CN116716424A - Fluorescent in-situ hybridization identification probe and identification method for Cephalosporium falciparum - Google Patents
Fluorescent in-situ hybridization identification probe and identification method for Cephalosporium falciparum Download PDFInfo
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Abstract
A fluorescent in situ hybridization identification probe and an identification method for a Blastomyces falciparum relate to the field of gene detection, and the sequence of the probe is as follows: 5'Cy3-ACGCATGGTAAAATTGCAAGA-3'. The probe is used for detecting the colonization of the Cephalomyces falciparum. The fluorescence in situ hybridization identification probe of the Fuscoporia obliqua designed by the invention has higher sensitivity and stronger specificity, and can detect the colonization of fungi in root systems. According to the specific probe designed by the IGS1 fragment, the distribution of fungi in the root system is analyzed, and the accuracy is high. By inoculating the fungus, the invasion dynamics of the fungus can be known by using in situ hybridization techniques.
Description
Technical Field
The invention belongs to the field of gene detection, and particularly relates to a fluorescent in-situ hybridization identification probe for Cephalosporium falciparum (Phialocephala fortinii) and an identification method.
Background
Dark-colored compartmentalized endophytes (Dark septate endophytes, DSE) broadly designate a group of small-sized soil fungi colonized in plant roots, which have more pronounced similar characteristics in different plant roots, are generally darker in hyphae color, can observe distinct transverse septa, are often present in the cells or interstices of the epidermis, cortex and even vascular bundle tissue of healthy plant roots, can observe shuttle hyphae, and can form "microsclerotia" in plant cells or interstices, which is a broad-range class endophyte under investigation, and is also a major group of endophytes. Cephalomyces falciparum (Phialocephala fortinii) is a dark-colored isolated endophyte with high separation frequency, and is helpful for increasing plant biomass, chlorophyll content, relative Water Content (RWC) and the like, improving antioxidant enzyme activity, enhancing drought adaptation capability of plants and improving stress resistance of hosts.
Plant endophytic fungi are of a plurality of types, and currently, a lot of research surrounds the separation of excellent strains with growth promoting or stress resisting effects, and further preparation of biological agents is carried out in fields. However, the isolated excellent endophytic fungus strain can not colonize the field, and is a key link of mycorrhizal fungi to exert biological functions, so that the detection of the colonization of the endophytic fungi is extremely key. Currently, common endophytic fungus colonization detection techniques include trypan blue staining, in situ hybridization techniques, and the like.
Trypan blue staining was first applied by Phillips and Hayman in 1970. In 1989, koske and Gemma were further applied and optimized. Trypan blue is a cell reactive dye, commonly used to detect the integrity of cell membranes. The trypan blue coloring agent comprises trypan blue, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride and sodium hydroxide for regulating pH. The trypan blue dye liquor can dye fungus hyphae in the root system into dark blue, and the plant root system is light or not dyed, thereby being beneficial to distinguishing the root system from fungi in the root system.
The trypan blue dyeing method is simple to operate, less in time consumption and obvious in effect, the color of the plant root system can be decolorized to be light or colorless, background color interference is less in the observation process, and the result reliability is high. The infection state of the fungus in the root system can be intuitively known under a microscope, and the infection state comprises special hypha forms such as extracellular shuttle hypha, intracellular hypha curling and the like, vesicle structures and the like. In addition, the co-infection state of different endophytes in plant root systems is also beneficial to be observed.
Although the trypan blue dyeing effect is reliable and stable, the root cortex is dyed with the same or slightly light color, the color contrast between fungi and the root cortex tissue is small, and the toxicity is large.
In situ hybridization is a molecular cytogenetic technique that uses labeled DNA or RNA probes of known sequences to hybridize directly to nucleic acids on tissue sections to locate specific target gene sequences. This technique was found by galls and Pardue in 1969. When in situ hybridization techniques are applied to the localization of fungi in the root system, it is often necessary to obtain a nucleotide sequence of the genome of the fungi, prepare DNA or RNA probes based thereon, and finally use the probes to localize the fungi in the root system. There is no report of using in situ hybridization technology to locate the Cephalosporium falciparum in the root system. Limiting the in-depth research and development and utilization of the fossa bottle head mould.
Disclosure of Invention
The invention aims to provide a fluorescent in-situ hybridization identification probe and an identification method for the fossa bottle head mould, aiming at the defects, so as to more accurately perform the colonisation detection of the fossa bottle head mould and position the fossa bottle head mould in a root system.
The invention relates to a fluorescent in situ hybridization identification probe for Huntington's bottle head mould, which has the following sequence:
5'Cy3-ACGCATGGTAAAATTGCAAGA-3'。
further, the probe is designed based on IGS1 fragment amplified by gene of the Cephalosporium falciparum; the IGS1 fragment is obtained by PCR amplification by taking the genome of the Fuscoporia obliqua as a template through an upstream primer LR12R and a downstream primer 5 SRNA;
the sequences of the upstream primer LR12R and the downstream primer 5SRNA are as follows:
the upstream primer LR12R: CTGAACGCCTCTAAGTCAGAA;
downstream primer 5SRNA: ATCAGACGGGATGCGGT.
The invention designs a specific fluorescent probe based on the sequence information of the fungus IGS1, and the fluorescent probe is marked by Shanghai biological engineering Co.
Further, the PCR amplification PCR system is as follows:
PCR amplification conditions: pre-denaturation at 94℃for 5min; denaturation at 94℃for 1min, annealing at 56℃for 50s, extension at 72℃for 100s,34 cycles, and extension at 72℃for 10min.
The invention relates to an application of a fluorescent in situ hybridization identification probe for the Blastomyces falciparum, which is used for detecting the colonization of the Blastomyces falciparum. The Fuscoporia obliqua belongs to a dark-colored endophytic fungus.
Further, the probe is used for the colonisation detection of the fostina bottle head mould, and the specific operation is as follows:
step one, taking a sample infected with the Huntington's Cephalosporium cucumerinum, and carrying out pretreatment;
and step two, carrying out prehybridization, probe denaturation, hybridization and washing on the pretreated sample to finish the colonisation detection.
Further, the pretreatment is slice hybridization pretreatment and whole-tissue hybridization pretreatment; wherein, slicing hybridization is to drop 10 mug/mL proteinase K on the slice, digest tissue for 6min, suck liquid, drop 0.2% glycine solution prepared by PBS, stop reaction for 3min; the whole-tissue hybridization is treated by proteinase K in a water bath at 35-37 ℃ for at least 4 hours, then the reaction is stopped for 3 minutes by using 2% glycine PBS buffer solution, and the pretreatment is completed by flushing with PBS.
Further, the prehybridization is slice prehybridization and whole-tissue prehybridization; wherein, slicing prehybridization is to drop high-efficiency liquid phase hybridization solution preheated to 50 ℃ on a glass slide, and then placing the glass slide in a hybridization furnace at 45 ℃ for prehybridization for 60min; the whole tissue prehybridization is to directly soak the root segments in hybridization mixed solution for hybridization; the volume ratio of formamide to water in the high performance liquid phase hybridization solution is 3.5:1.5.
further, the probe denaturation is as follows: the probe was placed in boiling water at 100deg.C for 10min, and then cooled on ice for 10min.
Further, the hybridization is slice hybridization and full-tissue hybridization; the slice hybridization is to take out the pre-hybridized glass slide, remove the pre-hybridization liquid, mix the Hyb high-efficiency hybridization liquid preheated to 65 ℃ with the denatured probe uniformly, drop the mixture onto the glass slide, place the glass slide in a wet box, and then transfer the glass slide to a hybridization furnace at 45 ℃ for hybridization overnight; the whole-tissue hybridization is that the sample is placed in a centrifuge tube, hyb high-efficiency hybridization solution preheated to 65 ℃ is added, and denatured probe is added under dark condition to be transferred to a hybridization furnace at 45 ℃ for hybridization overnight. The final concentration of the probe in the Hyb efficient hybridization solution is 0.8-0.9 ng/. Mu.L. The final concentration of the probe in the Hyb high efficiency hybridization solution is preferably 0.825 ng/. Mu.l.
The kit for fluorescent in situ hybridization identification of the Huntington's bottle head mould comprises the fluorescent in situ hybridization identification probe for the Huntington's bottle head mould.
The invention has the following beneficial effects:
the fluorescence in situ hybridization identification probe of the Fuscoporia obliqua designed by the invention has higher sensitivity and stronger specificity, and can detect the colonization of fungi in root systems. According to the specific probe designed by the IGS1 fragment, the distribution of fungi in the root system is analyzed, and the accuracy is high. By inoculating the fungus, the invasion dynamics of the fungus can be known by using in situ hybridization techniques.
Drawings
FIG. 1 is an electrophoresis diagram of fungal IGS1 fragments;
FIG. 2 is a graph of fluorescence in situ hybridization results; wherein a and c are in-situ hybridization results of the fluorescent probes based on the sections and negative control; e is the in situ hybridization result of the whole tissue of the fluorescent probe; b. d and f are the shapes of the corresponding slices and root sections under the light microscope.
Detailed Description
For the purposes of clarity, technical solutions and advantages of embodiments of the present invention, the spirit of the present disclosure will be described in detail below, and any person skilled in the art, after having appreciated the embodiments of the present disclosure, may make changes and modifications to the techniques taught by the present disclosure without departing from the spirit and scope of the present disclosure.
The exemplary embodiments of the present invention and the descriptions thereof are intended to illustrate the present invention, but not to limit the present invention.
Examples
1) Experimental test material
Tissue culture seedling: blueberry seedling
Blueberry seedlings are aseptic blueberry tissue culture seedlings and are of the trade name "rickettsia".
2) Strain
A strain H5 of conidiomycete, isolated from the root of cowberry.
Symbiotic culture of tissue culture seedlings and Funtingtian bottle head mold: inoculating fungus slices into a tissue culture Miao Turang culture medium with good growth vigor, inoculating 3 fungus slices with the diameter of 1cm into each bottle, and placing the fungus slices into root soil of the tissue culture seedling.
3) Experimental procedure
3.1 PCR amplification
The extracted genome DNA of the strain H5 of the Cephalomyces falciparum is used as a template, and an upstream primer LR12R is designed by using the IGS1 region of the strain: CTGAACGCCTCTAAGTCAGAA; downstream primer 5SRNA: ATCAGACGGGATGCGGT; the rDNA IGS1 specific fragment of the strain H5 is amplified by a PCR reaction, and the PCR reaction system is as follows:
TABLE 1 PCR reaction System for amplifying specific fragment of H5 rDNA IGS1 of Strain
The PCR parameters were as follows:
TABLE 2 PCR reaction parameters for H5 rDNA IGS1 specific segments of strains
3.2 recovery of the fragment of interest by agarose gel
The target fragment was recovered by using the agarose gel DNA recovery kit of TIANGEN company, and the operation method was as follows:
(1) About 0.1g of agarose gel containing the target band was cut under an ultraviolet lamp, placed in a pre-weighed 1.5mL centrifuge tube, and labeled.
(2) Column balance: to the adsorption column CA2, 500. Mu.L of the balance liquid BL was added at 12000rpm/min, followed by centrifugation for 1min. The waste liquid in the collecting pipe is poured out, and the adsorption column is put back into the collecting pipe.
(3) An equal volume of solution PN (100. Mu.L of PN solution if the gel weight is 0.1g, and the volume can be regarded as 100. Mu.L) is added into a centrifuge tube, the gel blocks are melted in a constant temperature water bath at 50 ℃, and the centrifuge tube is continuously and gently turned up and down during the period, so that the gel blocks are fully melted.
(4) Adding the solution obtained in the previous step into an adsorption column CA2 with good balance, standing at room temperature for 2min at 12000rpm/min, centrifuging for 1min, pouring out the waste liquid in the collecting pipe, and placing the adsorption column into the collecting pipe.
(5) 600. Mu.L of the rinse PW was added to the column CA2, centrifuged at 12000rpm/min for 1min, and the waste liquid was discarded.
(6) Repeating the above steps.
(7) After discarding the filtrate, the adsorption column CA2 was put back into the collection tube, centrifuged at 12000rpm/min for 2min, and the rinse solution was removed as much as possible. The column was left at room temperature for several minutes and dried thoroughly.
(8) Placing the adsorption column CA2 into a clean centrifuge tube, suspending and dripping 50 mu L of sterile water into the middle position of the adsorption film, standing for 2min at room temperature, centrifuging for 2min at 12000rpm, and collecting a DNA solution.
(9) The solution obtained by centrifugation was added back to the column, left at room temperature for 2min at 12000rpm/min, centrifuged for 2min, and the DNA solution was collected.
(10) After 1% agarose electrophoresis detection result, preserving in a refrigerator at-20deg.C, and if preserving for a long time, placing in a refrigerator at-80deg.C.
3.3 determination of fragments of interest and sequence analysis
And (3) delivering the target product recovered by cutting the gel to Shanghai biological engineering Co.
3.4 preparation of probes
Based on the fungal IGS1 sequence information, specific fluorescent probes were designed and labeled by Shanghai Biotechnology Inc.
3.5 preparation of the sections
(1) Drawing materials: and respectively selecting tissue culture seedlings which are not inoculated and inoculated with fungi H5 for two weeks, cleaning root systems, selecting young roots with similar thickness, cutting the young roots into root sections with about 0.5cm, fixing the root sections in 4% FAA at the temperature of 4 ℃ for 6 hours, and taking out the root sections and putting the root sections into a solution containing 30% sucrose-PBS overnight.
(2) Embedding a fixing material: a pencil is used as a model, tinfoil paper is wound at the tail end of the pencil to form a cylindrical barrel with the height of about 1cm, a sakura embedding agent is added into a mould, root sections are vertically inserted into the embedding agent by forceps, a temperature reducing switch of a slicing machine is regulated, the temperature of a box body is regulated to minus 25 ℃ from minus 4 ℃, the tinfoil mould with the embedded root sections is put into the slicing machine for freezing, and the temperature of a freezing table is reduced to minus 25 ℃. Adding an appropriate amount of OTC embedding agent on a sample fixing sheet of the slicer, stripping a tinfoil mould, vertically inserting a root section into a central position, cooling and fixing, and preparing the frozen slice after the embedding agent is completely solidified.
(3) Slicing: the thickness of the slice is adjusted to 8 mu m, the slicing process is required to be completed in a short time, the sample material can be frozen and hard for a long time, the slice is fragile, and the tissue is incomplete. If the material is frozen and hard, the temperature can be returned to the room temperature for 15-30s, and then slicing is carried out.
(4) Spreading: the cut slices are stuck on a glass slide, and naturally dried for a period of time at room temperature, so that a small amount of embedding agent stuck on the tissues is melted.
(5) And (3) preserving: the cut frozen slices can be immediately used or placed in a refrigerator and stored for several days at-20 ℃.
(6) And (5) microscopic examination: the excised tissue sections were observed under an inverted microscope for the integrity of the plant tissue.
(7) Baking slices: frozen slices stored in a refrigerator at the temperature of minus 20 ℃ are required to be warmed to about 20 minutes at room temperature, and the slices are put into an oven for baking.
(8) Alcohol dehydration: the sections were removed, 30%, 50%, 70%, 90% and 100% ethanol solutions were prepared and carefully added dropwise to the sections in order, and the plant tissue was dehydrated and carefully discarded after 1min of treatment per concentration.
(9) Pouring the alcohol clean, and carefully dripping 3% H on the tissue 2 O 2 PBS blocks endogenous peroxidase and is carefully aspirated after 10min.
(10) Digesting tissue cells: will H 2 O 2 The PBS was blotted clean with filter paper, proteinase K (10. Mu.g/mL of exposed nucleic acid) prepared with PBS was added dropwise to the sections, and the tissue was digested for 6min and blotted off.
(11) Terminating the digestion reaction: all proteinase K was blotted off and a 0.2% glycine solution in PBS was added dropwise to terminate the reaction for 3min.
3.6 Whole tissue fluorescence in situ hybridization treatment
(1) Selecting tiny and tender small roots, fixing the small roots by FAA, and then flushing the small roots by distilled water;
(2) Cutting into root sections of 2-3mm along the root by using a sterile scalpel, and placing the root sections into a 2mL centrifuge tube;
(3) Adding proteinase K, and placing in a water bath kettle at 36 ℃ for at least 4 hours;
(4) After stopping the reaction with 2% glycine PBS for 3min, the reaction was washed once with PBS.
3.7 fluorescence in situ hybridization
(1) Prehybridization: and (3) dripping the high-efficiency liquid-phase hybridization solution which is preheated in a water bath at 50 ℃ on the glass slide, putting the glass slide into a hybridization furnace with the temperature adjusted to 45 ℃ in advance, and sucking the hybridization solution after 60 minutes of prehybridization, wherein the glass slide cannot be dried. Full-tissue fluorescence in situ hybridization directly immerses the root segments in the hybridization mixture for hybridization. The volume ratio of formamide to water in the high performance liquid phase hybridization solution is 3.5:1.5.
(2) Denaturation probe: the labeled probe was placed in a water bath at 100deg.C in boiling water for 10min, during which time care was taken to observe that the lid of the PCR tube could not be opened, otherwise the probe would evaporate, and then cooled on ice for 10min.
(3) Hybridization: taking out the slide glass from the hybridization furnace, pouring out the prehybridization solution, taking a proper amount of Hyb efficient hybridization solution (preheated at 65 ℃ in advance) with formamide, then adding a denatured probe (2 mL of hybridization solution is added with 2 mu L of probe), blowing and sucking by a pipettor, mixing uniformly, carefully dripping the mixture into the slide glass, putting the slide glass into a wet box, transferring the wet box into the hybridization furnace at the set temperature, and hybridizing overnight. Full-tissue fluorescence in situ hybridization, putting the root segments into a new centrifuge tube, adding a proper amount of hybridization solution, simultaneously adding a probe under dark condition, putting into a hybridization furnace, and hybridizing overnight.
(4) The slides or sections were rinsed 2 times with distilled water and observed with a fluorescent inverted microscope.
4) Experimental results
4.1 fluorescent in situ hybridization positioning analysis of fungi
4.1.1 agarose gel electrophoresis
The IGS1 region of the strain H5 of the Cephalomyces falciparum is amplified by using a PCR technology, and the obtained fungus H5 target fragment size is 750-1000 bp. The results of gel electrophoresis are shown in FIG. 1.
4.1.2 preparation of fluorescent probes
Based on the IGS1 segment sequencing result, designing a specific primer sequence of the strain H5 of the Cephalomyces falciparum, and preparing a fluorescence specific probe. Finally, the length of the fluorescent probe of the strain H5 is determined to be 21bp, and a red fluorescent group (Cy 3) is added at the 5' end, and the base sequence is 5' -Cy3-ACG-CAT-GGT-AAA-ATT-GCA-AGA-3'.
4.1.3 fluorescence in situ hybridization detection of endophytic fungi
The specific fluorescent labeling probe can be combined with a sequence corresponding to a fungus rDNA IGS1 segment according to the base complementation pairing principle, the position of the fungus in root tissues is labeled, and a visible fluorescent signal is visible under excitation fluorescence.
As shown in FIG. 2, the red fluorescent signal of the strain H5 of Cephalosporium falciparum was observed in frozen section tissues mainly concentrated between epidermal cells, presumably as intercellular shuttle hyphae of Cephalosporium falciparum; in addition, the fluorescence signal region is also visible in the center pillar region. No significant fluorescent signal was seen for the negative control. A large number of red fluorescent signals can also be observed in the whole tissue fluorescence in situ hybridization, and the signals are widely distributed in each tissue, wherein the signals of the epidermis area are more.
The hybridization result based on fluorescent markers shows that the Cephalosporium falciparum is more distributed in the tissues and the proliferation amount is larger after 2 weeks of inoculation.
Claims (10)
1. A fluorescent in situ hybridization identification probe for fostina, characterized in that the probe has the following sequence:
5'Cy3-ACGCATGGTAAAATTGCAAGA-3'。
2. the fluorescent in situ hybridization identification probe for the trichoderma favum according to claim 1, wherein the probe is designed based on the gene amplified IGS1 fragment of the trichoderma favum; the IGS1 fragment is obtained by PCR amplification by taking the genome of the Fuscoporia obliqua as a template through an upstream primer LR12R and a downstream primer 5 SRNA;
the sequences of the upstream primer LR12R and the downstream primer 5SRNA are as follows:
the upstream primer LR12R: CTGAACGCCTCTAAGTCAGAA;
downstream primer 5SRNA: ATCAGACGGGATGCGGT.
3. The fluorescent in situ hybridization identification probe for the Cephalomyces falciparum according to claim 2, wherein the PCR amplification is carried out by the following PCR system:
PCR amplification conditions: pre-denaturation at 94℃for 5min; denaturation at 94℃for 1min, annealing at 56℃for 50s, extension at 72℃for 100s,34 cycles, and extension at 72℃for 10min.
4. Use of a fluorescent in situ hybridization identification probe for leptospirillum falciparum according to claim 1, characterized in that said probe is used for the colonisation detection of leptospirillum falciparum.
5. The use according to claim 4, wherein the probe is used for the colonisation detection of the group of Cephalosporium falciparum, and the following procedure is followed:
step one, taking a sample infected with the Huntington's Cephalosporium cucumerinum, and carrying out pretreatment;
and step two, carrying out prehybridization, probe denaturation, hybridization and washing on the pretreated sample to finish the colonisation detection.
6. The use according to claim 5, wherein the pretreatment is a slice hybridization pretreatment and a whole tissue hybridization pretreatment; wherein, slicing hybridization is to drop 10 mug/mL proteinase K on the slice, digest tissue for 6min, suck liquid, drop 0.2% glycine solution prepared by PBS, stop reaction for 3min; the whole-tissue hybridization is treated by proteinase K in a water bath at 35-37 ℃ for at least 4 hours, then the reaction is stopped for 3 minutes by using 2% glycine PBS buffer solution, and the pretreatment is completed by flushing with PBS.
7. The use according to claim 5, characterized in that said prehybridization is a slice prehybridization and a whole-tissue prehybridization; wherein, slicing prehybridization is to drop high-efficiency liquid phase hybridization solution preheated to 50 ℃ on a glass slide, and then placing the glass slide in a hybridization furnace at 45 ℃ for prehybridization for 60min; the whole tissue prehybridization is to directly soak the root segments in hybridization mixed solution for hybridization; the volume ratio of formamide to water in the high performance liquid phase hybridization solution is 3.5:1.5.
8. the use according to claim 5, wherein the probe denaturation is: the probe was placed in boiling water at 100deg.C for 10min, and then cooled on ice for 10min.
9. The use according to claim 5, characterized in that said hybridization is a slice hybridization and a whole tissue hybridization; the slice hybridization is to take out the pre-hybridized glass slide, remove the pre-hybridization liquid, mix the Hyb high-efficiency hybridization liquid preheated to 65 ℃ with the denatured probe uniformly, drop the mixture onto the glass slide, place the glass slide in a wet box, and then transfer the glass slide to a hybridization furnace at 45 ℃ for hybridization overnight; placing the sample in a centrifuge tube, adding Hyb high-efficiency hybridization solution preheated to 65 ℃, adding denatured probe under dark condition, and transferring to a hybridization furnace at 45 ℃ for hybridization overnight; the final concentration of the probe in the Hyb efficient hybridization solution is 0.8-0.9 ng/. Mu.L.
10. A kit for fluorescent in situ hybridization assay comprising a fluorescent in situ hybridization assay probe for leptospirillum falciparum, characterized in that said kit comprises a fluorescent in situ hybridization assay probe for leptospirillum falciparum of claim 1.
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| CN103409504A (en) * | 2013-06-26 | 2013-11-27 | 武汉康录生物技术有限公司 | FISH (fluorescence in situ hybridization) probe, kit and detection method for detecting Her2 (human epidermal growth factor receptor 2) gene free from repetitive sequence |
| CN103981265A (en) * | 2014-05-15 | 2014-08-13 | 董志平 | Nested PCR (Polymerase Chain Reaction) high-efficiency detection method for millet rust fungus |
| CN106916886A (en) * | 2017-01-18 | 2017-07-04 | 中国人民解放军第四军医大学 | A kind of gene primer of dynamin-related proteins 1 and its preparation method of cRNA in situ hybridization probes for FISH |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103409504A (en) * | 2013-06-26 | 2013-11-27 | 武汉康录生物技术有限公司 | FISH (fluorescence in situ hybridization) probe, kit and detection method for detecting Her2 (human epidermal growth factor receptor 2) gene free from repetitive sequence |
| CN103981265A (en) * | 2014-05-15 | 2014-08-13 | 董志平 | Nested PCR (Polymerase Chain Reaction) high-efficiency detection method for millet rust fungus |
| CN106916886A (en) * | 2017-01-18 | 2017-07-04 | 中国人民解放军第四军医大学 | A kind of gene primer of dynamin-related proteins 1 and its preparation method of cRNA in situ hybridization probes for FISH |
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