CN103882009B - The little extracting method passing through smaller green leaf hopper adult total serum IgE - Google Patents
The little extracting method passing through smaller green leaf hopper adult total serum IgE Download PDFInfo
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- CN103882009B CN103882009B CN201410102150.3A CN201410102150A CN103882009B CN 103882009 B CN103882009 B CN 103882009B CN 201410102150 A CN201410102150 A CN 201410102150A CN 103882009 B CN103882009 B CN 103882009B
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Abstract
The present invention provides a kind of little extracting method passing through smaller green leaf hopper adult total serum IgE, comprises the following steps: step 1, be placed in liquid nitrogen by the little smaller green leaf hopper adult Heat thermostability that passes through; Do step 2, taking-up centrifuge tube add Total RNAs extraction liquid RNAiso? Plus, homogenate; Step 3, homogenised sample room temperature is placed, centrifugal; Step 4, Aspirate supernatant, move in another centrifuge tube; Step 5, adding chloroform in supernatant, room temperature stands, centrifugal; Step 6, taking-up centrifuge tube, Aspirate supernatant proceeds in the 3rd centrifuge tube, the isopropanol of addition, stands, is centrifuged; Step 7, carefully abandon supernatant, add ethanol, centrifugal after washing, discard ethanol, dry, add appropriate RNase-free water and obtain highly purified total serum IgE. The extracting method of the present invention is simply effective, solves total serum IgE and easily degrades in extraction process and be not easy the problem that high-purity extracts total serum IgE.
Description
Technical field
The present invention relates to the extracting method of a kind of small-sized leafhopper class insecticide total serum IgE, the specifically little extracting method passing through smaller green leaf hopper adult total serum IgE.
Background technology
The little smaller green leaf hopper EmpoascaonukiiMatsuda that passes through is a kind of global Camellia sinensis primary pest, is also that the distribution of current China is the widest, the heaviest Pests of Tea-Plants of causing harm. Recently as being continuously increased of tea tree planting area, this worm constantly spreads, and has had a strong impact on the yield and quality of Folium Camelliae sinensis. Bud-leaf after being caused harm by this worm is frangible in the course of processing, produces the cigarette smell of burning, becomes bar rope of sampling tea loose, unqualified increases, and brews rear soup color contamination turbid, and flavour is pained, and fragrance is not normal. The general time Summer-autumn tea underproduction 10%-15% that is injured, Serious year part underproduction reaches more than 50%. Not only cause huge economic loss to tea grower, and seriously dampened the enthusiasm for production of numerous tea growers, it has also become restrict one of key factor of Folium Camelliae sinensis high-quality, high yield.
Domestic and the world other mainly produce tea country, India, Sri Lanka, Vietnam, the state scholar such as Japanese and African to little pass through the artificial diet technique of smaller green leaf hopper, bioecology, integrated control technique and resistance etc. in compare comprehensive and systematic research. But at high temperature to it, a situation arises has not yet to see someone with the research of the aspect such as ecological habit and relate to.
Little passing through smaller green leaf hopper on the Anhui of China, Jiangsu, Fujian, Guangxi, Guizhou and other places, often have twice peak of occurrence every year, the factor impact such as peak of occurrence climate is substantially. By 2010-2012, in tea place, Xinyang, to smaller green leaf hopper, a situation arises is monitored in this project team, it has been found that this area is often only and occurs peak of occurrence 10-11 month, and these are different from other regional little pests occurrence rules passing through smaller green leaf hopper. Research finds, temperature is to affect the key factor of its generation, and maximum temperature natural law more than 35 DEG C is more many, and little smaller green leaf hopper generation quantity of passing through is more few, and different year, along with the postponement of high temperature time of occurrence, occurs low peak period also with postponement. And temperature is a significant variable in insect growth process, the growth of insecticide, breeding and survival can be produced strong impact by variations in temperature.
Therefore, researching high-temperature coerces growth promoter and the molecular adaptation mechanism of lower false eye leafhopper, it is possible to for accurate prediction, it occurs to provide more foundations.Research shows, heatshock protein (Heatshockprotein, Hsp) is animal under stimulating higher than normal growth temperature, the new albumen of induction synthesis. It is prevented from protein denaturation so that it is recover original space conformation and biological activity. Academic circles at present trends towards heatshock protein divides Hsp90,70,60,40 and little Hsps(sHsps) family. Research finds, heatshock protein not only plays an important role on environmental suitability at organism, it is likely to also play an important role in insecticide normal growth is grown, this result of study further increases people's understanding to heat shock protein gene function, and the growth promoter for research insecticide provides new thinking. At present, the little research passing through smaller green leaf hopper growth promoter and heatshock protein expression is related to by high temperature stress there are no people, it is therefore desirable to the expression of the little impact passing through smaller green leaf hopper resistance material and heatshock protein thereof is changed by researching high-temperature, thus pass through smaller green leaf hopper provide fundamental basis for scientific forecasting forecast is little.
The classical way obtaining insect protein is directly to extract crude protein from insecticide, the method such as polyacrylamide gel electrophoresis, gel chromatography ion exchange column that then passes through is easily separated purification and obtains insect protein, but major part insecticide is only small, separate purification relatively difficult, and this process is comparatively laborious. In recent years, scientists utilizes the Protocols in Molecular Biology grown up clone to obtain the gene of albumen, the method of recycling vivoexpression obtains enough destination proteins, and this alleviates the difficulty of research purpose albumen to a certain extent, but the method also first must extract total serum IgE from insecticide. Bigger insecticide is not difficult to accomplish, pass through that smaller green leaf hopper is this kind of small-sized and to be good at the leafhopper class insecticide of jump be not but easy thing as little, it is easy to make RNA degraded also be not readily available satisfactory high-purity total serum IgE without the effective extracting method of complete set, makes subsequent experimental cannot be carried out implementing.
In order to solve above Problems existing, people are seeking a kind of desirably technical solution always.
Summary of the invention
It is an object of the invention to for the deficiencies in the prior art, thus providing the little extracting method passing through smaller green leaf hopper adult total serum IgE, with solve prior art exists as the little Total RNAs extraction passing through this kind of small insect of smaller green leaf hopper adult not easily and the purity problem that do not reach requirement.
To achieve these goals, the technical solution adopted in the present invention is: a kind of little extracting method passing through smaller green leaf hopper adult total serum IgE, comprises the following steps:
Step 1, collect and little pass through smaller green leaf hopper adult in the centrifuge tube being placed with fresh tea leaf bud head, smaller green leaf hopper adult Heat thermostability is passed through by little, make little to pass through smaller green leaf hopper adult heat shock stupor, from described centrifuge tube, then take out fresh tea leaf bud head, and described centrifuge tube is immediately placed in liquid nitrogen;
Step 2, from liquid nitrogen, take out centrifuge tube and add Total RNAs extraction liquid RNAisoPlus at once, with adding Total RNAs extraction liquid RNAisoPlus after the abundant homogenate of plastics grinding rod to enough, then with pipettor, serosity being blown and beaten for several times;
Step 3, will fill enough Total RNAs extraction liquid RNAisoPlus homogenised sample room temperature place 5min, 12000g, 4 DEG C of centrifugal 5min;
Step 4, careful Aspirate supernatant, move in another centrifuge tube by supernatant;
Step 5, adding the chloroform of 1/5 volume of Total RNAs extraction liquid in supernatant, cover tightly centrifuge tube, acutely shake, after solution is fully emulsified, room temperature stands 5min, 12000g, 4 DEG C of centrifugal 15min;
Step 6, from centrifuge take out centrifuge tube, Aspirate supernatant proceeds in the 3rd centrifuge tube, add and the isopyknic isopropanol of supernatant in the 3rd centrifuge tube, turn upside down test tube, fully mixing, stands 10min, 12000g in the environment of 15-30 DEG C,, there is precipitation in 4 DEG C of centrifugal 10min bottom test tube after centrifugal;
Step 7, carefully abandon supernatant, slowly adding concentration along centrifugal tube wall is the ethanol of 75%, turn upside down centrifuge tube gently, washing centrifuge tube wall, 12000g, discards ethanol, drying at room temperature 2-5min after 4 DEG C of centrifugal 5min, it is subsequently adding appropriate RNase-free water and carrys out dissolution precipitation, obtain highly purified total serum IgE.
Based on above-mentioned, the medium and small temperature conditions passing through smaller green leaf hopper adult Heat thermostability of step 1 is 34-36 DEG C, and the Heat thermostability time is 50-70min.
Based on above-mentioned, after adding the chloroform of 1/5 volume of Total RNAs extraction liquid in step 5 in supernatant and covering tightly centrifuge tube, acutely the time of concussion is 15s.
Based on above-mentioned, each centrifuge tube is the centrifuge tube of 1.5ml.
Based on above-mentioned, the medium and small quantity passing through smaller green leaf hopper adult of step 1 is 5; The total amount adding described Total RNAs extraction liquid RNAisoPlus in step 2 is 1000ul, and is initially charged after the abundant homogenate of 200ul and adds 800ul; Step 5 adds the chloroform of 200ul; Step 6 adds the isopropanol of 700ul; Step 7 adds the ethanol 1ml that concentration is 75%, and the amount of the RNASE-FREE water of addition is 15ul.
Based on above-mentioned, Heat thermostability in step 1 is in growth cabinet and at light intensity 4000lx, carry out under the illumination condition of RH60% ± 5%, little pass through smaller green leaf hopper adult collecting and enter in centrifuge tube and shift in the process in described growth cabinet, by centrifuge tube oral area down, utilize little when passing through smaller green leaf hopper activity habit upwards be collected.
Hinge structure of the present invention has substantive distinguishing features and progress, specifically, smaller green leaf hopper adult heat shock stupor is passed through by little, and it is immediately placed in liquid nitrogen, then from liquid nitrogen, take out centrifuge tube and add a small amount of Total RNAs extraction liquid RNAisoPlus, Total RNAs extraction liquid RNAisoPlus will be added after its abundant homogenate to enough with plastics grinding rod, then according to method for extracting total RNA extracts total serum IgE after pipettor piping and druming for several times, through chloroform, isopropanol and ethanol precipitate and separate obtain highly purified total serum IgE, extracting method is simply effective, solve total serum IgE in extraction process, hold problem of easy degradation, particularly before adding chloroform, first pass through the centrifugal of step 3 and step 4 extracts supernatant, the precipitation of bottom is cast out, containing substantial amounts of fat and macromole impurity in these precipitations, some of them cannot remove in subsequent step again, and these impurity can be removed in advance by step 3 and step 4, reduce contaminating impurity as far as possible, for follow-up further purification, lay the foundation, solve the problem that high-purity extracts total serum IgE.
Further, utilize little when passing through smaller green leaf hopper adult activity habit upwards pass through smaller green leaf hopper collect in centrifuge tube by little, convenient easily operation.
Further, fresh tea leaf bud head is placed in centrifuge tube before Heat thermostability, and after Heat thermostability and described centrifuge tube be placed in liquid nitrogen before by fresh tea leaf bud head take out, purpose is the little land for growing field crops ecological environment passing through smaller green leaf hopper adult of true simulation, make to test the data obtained and can more effectively support in the little study on prevention passing through smaller green leaf hopper adult, on the other hand, owing to the time of Heat thermostability is long, smaller green leaf hopper adult offer food is passed through for little, prevent from little passing through smaller green leaf hopper adult and affecting the accuracy of result of the test because the situations such as Heat thermostability early stage is dead occur.
Accompanying drawing explanation
Fig. 1 little passes through smaller green leaf hopper adult total serum IgE integrity detection electrophoretogram.
Fig. 2 is the little cDNA template the passing through smaller green leaf hopper adult total serum IgE reverse transcription detection electrophoretogram to heatshock protein pane gene pcr amplification result.
Fig. 3 is the little cDNA template the passing through smaller green leaf hopper adult total serum IgE reverse transcription detection electrophoretogram to Heat shock protein 70 gene pcr amplification result.
Fig. 4 is the little cDNA template the passing through smaller green leaf hopper adult total serum IgE reverse transcription detection electrophoretogram to internal reference Gene A ctinPCR amplification.
Detailed description of the invention
Below by detailed description of the invention, technical scheme is described in further detail.
The little extracting method passing through smaller green leaf hopper adult total serum IgE, comprises the following steps:
Step 1, collect and little pass through smaller green leaf hopper adult in 1.5ml centrifuge tube, smaller green leaf hopper adult Heat thermostability 50-70min under the temperature conditions of 34-36 DEG C is passed through by little, make little pass through smaller green leaf hopper adult heat shock stupor, then described centrifuge tube is immediately placed in liquid nitrogen;
Step 2, from liquid nitrogen, take out centrifuge tube and add Total RNAs extraction liquid RNAisoPlus at once, with the abundant homogenate of plastics grinding rod, in without granule transparence, being subsequently adding Total RNAs extraction liquid RNAisoPlus to enough, then with 1ml pipettor, serosity is blown and beaten for several times;
Step 3, will fill enough Total RNAs extraction liquid RNAisoPlus homogenised sample room temperature place 5min, 12000g, 4 DEG C of centrifugal 5min;
Step 4, careful Aspirate supernatant, move into supernatant in another 1.5ml centrifuge tube, is sure not to draw precipitation;
Step 5, adding the chloroform of 1/5 volume of Total RNAs extraction liquid in supernatant, cover tightly centrifuge tube, acutely shake 15s, after solution is fully emulsified, room temperature stands 5min, 12000g, 4 DEG C of centrifugal 15min;
Step 6, from centrifuge take out centrifuge tube, Aspirate supernatant proceeds in the 3rd centrifuge tube, add and the isopyknic isopropanol of supernatant in the 3rd centrifuge tube, turn upside down test tube, fully mixing, stands 10min, 12000g in the environment of 15-30 DEG C,, there is precipitation in 4 DEG C of centrifugal 10min bottom test tube after centrifugal;
Step 7, carefully abandon supernatant, slowly adding concentration along centrifugal tube wall is the ethanol of 75%, turn upside down centrifuge tube gently, washing centrifuge tube wall, 12000g, discards ethanol, drying at room temperature 2-5min after 4 DEG C of centrifugal 5min, it is subsequently adding appropriate RNase-free water and carrys out dissolution precipitation, obtain highly purified total serum IgE.
Wherein, described in step 1, the little quantity passing through smaller green leaf hopper adult is 5, the total amount adding described Total RNAs extraction liquid RNAisoPlus in step 2 is 1000ul, and is initially charged after the abundant homogenate of 200ul and adds 800ul, step 5 adds the chloroform of 200ul, step 6 adds the isopropanol of 700ul, step 7 adds the ethanol 1ml that concentration is 75%, and the amount of the RNASE-FREE water of addition is 15ul, the little consumption passing through smaller green leaf hopper adult and reagent of above-mentioned quantity and proportioning, can save cost, and the situation reducing reagent dosage is issued to test requirements document, additionally, Heat thermostability in step 1 is in growth cabinet and at light intensity 4000lx, carry out under the illumination condition of RH60% ± 5%, little pass through smaller green leaf hopper adult collecting and enter in centrifuge tube and shift in the process in described growth cabinet, by centrifuge tube oral area down, it is collected, to pass through smaller green leaf hopper build only small due to little, jump everywhere, if by conventional method, it is difficult under injury-free premise, move into it centrifuge tube, technical staff is by careful observation and ponders, utilize little when passing through smaller green leaf hopper activity habit upwards by downward for the centrifuge tube mouth of pipe, their own is allowed to enter in centrifuge tube, operation is very easy, collect simple effective,In step 1, fresh tea leaf bud head is placed in described centrifuge tube before Heat thermostability, and after Heat thermostability and described centrifuge tube be placed in liquid nitrogen before by fresh tea leaf bud head take out, purpose is the little land for growing field crops ecological environment passing through smaller green leaf hopper adult of true simulation, make to test the data obtained and can more effectively support in the little study on prevention passing through smaller green leaf hopper adult, on the other hand, owing to the time of Heat thermostability is long, smaller green leaf hopper adult offer food is passed through for little, prevent from little passing through smaller green leaf hopper adult and affecting the accuracy of result of the test because the situations such as Heat thermostability early stage is dead occur.
It is simply effective that the present embodiment extracts the little method passing through smaller green leaf hopper adult total serum IgE, solve total serum IgE in extraction process, hold problem of easy degradation, particularly before adding chloroform, first pass through the centrifugal of step 3 and step 4 extracts supernatant, the precipitation of bottom is cast out, containing substantial amounts of fat and macromole impurity in these precipitations, some of them cannot remove in subsequent step again, and these impurity can be removed in advance by step 3 and step 4, reduce contaminating impurity as far as possible, for follow-up further purification, lay the foundation, solve the problem that high-purity extracts total serum IgE.
After obtaining highly purified total serum IgE, taking small part for test experience, all the other are in-80 DEG C of preservations, or are directly used in subsequent experimental.
Experiment one
Carry out extracting test according to the extracting method of above-mentioned total serum IgE, in order to prevent single sample from occasionality result occurring, this extraction have selected two samples, all selects five and little passes through smaller green leaf hopper adult, obtain little to pass through smaller green leaf hopper worm adult total serum IgE quality as shown in table 1:
Table 1 is little passes through smaller green leaf hopper adult total serum IgE quality
| The sample processed | Concentration (ng/ul) | OD260/280 | OD260/230 |
| Sample 1 | 301.6 | 1.99 | 1.32 |
| Sample 2 | 295.8 | 1.98 | 1.30 |
Electrophoresis result is as follows:
Passing through smaller green leaf hopper adult total serum IgE integrity detection electrophoretogram as it is shown in figure 1, little, in figure, swimming lane is little pass through smaller green leaf hopper adult total serum IgE.
As shown in Figure 2, the little cDNA template the passing through smaller green leaf hopper adult total serum IgE reverse transcription detection electrophoretogram to heatshock protein pane gene pcr amplification result, in figure, M is DNALD2000Marker, and swimming lane is that adult total serum IgE reverse transcription cDNA template passes through smaller green leaf hopper adult heat shock protein gene 90PCR amplification to little.
As shown in Figure 3, the little cDNA template the passing through smaller green leaf hopper adult total serum IgE reverse transcription detection electrophoretogram to Heat shock protein 70 gene pcr amplification result, in figure, M is DNALD2000Marker, and swimming lane is that adult total serum IgE reverse transcription cDNA template passes through smaller green leaf hopper adult heat shock protein gene 70PCR amplification to little.
As shown in Figure 4, the little cDNA template the passing through smaller green leaf hopper adult total serum IgE reverse transcription detection electrophoretogram to internal reference Gene A ctinPCR amplification, in figure, M is DNALD2000Marker, and swimming lane is that adult total serum IgE reverse transcription cDNA template passes through smaller green leaf hopper adult Actin gene PCR amplification to little.
Experiment two
One of them sample is extracted fully according to the extracting method of above-mentioned total serum IgE, the step 3 differed only in the extracting method lacking above-mentioned total serum IgE of the extracting method of another sample and step 4, testing result finds, each electrophoretogram band is very fuzzy, whole screen is almost completely black, do not show clear swimming lane, when illustrating to lack step 3 and step 4 the two step, it is impossible to extract satisfactory high-purity total serum IgE.
Experiment three
One of them sample extracts fully according to the extracting method of above-mentioned total serum IgE, another sample extracting method differ only in Heat thermostability before described centrifuge tube in do not place fresh tea leaf bud head, testing result finds, each electrophoretogram band definition reduces, swimming lane occurs a little fuzzy, when illustrating not place in centrifuge tube fresh tea leaf bud head, have a negative impact to extracting total serum IgE.
Finally should be noted that: above example is only in order to illustrate that technical scheme is not intended to limit; Although the present invention being described in detail with reference to preferred embodiment, those of ordinary skill in the field are it is understood that still can modify to the specific embodiment of the present invention or portion of techniques feature carries out equivalent replacement; Without deviating from the spirit of technical solution of the present invention, it all should be encompassed in the middle of the technical scheme scope that the present invention is claimed.
Claims (6)
1. the one kind little extracting method passing through smaller green leaf hopper adult total serum IgE, it is characterised in that comprise the following steps:
Step 1, collect and little pass through smaller green leaf hopper adult in the centrifuge tube being placed with fresh tea leaf bud head, smaller green leaf hopper adult Heat thermostability is passed through by little, make little to pass through smaller green leaf hopper adult heat shock stupor, from described centrifuge tube, then take out fresh tea leaf bud head, and described centrifuge tube is immediately placed in liquid nitrogen;
Step 2, from liquid nitrogen, take out centrifuge tube and add Total RNAs extraction liquid RNAisoPlus at once, with adding Total RNAs extraction liquid RNAisoPlus after the abundant homogenate of plastics grinding rod to enough, then with pipettor, serosity being blown and beaten for several times;
Step 3, will fill enough Total RNAs extraction liquid RNAisoPlus homogenised sample room temperature place 5min, 12000g, 4 DEG C of centrifugal 5min;
Step 4, careful Aspirate supernatant, move in another centrifuge tube by supernatant;
Step 5, adding the chloroform of 1/5 volume of Total RNAs extraction liquid in supernatant, cover tightly centrifuge tube, acutely shake, after solution is fully emulsified, room temperature stands 5min, 12000g, 4 DEG C of centrifugal 15min;
Step 6, from centrifuge take out centrifuge tube, Aspirate supernatant proceeds in the 3rd centrifuge tube, add and the isopyknic isopropanol of supernatant in the 3rd centrifuge tube, turn upside down test tube, fully mixing, stands 10min, 12000g in the environment of 15-30 DEG C,, there is precipitation in 4 DEG C of centrifugal 10min bottom test tube after centrifugal;
Step 7, carefully abandon supernatant, slowly adding concentration along centrifugal tube wall is the ethanol of 75%, turn upside down centrifuge tube gently, washing centrifuge tube wall, 12000g, discards ethanol, drying at room temperature 2-5min after 4 DEG C of centrifugal 5min, it is subsequently adding appropriate RNase-free water and carrys out dissolution precipitation, obtain highly purified total serum IgE.
2. the little extracting method passing through smaller green leaf hopper adult total serum IgE according to claim 1, it is characterised in that: the medium and small temperature conditions passing through smaller green leaf hopper adult Heat thermostability of step 1 is 34-36 DEG C, and the Heat thermostability time is 50-70min.
3. the little extracting method passing through smaller green leaf hopper adult total serum IgE according to claim 2, it is characterised in that: after adding the chloroform of 1/5 volume of Total RNAs extraction liquid in step 5 in supernatant and covering tightly centrifuge tube, acutely the time of concussion is 15s.
4. the little extracting method passing through smaller green leaf hopper adult total serum IgE according to claim 3, it is characterised in that: each centrifuge tube is the centrifuge tube of 1.5ml.
5. the little extracting method passing through smaller green leaf hopper adult total serum IgE according to claim 1-4 any one, it is characterised in that: the medium and small quantity passing through smaller green leaf hopper adult of step 1 is 5; The total amount adding described Total RNAs extraction liquid RNAisoPlus in step 2 is 1000ul, and is initially charged after the abundant homogenate of 200ul and adds 800ul; Step 5 adds the chloroform of 200ul; Step 6 adds the isopropanol of 700ul; Step 7 adds the ethanol 1ml that concentration is 75%, and the amount of the RNASE-FREE water of addition is 15ul.
6. the little extracting method passing through smaller green leaf hopper adult total serum IgE according to claim 5, it is characterized in that: the Heat thermostability in step 1 is in growth cabinet and at light intensity 4000lx, carry out under the illumination condition of RH60% ± 5%, little pass through smaller green leaf hopper adult collecting and enter in centrifuge tube and shift in the process in described growth cabinet, by centrifuge tube oral area down, utilize little when passing through smaller green leaf hopper activity habit upwards be collected.
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