CN101892308A - Specific amplification primer for detecting marssonina coronaria and detection method - Google Patents
Specific amplification primer for detecting marssonina coronaria and detection method Download PDFInfo
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- CN101892308A CN101892308A CN2010101708438A CN201010170843A CN101892308A CN 101892308 A CN101892308 A CN 101892308A CN 2010101708438 A CN2010101708438 A CN 2010101708438A CN 201010170843 A CN201010170843 A CN 201010170843A CN 101892308 A CN101892308 A CN 101892308A
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Abstract
The invention discloses a specific amplification primer for detecting marssonina coronaria. A method for quickly obtaining gene segments of the marssonina coronaria by using the specific primer aims to prevent obstruction brought to actual researches by the difficult separation of pathogenic bacteria. The invention also discloses a rapid molecular detection method for the marssonina coronaria by using the specific amplification primer, which comprises the following steps of: first collecting field diseased leaves and picking and placing the acervulus of the marssonina coronaria on a white paper sheet with a sterilized insect needle; then preparing a polymerase chain reaction (PCR) system; next transferring the acervulus into a small PCR amplification tube in which the PCR system is arranged and performing centrifugation to completely immerse the acervulus in the prepared PCR system; and finally performing PCR amplification and detecting the amplification product. By changing the PCR amplification reaction system and the amplification procedure, the target segment of the pathogenic bacteria can be directly amplified. The primer and the method prevent the separation of the pathogenic bacteria and additional DNA extraction, shorten a test flow and improve the detection efficiency.
Description
Technical field
The invention belongs to field of molecular biotechnology, relate to a kind of specific amplification primer that is used to detect marssonina coronaria, also relate to and utilize the rapid molecular detection method of this specific amplification primer marssonina coronaria.
Background technology
Apple marssonina leaf spot
Marssonina coronaria(Ellis ﹠amp; J.J. J. J. Davis Davis), different name
M. mali(Henn.) S. Ito(sexual generation
Diplocarpon maliHarada ﹠amp; Sawamura) be one of main disease of causing the apple tree early leaf fall.Apple marssonina leaf spot not only endangers blade, petiole, also causes storage period apple fruit morbidity, and then the yield and quality of apple caused has a strong impact on.In recent years, this disease had and increased the weight of trend year by year, more and more was subject to people's attention.
Accurately grasp the biology characteristics of cause of disease, disclose the hereditary property of pathogenic bacteria, seem particularly important exploring the rational and effective method of preventing and treating by molecular biology method.Rapid detection is distinguished the apple marssonina leaf spot germ and also is important one side to its further investigation, general germ Molecular Detection mainly through the separation and Culture of the collection of sample, object bacteria, extract thallus DNA, with primer the DNA that extracts carried out augmentation detection then and order-checking is compared.This method is lasted longer, and the trace routine complexity, and it is also higher to utilize test kit to extract the DNA expense sometimes.In addition, because the growth on general artificial medium of apple marssonina leaf spot pathogenic bacteria isolate is extremely slow, process of growth very easily is subjected to living contaminants, separate extremely difficult, this has caused very big obstruction for the research of carrying out molecular biological characteristic, so cause the research of present this respect also to be in the exploratory stage.
Summary of the invention
The purpose of this invention is to provide a kind of specific amplification primer that is used to detect apple marssonina leaf spot, utilize Auele Specific Primer to obtain the method for apple marssonina leaf spot gene fragment fast, avoid because the obstacle that the pathogenic bacteria separation difficulty is brought to practical study.
Another object of the present invention provides a kind of this specific amplification primer that utilizes marssonina coronaria is carried out the method that rapid molecular detects, and has solved prior art tested for pathogens long flow path, detection efficiency is low, detects the high problem of cost.
The technical solution adopted in the present invention is that a kind of specific amplification primer that is used to detect marssonina coronaria is characterized in that:
Upstream primer P
1: 5 ˊ-TCCCTGTTGGTTTCTTTTCCTCC-3 ˊ,
Downstream primer P
2: 5 ˊ-ACTCTTTGTGTGAATAACGTGATGT-3 ˊ;
Another technical scheme of the present invention is, utilizes above-mentioned specific amplification primer to the marssonina coronaria rapid molecular detection method, comprises following operation steps:
Gather the field incidence of leaf, clean dry after, it is standby with the insect needle of the bacterium of going out the apple marssonina leaf spot acervulus to be placed the white scraps of paper;
10×Taq?Buffer 6μL
DNTP 0.2mM
P
1 1μM
P
2 1μM
Taq archaeal dna polymerase 1U
Mg
2+Concentration
1.75mM
DTT concentration 1.25mM
Adding the sterilization ultrapure water mends reaction system to 50 μ L;
Wherein, P
1And P
2Be a pair of specific amplification primer according to the sequences Design of apple marssonina leaf spot pathogenic bacteria DNA, upstream primer P
1: 5 ˊ-TCCCTGTTGGTTTCTTTTCCTCC-3 ˊ, downstream primer P
2: 5 ˊ-ACTCTTTGTGTGAATAACGTGATGT-3 ˊ;
Insect needle with the bacterium of going out goes to the acervulus on the scraps of paper in the pcr amplification tubule that the PCR reaction system is housed, and whenever guarantees 1～2 acervulus of card; The PCR pipe that adds acervulus is centrifugal, acervulus is immersed in the PCR reaction system of preparation fully;
Response procedures is set to: 94 ℃ of pre-sex change 5min; Next 1min is extended in 94 ℃ of pre-sex change 1min, 48 ℃ of sex change 1min, 72 ℃ of annealing, so carries out 5 circulations; 1min is extended in 94 ℃ of pre-sex change 1min, 53 ℃ of sex change 1min, 72 ℃ of annealing then, so carries out 35 circulations; Extend 8min at 72 ℃ at last, and preserve the amplification sample in 4 ℃;
Earlier pcr amplification product is carried out electrophoresis detection, analytical results is taken a picture in the blob of viscose dyeing, the colour developing that electrophoresis are finished at last again.
Wherein, pcr amplification product is to be that 6% polyacrylamide gel electrophoresis detects with concentration in the step 5.
Wherein, the blob of viscose that in the step 5 electrophoresis is finished is 1% silver nitrate solution dyeing 20min through concentration, pass through developing solution color reaction identification band situation again, take a picture by the gel imaging instrument then, if the band of 400bp, by to amplified production order-checking, sequencing result sequence and the gene pool sequence submitted to are compared, judge whether to be the apple marssonina leaf spot gene order.
The invention has the beneficial effects as follows,, micro-thalline is directly added the method that the PCR reaction system replaces the dna profiling of extraction, just can reach direct amplification purpose the pathogenic bacteria target fragment by changing pcr amplification reaction system and amplification program.This technology has been avoided the other extraction to separation of pathogenic bacteria and DNA, has shortened experiment flow greatly, has improved detection efficiency, has saved cost.And directly utilize pathogenic bacteria on the incidence of leaf of field to amplify the method for germ target fragment, avoided the restriction that brings to practical study because of the pathogenic bacteria separation difficulty.In addition, utilize special primer that the apple marssonina leaf spot pathogenic bacteria is directly carried out pcr amplification, improved evaluation efficient greatly, also provide convenience for further studying its molecular biological characteristic to pathogenic bacteria.By custom-designed special primer, can finish the evaluation of apple blade brown spot pathogenic bacteria and Chinese rose, willow brown spot pathogenic bacteria and the common disease of apple is distinguished, and utilize micro-thalline rapid amplifying to go out the target fragment of marssonina coronaria.
Description of drawings
Fig. 1 be among the embodiment 1 pcr amplification product by the image behind the electrophoresis dying;
Fig. 2 be among the embodiment 2 pcr amplification product by the image behind the electrophoresis dying;
Fig. 3 be among the embodiment 3 pcr amplification product by the image behind the electrophoresis dying.
Embodiment
The present invention is described in detail below in conjunction with the drawings and specific embodiments.
Increase to Shaanxi Baishui County apple marssonina leaf spot collect specimen in July, 2007, and the concrete operations step is as follows:
Choose the orchard incidence of leaf, after cleaning is dried, use insect needle picking acervulus under Stereo microscope of the bacterium of going out also to go to standby respectively through the white scraps of paper of sterilising treatment;
10×Taq?Buffer 6μL
DNTP 0.2mM
P
1 1μM
P
2 1μM
Taq archaeal dna polymerase 1U
Mg
2+Concentration
1.75mM
DTT concentration 1.25mM
Adding the sterilization ultrapure water mends reaction system to 50 μ L;
Wherein, P
1And P
2Be sequence, utilize a pair of specific amplification primer of primer5.0 software design at this germ according to apple marssonina leaf spot pathogenic bacteria DNA,
Upstream primer P
1: 5 ˊ-TCCCTGTTGGTTTCTTTTCCTCC-3 ˊ,
Downstream primer P
2: 5 ˊ-ACTCTTTGTGTGAATAACGTGATGT-3 ˊ;
4 parts of PCR reaction systems are filled to respectively in 4 pcr amplification tubules, with the insect needle of the bacterium of going out the acervulus on the scraps of paper are gone to respectively wherein in 3 pcr amplification tubules, other 1 tubule and is carried out mark in contrast; Whenever guarantee 1～2 acervulus of card.With the PCR pipe that adds acervulus under the 12000rpm condition centrifugal 10 seconds, so that acervulus is immersed in the PCR reaction system fully;
Elder generation is 94 ℃ of pre-sex change 5min 1.;
2. 94 ℃ of pre-sex change 1min again,
3. 48 ℃ of sex change 1min,
4. 1min is extended in 72 ℃ of annealing,
Repeat 2., 3., 4. step is carried out 5 circulations;
5. 94 ℃ of pre-sex change 1min then,
6. 53 ℃ of sex change 1min,
7. 1min is extended in 72 ℃ of annealing,
Repeat 5., 6., 7. step is carried out 35 circulations;
Extend 8min at 72 ℃ at last, and preserve the amplification sample in 4 ℃.
Be that 6% polyacrylamide gel electrophoresis detects with pcr amplification product with 45mL concentration earlier, the blob of viscose that electrophoresis is finished is 1% silver nitrate solution dyeing 20min through concentration again, after blob of viscose cleaned twice with distilled water, put into the 400mL developing solution, color reaction identification band situation is taken a picture by the gel imaging instrument then, as shown in Figure 1: wherein, 1 road is Marker2000, and 2 roads are contrast, and 3,4,5 roads are for gathering different location apple marssonina leaf spot sample sample.
Wherein, 45mL concentration is that 6% polyacrylamide glue is prepared by following component: the 9mL volumetric concentration is 30% polyacrylamide, 10 * TBE Buffer electrophoretic buffer of 4.5mL, and the 0.6mL volumetric concentration is 10% ammonium persulphate, 0.055mL TEMEP, the ddH of 31.5mL
2O;
The 9mL volumetric concentration is that 30% polyacrylamide is that 29:1 prepares according to acrylamide and methylene diacrylamide according to volume ratio;
10 * TBE Buffer electrolytic buffer is by the Tris.base of 108.0g, the EDTA-Na of 9.3g
2, 55g boric acid fully dissolve and be settled to 1L in 4 ℃ of preservations.
The 400mL developing solution is the mixing solutions of NaOH and formaldehyde, and wherein the mass concentration of NaOH is 1.5%, and the volumetric concentration of formaldehyde is 0.2%, and solvent is a distilled water.
According to the augmentation detection result as can be known, an obvious master tape occurs, prove that the Auele Specific Primer of design has the amplification function to the apple marssonina leaf spot pathogenic bacteria, reach purpose fragment rapid amplifying purpose at 400bp.
The apple of in August, 2008 Shaanxi Yang Ling being gathered, Chinese rose, the amplification of willow brown spot germ, concrete steps are as follows:
Apple, Chinese rose, the willow incidence of leaf gathered after cleaning is dried, use insect needle picking acervulus under Stereo microscope of the bacterium of going out also to go to through the white scraps of paper of sterilising treatment standby respectively;
10×Taq?Buffer 6μL
DNTP 0.2mM
P
1 1μM
P
2 1μM
Taq archaeal dna polymerase 1U
Mg
2+Concentration
1.75mM
DTT concentration 1.25mM
Adding the sterilization ultrapure water mends reaction system to 50 μ L;
Wherein, P
1And P
2Be a pair of specific amplification primer according to the sequences Design of apple marssonina leaf spot pathogenic bacteria DNA, upstream primer P
1: 5 ˊ-TCCCTGTTGGTTTCTTTTCCTCC-3 ˊ, downstream primer P
2: 5 ˊ-ACTCTTTGTGTGAATAACGTGATGT-3 ˊ;
13 parts of PCR reaction systems are filled to respectively in 13 pcr amplification tubules; With the insect needle of the bacterium of going out the acervulus on the scraps of paper is gone to respectively wherein in 12 pcr amplification tubules, every kind of germ classification is established four repetitions, carries out mark; Other 1 tubule is carried out mark in contrast; Whenever guarantee 1～2 acervulus of card, with the PCR pipe that adds acervulus under the 12000rpm condition centrifugal 10 seconds, so that acervulus is immersed in the PCR reaction system fully.
Elder generation is 94 ℃ of pre-sex change 5min 1.;
2. 94 ℃ of pre-sex change 1min again,
3. 48 ℃ of sex change 1min,
4. 1min is extended in 72 ℃ of annealing,
Repeat 2., 3., 4. step is carried out 5 circulations;
5. 94 ℃ of pre-sex change 1min then,
6. 53 ℃ of sex change 1min,
7. 1min is extended in 72 ℃ of annealing,
Repeat 5., 6., 7. step is carried out 35 circulations;
Extend 8min at 72 ℃ at last, and preserve the amplification sample in 4 ℃.
Be that 6% polyacrylamide gel electrophoresis detects with pcr amplification product with 45mL concentration earlier, the blob of viscose that electrophoresis is finished is 1% silver nitrate solution, 20 min that dye through concentration again, pass through the 400mL developing solution again, color reaction identification band situation is taken a picture by the gel imaging instrument at last, and as shown in Figure 2: wherein 1 road is Marker2000,2 roads are contrast, 3-6 roads are apple marssonina leaf spot, and 7-10 roads are the willow brown spot, and 11-14 roads are the Chinese rose brown spot;
Wherein, 45mL concentration is that 6% polyacrylamide glue is prepared by following component: the 9mL volumetric concentration is 30% polyacrylamide, 10 * TBE Buffer electrophoretic buffer of 4.5mL, and the 0.6mL volumetric concentration is 10% ammonium persulphate, 0.055mL TEMEP, the ddH of 31.5mL
2O;
The 9mL volumetric concentration is that 30% polyacrylamide is that 29:1 prepares according to acrylamide and methylene diacrylamide according to volume ratio;
10 * TBE Buffer electrolytic buffer is by the Tris.base of 108.0g, the EDTA-Na of 9.3g
2, 55g boric acid fully dissolve and be settled to 1L in 4 ℃ of preservations.
The 400mL developing solution is the mixing solutions of NaOH and formaldehyde, and wherein the mass concentration of NaOH is 1.5%, and the volumetric concentration of formaldehyde is 0.2%, and solvent is a distilled water.
Conclusion: show by amplification to apple marssonina leaf spot, willow brown spot, Chinese rose brown spot germ, one obvious master tape appears at 400bp, the Auele Specific Primer of proof design can effectively increase to the apple marssonina leaf spot germ, reaches the purpose of differentiating fast and accurately with to the target fragment amplification.
Choose apple marssonina leaf spot, wheel line, anthrax, these apple common diseases of spot disease and fall ill that to make the more specific step of amplification as follows for bacterium:
Choose apple marssonina leaf spot blade, ring rot of apple evil, apple anthracnose disease, the common disease sample of these apples of alternaria leaf spot of apple of collection, use insect needle picking disease position thalline under Stereo microscope of the bacterium of going out also to go to standby respectively through the white scraps of paper of sterilising treatment.
10×Taq?Buffer 6μL
DNTP 0.2mM
P
1 1μM
P
2 1μM
Taq archaeal dna polymerase 1U
Mg
2+Concentration
1.75mM
DTT concentration 1.25mM
Adding the sterilization ultrapure water mends reaction system to 50 μ L;
Wherein, P
1And P
2Be a pair of specific amplification primer according to the sequences Design of apple marssonina leaf spot pathogenic bacteria DNA, upstream primer P
1: 5 ˊ-TCCCTGTTGGTTTCTTTTCCTCC-3 ˊ, downstream primer P
2: 5 ˊ-ACTCTTTGTGTGAATAACGTGATGT-3 ˊ;
13 parts of PCR reaction systems are filled to respectively in 13 pcr amplification tubules; With the insect needle of the bacterium of going out the acervulus on the scraps of paper is gone to respectively wherein in 12 pcr amplification tubules, every kind of germ classification is established three repetitions, carries out mark; Other 1 tubule is carried out mark in contrast; Whenever guarantee 1～2 acervulus of card, with the PCR pipe that adds acervulus under the 12000rpm condition centrifugal 10 seconds, so that acervulus is immersed in the PCR reaction system fully.
Elder generation is 94 ℃ of pre-sex change 5min 1.;
2. 94 ℃ of pre-sex change 1min again,
3. 48 ℃ of sex change 1min,
4. 1min is extended in 72 ℃ of annealing,
Repeat 2., 3., 4. step is carried out 5 circulations;
5. 94 ℃ of pre-sex change 1min then,
6. 53 ℃ of sex change 1min,
7. 1min is extended in 72 ℃ of annealing,
Repeat 5., 6., 7. step is carried out 35 circulations;
Extend 8min at 72 ℃ at last, and preserve the amplification sample in 4 ℃.
Be that 6% polyacrylamide gel electrophoresis detects with pcr amplification product with 45mL concentration earlier, the blob of viscose that electrophoresis is finished is 1% silver nitrate solution, 20 min that dye through concentration again, pass through 400mL developing solution color reaction identification band situation again, take a picture by the gel imaging instrument then, as shown in Figure 3: 1 road is Marker2000, and 2 roads are contrast, 3,4,5 roads are apple anthracnose, 6,7,8 roads are apple marssonina leaf spot, and 9,10,11 roads are ring rot of apple, and 12,13,14 roads are alternaria leaf spot of apple.
Wherein, 45mL concentration is that 6% polyacrylamide glue is prepared by following component: the 9mL volumetric concentration is 30% polyacrylamide, 10 * TBE Buffer electrophoretic buffer of 4.5mL, and the 0.6mL volumetric concentration is 10% ammonium persulphate, 0.055mL TEMEP, the ddH of 31.5mL
2O.
The 9mL volumetric concentration is that 30% polyacrylamide is that 29:1 prepares according to acrylamide and methylene diacrylamide according to volume ratio.
10 * TBE Buffer electrolytic buffer is by the Tris.base of 108.0g, the EDTA-Na of 9.3g
2, 55g boric acid fully dissolve and be settled to 1L in 4 ℃ of preservations.
The 400mL developing solution is the mixing solutions of NaOH and formaldehyde, and wherein the mass concentration of NaOH is 1.5%, and the volumetric concentration of formaldehyde is 0.2%, and solvent is a distilled water.
Conclusion: show by amplification, an obvious master tape occurs, prove that the Auele Specific Primer of design can effectively be distinguished apple marssonina leaf spot and other common diseases of apple, reach purpose to the target fragment rapid amplifying at 400bp to the common disease of apple.
SEQUENCE LISTING
＜110〉Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology
＜120〉be used to detect the specific amplification primer and the detection method of marssonina coronaria
＜130〉do not have
<160> 2
<170> PatentIn version?3.3
<210> 1
<211> 23
<212> DNA
＜213〉apple Marssonina (Marssonina coronaria)
<400> 1
tccctgttgg tttcttttcc tcc 23
<210> 2
<211> 25
<212> DNA
＜213〉apple Marssonina (Marssonina coronaria)
<400> 2
actctttgtg tgaataacgt gatgt 25
Claims (4)
1. specific amplification primer that is used to detect marssonina coronaria is characterized in that:
Upstream primer P
1: 5 ˊ-TCCCTGTTGGTTTCTTTTCCTCC-3 ˊ,
Downstream primer P
2: 5 ˊ-ACTCTTTGTGTGAATAACGTGATGT-3 ˊ.
2. one kind is utilized the described specific amplification primer of claim 1 to the method that marssonina coronaria detects, and it is characterized in that: comprise following operation steps:
Step 1,
Gather the field incidence of leaf, clean dry after, it is standby with the insect needle of the bacterium of going out the apple marssonina leaf spot acervulus to be placed the white scraps of paper;
Step 2, preparation PCR reaction system:
10×Taq?Buffer 6μL
DNTP 0.2mM
P
1 1μM
P
2 1μM
Taq archaeal dna polymerase 1U
Mg
2+Concentration
1.75mM
DTT concentration 1.25mM
Adding the sterilization ultrapure water mends reaction system to 50 μ L;
Wherein, P
1And P
2Be a pair of specific amplification primer according to the sequences Design of apple marssonina leaf spot pathogenic bacteria DNA, upstream primer P
1: 5 ˊ-TCCCTGTTGGTTTCTTTTCCTCC-3 ˊ, downstream primer P
2: 5 ˊ-ACTCTTTGTGTGAATAACGTGATGT-3 ˊ;
Step 3, the adding of template:
Insect needle with the bacterium of going out goes to the acervulus on the scraps of paper in the pcr amplification tubule that the PCR reaction system is housed, and whenever guarantees 1～2 acervulus of card; The PCR pipe that adds acervulus is centrifugal, acervulus is immersed in the PCR reaction system of preparation fully;
Step 4, carry out pcr amplification:
Response procedures is set to: 94 ℃ of pre-sex change 5min; Next 1min is extended in 94 ℃ of pre-sex change 1min, 48 ℃ of sex change 1min, 72 ℃ of annealing, so carries out 5 circulations; 1min is extended in 94 ℃ of pre-sex change 1min, 53 ℃ of sex change 1min, 72 ℃ of annealing then, so carries out 35 circulations; Extend 8min at 72 ℃ at last, and preserve the amplification sample in 4 ℃;
Step 5, the detection of pcr amplification product:
Earlier pcr amplification product is carried out electrophoresis detection, analytical results is taken a picture in the blob of viscose dyeing, the colour developing that electrophoresis are finished at last again.
3. detection method according to claim 2 is characterized in that: pcr amplification product is to be that 6% polyacrylamide gel electrophoresis detects with concentration in the described step 5.
4. detection method according to claim 2, it is characterized in that: the blob of viscose that in the described step 5 electrophoresis is finished is 1% silver nitrate solution dyeing 20min through concentration, pass through developing solution color reaction identification band situation again, take a picture by the gel imaging instrument then, if the band of 400bp, by to amplified production order-checking, sequencing result sequence and the gene pool sequence submitted to are compared, judge whether to be the apple marssonina leaf spot gene order.
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|---|---|---|---|
| CN2010101708438A CN101892308B (en) | 2010-05-13 | 2010-05-13 | Specific amplification primer for detecting marssonina coronaria and detection method |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106566868A (en) * | 2015-10-12 | 2017-04-19 | 李保华 | Method for rapidly detecting ascospores of marssonina mali |
| CN107541560A (en) * | 2017-10-31 | 2018-01-05 | 青岛农业大学 | Nucleic acid, kit and method for the Multiple detection of ulcer bacteria, brown patch germ and withes broom phytoplasma |
| CN111100947A (en) * | 2020-01-22 | 2020-05-05 | 青岛农业大学 | LAMP primer for rapidly detecting alternaria mali, detection method and kit thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101457259B (en) * | 2009-01-15 | 2011-12-28 | 中国检验检疫科学研究院 | Primer for detecting orchid bacterial speckle brown spot pathogen and detection method thereof |
-
2010
- 2010-05-13 CN CN2010101708438A patent/CN101892308B/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106566868A (en) * | 2015-10-12 | 2017-04-19 | 李保华 | Method for rapidly detecting ascospores of marssonina mali |
| CN107541560A (en) * | 2017-10-31 | 2018-01-05 | 青岛农业大学 | Nucleic acid, kit and method for the Multiple detection of ulcer bacteria, brown patch germ and withes broom phytoplasma |
| CN107541560B (en) * | 2017-10-31 | 2020-06-16 | 青岛农业大学 | Nucleic acid, kit and method for multiple detection of ulcer germs, brown spot germs and arbuscular disease phytoplasma |
| CN111100947A (en) * | 2020-01-22 | 2020-05-05 | 青岛农业大学 | LAMP primer for rapidly detecting alternaria mali, detection method and kit thereof |
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| CN101892308B (en) | 2012-09-26 |
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