. the PCR-DHPLC detection kit simultaneously differentiating cashmere and sheep's wool composition according to claim 3, is characterized in that: the Taq archaeal dna polymerase and the PCR reaction solution that comprise 5U/ μ l;
Wherein, described PCR reaction solution is by each 10 μMs of primer SEQ ID NO.1 ~ 4 described in claim 1 and 2,10mM TrisCl, 50mM KCl, 25mM MgCl
2, dNTP each 2.5mM composition.
5. differentiate a PCR-DHPLC detection method for cashmere and sheep's wool composition in textiles simultaneously, it is characterized in that: comprise the detection kit utilized described in claim 3, PCR method detection is carried out to textiles DNA.
6. differentiate the PCR-DHPLC detection method of cashmere and sheep's wool composition in textiles according to claim 5 simultaneously, it is characterized in that: described the step that sample DNA carries out PCR method detection to be comprised:
1. PCR reaction system is 50 μ l, wherein: Taq archaeal dna polymerase 0.4 μ l, PCR reaction solution 24 μ l of sample DNA 4 μ l, 5U/ μ l, sterilizing ultrapure water 21.6 μ l;
2. PCR reaction conditions: denaturation: 94 DEG C, 3min; Enter circulation: 94 DEG C of sex change 30s, 56 DEG C of annealing 30s, 72 DEG C extend 30s, 35 circulations; Stop extending: 72 DEG C, 7min.
7. differentiate the PCR-DHPLC detection method of cashmere and sheep's wool composition in textiles according to claim 6 simultaneously, it is characterized in that: also comprise following operation steps:
Carry out DHPLC analysis to the pcr amplification product that method described in claim 6 obtains, condition is as follows:
Chromatographic column: PS-DVB & C18DNASep chromatographic column (4.6mm × 50mm, granularity 3 μm);
Column temperature: 50 DEG C;
Moving phase (volume ratio): 0.0min, 48.0% buffered soln A, 52.0% buffered soln B;
0.5min, 42.9% buffered soln A, 57.1% buffered soln B;
3.6min, 38.8% buffered soln A, 61.2% buffered soln B;
6.8min, 36.6% buffered soln A, 63.4% buffered soln B
9.9min, 35.2% buffered soln A, 64.8% buffered soln B;
13min, 34.3% buffered soln A, 65.7% buffered soln B;
Wherein, buffered soln A is 50ml TEAA and the mixing of 250 μ l acetonitriles, adds sterilizing ultrapure water and is settled to 1000ml; Buffered soln B is 50ml TEAA and the mixing of 250ml acetonitrile, adds sterilizing ultrapure water and is settled to 1000ml;
Flow velocity: 0.9mL/min;
Detector: fluorimetric detector, light source: 150W Xenon lamp; Excitation spectrum bandwidth: 15nm; Emission spectrum bandwidth: 15.3nm; Detection sensitivity: at wavelength 350nm integration 2s;
Applied sample amount: PCR primer 5 μ L.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106967817A (en) * | 2017-05-02 | 2017-07-21 | 嘉兴学院 | A kind of rapid molecular authentication method of wool product |
| CN113430276A (en) * | 2021-07-19 | 2021-09-24 | 浙江大学 | Method for identifying sheep wool and goat wool based on CO I gene |
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| CN101624624A (en) * | 2009-03-20 | 2010-01-13 | 曹际娟 | Detection kit for salmonella and shigella in feeds and detection method thereof |
| CN102304579A (en) * | 2011-08-31 | 2012-01-04 | 福建出入境检验检疫局检验检疫技术中心 | Real-time fluorescent polymerase chain reaction (PCR) identification method for cashmere and sheep wool |
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2015
- 2015-05-12 CN CN201510240326.6A patent/CN104894245A/en active Pending
Patent Citations (2)
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| CN101624624A (en) * | 2009-03-20 | 2010-01-13 | 曹际娟 | Detection kit for salmonella and shigella in feeds and detection method thereof |
| CN102304579A (en) * | 2011-08-31 | 2012-01-04 | 福建出入境检验检疫局检验检疫技术中心 | Real-time fluorescent polymerase chain reaction (PCR) identification method for cashmere and sheep wool |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106967817A (en) * | 2017-05-02 | 2017-07-21 | 嘉兴学院 | A kind of rapid molecular authentication method of wool product |
| CN106967817B (en) * | 2017-05-02 | 2020-05-15 | 嘉兴学院 | Rapid molecular identification method of plush product |
| CN113430276A (en) * | 2021-07-19 | 2021-09-24 | 浙江大学 | Method for identifying sheep wool and goat wool based on CO I gene |
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Application publication date: 20150909 |