CN104694532A - Extracting method of RNA of rubber tree powdery mildew - Google Patents
Extracting method of RNA of rubber tree powdery mildew Download PDFInfo
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Abstract
The invention relates to the field of biotechnology and discloses an extracting method of RNA of rubber tree powdery mildew. The powdery mildew is induced to germinate on hydrophobic media and form appressoria, different growth periods of the powdery mildew are convenient to distinguish, and the purpose of extracting the RNA in a grading mode is achieved. By extracting the RNA of the rubber tree powdery mildew at different periods, it is displayed that the concentration of the RNA and integrity meet the Illumina transcriptome library requirement through quantitative determination, 18SrRNA genes are amplified through RT-PCR, and a foundation is further provided for transcriptome sequencing analysis and gene expression research.
Description
Technical field
The present invention relates to biological technical field, refer to the extracting method of a kind of rubber tree powdery mildew bacterium RNA especially.
Background technology
Rubber tree powdery mildew be belonged to by powder spore rubber powder spore (
oidium heveaeb.A.Steinmann) infect the disease caused, this germ is distributed widely in global each rubber planting district, the emphasis in Zhi Jiao district of Ye Shi China control in early spring " two sick worms ".Rubber tree powdery mildew is obligate parasite, cannot in artificial medium isolated culture, difficult genetic manipulation, more research concentrates on cytological observation and control, not yet cultivate the anti-powdery mildew rubber tree strain of producing upper large-scale promotion at present, molecule experiments system about the functional verification of rubber tree powdery mildew Disease-causing gene is not yet set up, and pathogenic bacteria pathogenesis lacks scrutiny.10003 even wait the powdery mildew RNA obtained by improvement Trizol extracting method to meet builds the requirement of transcript profile library.Improvement Trizol extracts RNA method, is the salt concn improving extracting solution on Trizol fungal rna extraction method basis, and the salt concn increased in solution can reduce RNA and be dissolved in extracting solution, promotes that polysaccharide and albumen etc. are dissolved in solution.Meanwhile, adsorb RNA in conjunction with the pipe of preparing in OMEGA fungal rna RNA isolation kit, the time of volatilization ethanol can be reduced, prevent RNA from degrading.
The Trizol method improved is collected the powdery mildew extracted and is actually spore and hypohostroma mixture RNA, powdery mildew cannot be obtained at the spore sprouted and note fields state is homogeneous, lack the cultivation of infecting powdery mildew tissue in growth course to collect and RNA extraction means, the expression study of functional gene in infection processs cannot be carried out.
Summary of the invention
This present invention proposes the extracting method of a kind of rubber tree powdery mildew bacterium RNA, and the RNA concentration of gained and integrity meet the library requirement of Illumina transcript profile.
Technical scheme of the present invention is achieved in that
An extracting method of the RNA of rubber tree powdery mildew bacterium, comprises the following steps:
(1) adopt and connect bacterium and to cultivate after 10 ~ 12d the fresh conidium in 24 h ages as experimental bacteria source; Hydrophobic medium is placed in the crisper that moistening filter paper is arranged at bottom, gently brush in hydrophobic medium surface with banister brush by the powdery mildew spores on rubber tree leaf, culture temperature 21 ~ 23 DEG C, relative humidity 50% ~ 80%, the sealing of crisper lucifuge is preserved;
(2) after cultivating 0 ~ 24 h in crisper, add the Trizol lysate of precooling on medium, dip lysate with paint daubs to be repeatedly coated with to scrape and to connect bacterium dielectric surface, in lysate on medium when visible white meal or appearance muddiness, collecting lysate with pipettor transfers in the centrifuge tube of precooling, concussion mixing, 20 ~ 25 DEG C leave standstill 8 ~ 10 min;
(3) centrifuge tube adds chloroform, vibration mixing, and 20 ~ 25 DEG C leave standstill rear centrifugal;
(4), after centrifugal, transfer upper strata aqueous phase, in new RNase free centrifuge tube, adds Potassium ethanoate, puts upside down mixing, centrifugal, and 20 ~ 25 DEG C leave standstill to obtain mixing solutions;
(5) isopyknic Virahol is added with mixed solution, mixing, sedimentation RNA sample;
(6) after step (5) process, after centrifugal, remove supernatant liquor, add DEPC water dissolution precipitation; Add RB, 70% ethanol and beta-mercaptoethanol subsequently, put upside down mixing;
(7) after step (6) process, add DEPC water elution RNA after centrifugal, obtain rubber tree powdery mildew bacterium RNA.
Further, the front 24 h shake blades of described step (1) inoculation shake off old spore, using newborn 24 h conidiums in age as inoculation material; Described hydrophobic medium is Parafilm film.
Further, guarantee in centrifuge tube without fungal mass after described step (2) concussion.
Further, described step (3) add that chloroform volume is liquid volume in centrifuge tube 1/4 ~ 1/3; Duration of oscillation is 15 ~ 20 s, and 0 ~ 25 DEG C of time of repose is 5 ~ 7 min, and centrifugal condition is 4 DEG C, 10000 ~ 12000 g, centrifugal 15 ~ 20 min; 2.
Further, the volume that described step (4) adds liquor kalii acetici is 1/3 of aqueous phase volume, and pH value is 6.0; Centrifugal condition is 4oC, 8000 ~ 10000 g, centrifugal 1.0 ~ 1.5 min; Time of repose is 5 ~ 7 min.
Further, described step (5) the RNA sample settling time is 2 ~ 10 h, control temperature-80 DEG C in settling process.
Further, described step (6) centrifugally operated condition is 4 DEG C, 12000 ~ 14000 g, centrifugal 10 ~ 12 min; Volume ratio=100 ~ 120:350 ~ 400:250 ~ 300:7 ~ 9 of the DEPC water added, RB, 70% ethanol and beta-mercaptoethanol.
Further, described step (7) centrifugally operated condition is 4 oC, 10000 ~ 12000 g, centrifugation time 30 ~ 40 s; The add-on of DEPC water be step (6) gained population of samples long-pending 1/7 ~ 1/6.
Beneficial effect of the present invention:
Before inoculation, after cultivating 12 d, fresh
o.heveaespore shape is full, flushes, and chooses same batch
o.heveaefresh conidium inoculation Parafilm film, ensures
o.heveaespore infects the consistence of developmental condition on medium.After parafilm hydrophobic medium is cultivated, observe powdery mildew and infect without the need to steps such as dyeing-decolorzings, save pathogenic bacteria original form, be convenient to the different steps distinguishing powdery mildew growth, thus reach the object extracting RNA stage by stage.Observe and find
o.heveae0 ~ 24 hpi infect growth course can be divided into do not sprout (0 hpi), sprout (4 hpi), note fields (6,8 hpi) and secondary note fields (16,24 hpi) etc. 4 form significant etap, extract under namely respective stage total serum IgE obtain this state
o.heveaetranscript, meets the library requirement of Illumina transcript profile through quantitative assay display RNA concentration and integrity, adopts RT-PCR to increase 18SrRNA gene, for carrying out transcript profile sequencing analysis further and gene expression research provides basis.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings.
Fig. 1 is the morphological specificity schematic diagram that rubber tree powdery mildew difference grows the period;
In figure, A:0 hpi conidium (200 ×); B:4 hpi spore germination (200 ×); C:6 hpi note fields (200 ×); D:8 hpi note fields (400 ×); The secondary note fields of E:16 hpi (400 ×); The secondary note fields of F:24 hpi (200 ×).CO: conidium; Gt: germ tube; AP: appressorium
Fig. 2 is that Different periods extracts the RNA sample electrophoresis obtained;
Fig. 3 is the expression electrophoresis of 18S gene at Different periods.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment 1
(1) rubber 7-33-97 strain Graft 7-10 days leaf age blades are chosen as cultivation
o.heveaematerial, adopts and connects bacterium and to cultivate after 12d HO-73 24h age fresh conidium as experimental bacteria source.Inoculate front 24 h jog blades and shake off old spore, using newborn 24 h conidiums in age as inoculation material, guarantee that experiment miospore vigor and Infection structure grow homogeneity; Parafilm (U.S. BEMIS product) film is placed in the crisper (40 cm × 30 cm) that moistening filter paper is arranged at bottom, with banister brush, the powdery mildew spores on rubber tree leaf is gently brushed in Parafilm film surface, culture temperature 22 DEG C, relative humidity 50% ~ 80%, the sealing of crisper lucifuge is preserved;
(2) after being transferred to the cultivation of Parafilm film surface, adding the Trizol(American I nvitrogen company of 2 mL precoolings immediately) lysate is on Parafilm film, dip lysate with paint daubs to be repeatedly coated with to scrape and to connect bacterium dielectric surface, in lysate on medium when visible white meal or appearance muddiness, collecting lysate with pipettor transfers in 2.0 mL centrifuge tubes of precooling, often pipe packing 1 mL, concussion mixing 30 s, and guarantee without fungal mass in centrifuge tube, 20 ~ 25 DEG C leave standstill 10 min;
(3) every centrifuge tube adds 250 μ L chloroforms, thermal agitation 15 s, and 20 ~ 25 DEG C leave standstill, after 5 min, at 4 DEG C, and 12000 g, centrifugal 15 min;
(4), after centrifugal, transfer upper strata aqueous phase is in new RNase free 2 mL centrifuge tube; Add the Potassium ethanoate (pH=6.0) of 1/3 liquor capacity, put upside down mixing and 4 oC, 8000 g, centrifugal 1 min, 20 ~ 25 DEG C leave standstill 5 min, obtain mixing solutions;
(5) mixing solutions adds isopyknic Virahol, fully mixes, and is placed in-80 DEG C of sedimentation RNA sample 2 h;
(6) after step (5) process, at 4 DEG C, 14000 g, centrifugal 10 min; (7) after step (6) process, carefully remove supernatant, add 100 μ L DEPC water dissolution precipitations; Carry out with reference to Omega fungal rna test kit (Omega company of the U.S.) extracting method elution step subsequently, add 350 μ L RB, 250 μ L 70% ethanol, 7 μ L beta-mercaptoethanols, put upside down mixing.
(7) be transferred in RNA mini column pipe by cumulative volume 700 μ L sample, 4 oC, 10000 g, 30 s are centrifugal; Final 100 μ L DEPC water elution RNA, obtain rubber tree powdery mildew bacterium RNA; RNA-80 DEG C of preservation of gained.
Embodiment 2
(1) rubber 7-33-97 strain Graft 7-10 days leaf age blades are chosen as cultivation
o.heveaematerial, adopts and connects bacterium and to cultivate after 10 d HO-73 24h age fresh conidium as experimental bacteria source.Inoculate front 24 h jog blades and shake off old spore, using newborn 24 h conidiums in age as inoculation material, guarantee that experiment miospore vigor and Infection structure grow homogeneity; Parafilm (U.S. BEMIS product) film is placed in the crisper (40 cm × 30 cm) that moistening filter paper is arranged at bottom, with banister brush, the powdery mildew spores on rubber tree leaf is gently brushed in Parafilm film surface, culture temperature 21 DEG C, relative humidity 50% ~ 80%, the sealing of crisper lucifuge is preserved;
(2) be transferred to Parafilm film surface and cultivate rear 24 h, adding the Trizol(American I nvitrogen company of 2 mL precoolings immediately) lysate is on Parafilm film, dip lysate with paint daubs to be repeatedly coated with to scrape and to connect bacterium dielectric surface, in lysate on medium when visible white meal or appearance muddiness, collecting lysate with pipettor transfers in 2.0 mL centrifuge tubes of precooling, often pipe packing 1 mL, concussion mixing, and guarantee without fungal mass in centrifuge tube, 20 ~ 25 DEG C leave standstill 8 min;
(3) every centrifuge tube adds 330 μ L chloroforms, thermal agitation 20 s, after 20 ~ 25 DEG C of standing 7min, at 4 DEG C, and 10000 g, centrifugal 20 min;
(4), after centrifugal, transfer upper strata aqueous phase is in new RNase free 2 mL centrifuge tube; Add the Potassium ethanoate (pH=6.0) of liquor capacity 1/4, put upside down mixing and 4 oC, 8000 g, centrifugal 1.5 min, 20 ~ 25 DEG C leave standstill 7 min, obtain mixing solutions;
(5) mixing solutions adds isopyknic Virahol, fully mixes, and is placed in-80 DEG C of sedimentation RNA sample 10 h;
(6) after step (5) process, at 4 DEG C, 12000 g, centrifugal 12 min; (7) after step (6) process, carefully remove supernatant, add 120 μ L DEPC water dissolution precipitations; Carry out with reference to Omega fungal rna test kit (Omega company of the U.S.) extracting method elution step subsequently, add 400 μ L RB, 300 μ L 70% ethanol, 9 μ L beta-mercaptoethanols, put upside down mixing.
(7) be transferred in RNA mini column pipe by cumulative volume 829 μ L sample, 4 oC, 10000 g, 30 s are centrifugal; Final 138 μ L DEPC water elution RNA, obtain rubber tree powdery mildew bacterium RNA; RNA-80 DEG C of preservation of gained.
Embodiment 3
(1) rubber 7-33-97 strain Graft 7-10 days leaf age blades are chosen as cultivation
o.heveaematerial, adopts and connects bacterium and to cultivate after 10 d HO-73 24h age fresh conidium as experimental bacteria source.Inoculate front 24 h jog blades and shake off old spore, using newborn 24 h conidiums in age as inoculation material, guarantee that experiment miospore vigor and Infection structure grow homogeneity; Parafilm (U.S. BEMIS product) film is placed in the crisper (40 cm × 30 cm) that moistening filter paper is arranged at bottom, with banister brush, the powdery mildew spores on rubber tree leaf is gently brushed in Parafilm film surface, culture temperature 21 DEG C, relative humidity 50% ~ 80%, the sealing of crisper lucifuge is preserved;
(2) after being transferred to Parafilm film surface cultivation 8 h, adding the Trizol(American I nvitrogen company of 2 mL precoolings) lysate is on Parafilm film, dip lysate with paint daubs to be repeatedly coated with to scrape and to connect bacterium dielectric surface, in lysate on medium when visible white meal or appearance muddiness, collecting lysate with pipettor transfers in 2.0 mL centrifuge tubes of precooling, often pipe packing 1 mL, concussion mixing, and guarantee without fungal mass in centrifuge tube, 20 ~ 25 DEG C leave standstill 9 min;
(3) every centrifuge tube adds 300 μ L chloroforms, thermal agitation 17 s, after 20 ~ 25 DEG C of standing 6 min, at 4 DEG C, and 11000 g, centrifugal 18 min;
(4), after centrifugal, transfer upper strata aqueous phase is in new RNase free 2 mL centrifuge tube; Add the Potassium ethanoate (pH=6.0) of liquor capacity 1/4, put upside down mixing and 4 oC, 9000 g, centrifugal 1.3 min, 20 ~ 25 DEG C leave standstill 6 min, obtain mixing solutions;
(5) mixing solutions adds isopyknic Virahol, fully mixes, and is placed in-80 DEG C of sedimentation RNA sample 5 h;
(6) after step (5) process, at 4 DEG C, 12000 g, centrifugal 12 min; (7) after step (6) process, carefully remove supernatant, add 110 μ L DEPC water dissolution precipitations; Carry out with reference to Omega fungal rna test kit (Omega company of the U.S.) extracting method elution step subsequently, add 370 μ L RB, 280 μ L 70% ethanol, 8 μ L beta-mercaptoethanols, put upside down mixing;
(7) be transferred in RNA mini column pipe by cumulative volume 829 μ L sample, 4 oC, 10000 g, 30 s are centrifugal; Final 115 μ L DEPC water elution RNA, obtain rubber tree powdery mildew bacterium RNA; RNA-80 DEG C of preservation of gained.
powdery mildew is in the extraction of the RNA of day part
Cultivate rubber tree powdery mildew, according to cultural method provided by the invention and RNA extraction method, after being cultured to Different periods (0,4,6,8,16,24 hpi) respectively, medium is taken out.Each period is all cut small pieces (1.5 cm × 1.5 cm) and connects bacterium medium, counted under microscope spore germination and note fields rate, and observes the development characteristics of powdery mildew at day part, extracts the RNA sample of each period simultaneously.Wherein, the cultural method of 0 hpi, 8 hpi and 24 hpi, tri-period rubber tree powdery mildews and RNA extraction method, carry out with reference to 3 embodiments respectively, 4hpi, 6hpi and incubation time in 16hpi period crisper be respectively 4,6, all the other operations of 16h are identical with embodiment 1.
The half reaching spore major diameter with germ tube length is designated as sprouting, adds up 100 conidial germination rates, note fields rate (spore count/total spore count of spore germination rate=sprouted; Note fields rate=appressorium number/spore germination number).
o.heveaeto sprout on Parafilm film surface and note fields feature and data statistics are shown in Fig. 1, table 1 respectively.
Note: mean value represents with mean value ± standard error, the different group difference of different letter representation significantly (
p< 0.05).
Note： Mean±SE express average，different letters mean significant difference between different groups(
P＜0.05).
Composition graphs 1 and table 1,
o.heveae0 ~ 24 hpi infects growth course and can be divided into and do not sprout (0 hpi), sprout (4 hpi), note fields (6,8 hpi) and 4 form significant etap of secondary note fields (16,24 hpi).Adopt method provided by the invention, observe powdery mildew and infect without the need to steps such as dyeing-decolorzings, save pathogenic bacteria original form, illustrate that Parafilm film can be used as and observe the ideal material that Spores infects growth metamorphosis in early stage.
RNA sample electrophoresis result display (see figure 2), there are 2 band in each stage sample that infects, respectively corresponding 28S and 18S rRNA, and wherein 28S band is approximately the twice of 18S band brightness, shows that the total serum IgE integrity degree extracted is better.Nucleic acid quantification detected result display A260/280 numerical value is between 1.95 ~ 2.25, and concentration reaches 150 ngul
-1above (see table 2), show that the RNA of extraction is pure and concentration is higher.General structure Illumina transcript profile sequencing library requires concentration >=200 ng/ μ l, RNA total amount >=10 μ g, adopt the program repeatedly to extract and concentrate and meet library requirement, detected result proves that this RNA extraction method can obtain and grows period corresponding high-quality powdery mildew total serum IgE with specific.
Table 2 powdery mildew infects the RNA quality examination of development characteristics period
Table 2. Detection of RNA quality isolated from characteristic development stages of
O.heveae
The people such as employing Magnus C. Lydolph were published in the SR7R-SR5 primer pair amplifies related in the paper " Beringian Paleoecology Inferred from Permafrost-Preserved Fungal DNA " on " Applied and Environmental Microbiology " in 2005
o. heveae18S gene fragment, 6 periods all can be cloned into the fragment (see figure 3) of about 500 bp, more consistent through the brightness of 30 amplification cycles 10 μ L point sample gained bands, prove that the RNA sample extracted can be used for rubber powdery mildew gene function equally and expresses confirmatory experiment.Rubber tree powdery mildew 18S sequence (518bp) of amplification order-checking is inputted NCBI and does Blastn comparison, result display homologous sequence is mainly from Erysiphales Erysiphaceae species.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (8)
1. an extracting method of the RNA of rubber tree powdery mildew bacterium, is characterized in that, comprises the following steps:
(1) adopt and connect bacterium and to cultivate after 10 ~ 12d the fresh conidium in 24 h ages as experimental bacteria source; Hydrophobic medium is placed in the crisper that moistening filter paper is arranged at bottom, gently brush in hydrophobic medium surface with banister brush by the powdery mildew spores on rubber tree leaf, culture temperature 21 ~ 23 DEG C, relative humidity 50% ~ 80%, the sealing of crisper lucifuge is preserved;
(2) after cultivating 0 ~ 24 h in crisper, add the Trizol lysate of precooling on medium, dip lysate with paint daubs to be repeatedly coated with to scrape and to connect bacterium dielectric surface, in lysate on medium when visible white meal or appearance muddiness, collecting lysate with pipettor transfers in the centrifuge tube of precooling, concussion mixing, 20 ~ 25 DEG C leave standstill 8 ~ 10 min;
(3) add chloroform in centrifuge tube, vibration mixing, 20 ~ 25 DEG C leave standstill rear centrifugal;
(4), after centrifugal, transfer upper strata aqueous phase, in new RNase free centrifuge tube, adds Potassium ethanoate, puts upside down mixing, centrifugal, and 20 ~ 25 DEG C leave standstill to obtain mixing solutions;
(5) isopyknic Virahol is added with mixed solution, mixing, sedimentation RNA sample;
(6) through the sample that step (5) processes, after centrifugal, remove supernatant liquor, add DEPC water dissolution precipitation; Add RB, 70% ethanol and beta-mercaptoethanol subsequently, put upside down mixing;
(7) through the sample that step (6) processes, after centrifugal, add DEPC water elution RNA, obtain rubber tree powdery mildew bacterium RNA.
2. the extracting method of the RNA of a kind of rubber tree powdery mildew bacterium according to claim 1, is characterized in that: front 24 h of described step (1) inoculation shake blade and shake off old spore, using newborn 24 h conidiums in age as inoculation material; Described hydrophobic medium is Parafilm film.
3. the extracting method of the RNA of a kind of rubber tree powdery mildew bacterium according to claim 1, is characterized in that: guarantee in centrifuge tube without fungal mass after described step (2) concussion.
4. the extracting method of the RNA of a kind of rubber tree powdery mildew bacterium according to claim 1, is characterized in that: described step (3) add that chloroform volume is liquid volume in centrifuge tube 1/4 ~ 1/3; Duration of oscillation is 15 ~ 20 s, and centrifugal condition is 4 DEG C, 10000 ~ 12000 g, centrifugal 15 ~ 20 min; 20 ~ 25 DEG C of time of repose are 6 ~ 7 min.
5. the extracting method of the RNA of a kind of rubber tree powdery mildew bacterium according to claim 1, is characterized in that: the volume that described step (4) adds liquor kalii acetici is 1/3 of aqueous phase volume, and pH value is 6.0; Centrifugal condition is 4oC, 8000 ~ 10000 g, centrifugal 1.0 ~ 1.5 min; Time of repose is 5 ~ 7 min.
6. the extracting method of the RNA of a kind of rubber tree powdery mildew bacterium according to claim 1, is characterized in that: described step (5) the RNA sample settling time is 2 ~ 10 h, control temperature-60 DEG C to-80 DEG C in settling process.
7. the extracting method of the RNA of a kind of rubber tree powdery mildew bacterium according to claim 1, is characterized in that: described step (6) centrifugally operated condition is 4 DEG C, 12000 ~ 14000 g, centrifugal 10 ~ 12 min; Volume ratio=100 ~ 120:350 ~ 400:250 ~ 300:7 ~ 9 of the DEPC water added, RB, 70% ethanol and beta-mercaptoethanol.
8. the extracting method of the RNA of a kind of rubber tree powdery mildew bacterium according to claim 1, is characterized in that: described step (7) centrifugally operated condition is 4 oC, 10000 ~ 12000 g, centrifugation time 30 ~ 40 s; The add-on of DEPC water be step (6) gained population of samples long-pending 1/7 ~ 1/6.
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| CN107338213A (en) * | 2017-07-31 | 2017-11-10 | 东北林业大学 | A kind of processing method of trichoderma conidium for being beneficial to extraction total serum IgE and its application |
| CN108192892A (en) * | 2018-03-01 | 2018-06-22 | 杭州比格飞序生物科技有限公司 | A kind of kit and extracting method of hydroxyl nanometer magnetic bead method extraction RNA |
| CN113755632A (en) * | 2021-10-08 | 2021-12-07 | 中国热带农业科学院环境与植物保护研究所 | SSR primer group for genetic structure analysis of rubber tree powdery mildew flora and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106399300A (en) * | 2016-11-11 | 2017-02-15 | 北京市农林科学院 | Method for extracting Botryosphaeria dothidea hypha RNA (ribonucleic acid) |
| CN106399300B (en) * | 2016-11-11 | 2019-03-05 | 北京市农林科学院 | A method of extracting grape ulcer bacterium mycelia RNA |
| CN107338213A (en) * | 2017-07-31 | 2017-11-10 | 东北林业大学 | A kind of processing method of trichoderma conidium for being beneficial to extraction total serum IgE and its application |
| CN108192892A (en) * | 2018-03-01 | 2018-06-22 | 杭州比格飞序生物科技有限公司 | A kind of kit and extracting method of hydroxyl nanometer magnetic bead method extraction RNA |
| CN108192892B (en) * | 2018-03-01 | 2021-12-28 | 杭州比格飞序生物科技有限公司 | Kit for extracting RNA by hydroxyl nano-magnetic bead method and extraction method |
| CN113755632A (en) * | 2021-10-08 | 2021-12-07 | 中国热带农业科学院环境与植物保护研究所 | SSR primer group for genetic structure analysis of rubber tree powdery mildew flora and application thereof |
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