CN104195254A - Method and primer composition for detecting fusarium equiseti based on loop-mediated isothermal amplification technology - Google Patents
Method and primer composition for detecting fusarium equiseti based on loop-mediated isothermal amplification technology Download PDFInfo
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- CN104195254A CN104195254A CN201410455196.3A CN201410455196A CN104195254A CN 104195254 A CN104195254 A CN 104195254A CN 201410455196 A CN201410455196 A CN 201410455196A CN 104195254 A CN104195254 A CN 104195254A
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Abstract
The invention discloses a method and primer composition for detecting fusarium equiseti based on a loop-mediated isothermal amplification technology. The LAMP primer composition for detecting soybean fusarium equiseti consists of a forward inner primer FIP shown in SEQ ID NO.2, a reverse inner primer BIP shown in SEQ ID NO.3, a forward outer primer F3 shown in SEQ ID NO.4, a reverse outer primer B3 shown in SEQ ID NO.5, a loop primer LF shown in SEQ ID NO.6 and a loop primer LB shown in SEQ ID NO.7. The primer composition disclosed by the invention can be applied to preparation of a LAMP detection reagent of soybean fusarium equiseti. The kit has the advantages of good specificity and sensitivity, rapid and high-efficiency amplification and easy and convenient identification operation. The detection system disclosed by the invention can rapidly, conveniently and efficiently detect the soybean fusarium equiseti with high specificity and high sensitivity under the isothermal condition of 62 DEG C.
Description
Technical field
The invention belongs to field of biological detection, relate to the method and the primer sets compound that detect scouring rush's Fusariumsp based on loop-mediated isothermal amplification technique.
Background technology
Soybean Fusarium is to be reported in the U.S. (1) in 1917 by Crommell the earliest, and main harm soybean root system, affects the absorption of crop to moisture and nutrient.The generation of this disease can make root activity, root weight, root nodule weight, nitrogenase activity and the lateral root number etc. of soybean plant strain all be subject to impact (23) in various degree.Disease can make the general underproduction 10%-30% of soybean according to investigations, when serious, can reach 60%.Existing known this disease is distributed in the states such as China, the U.S., India, Japan and Philippines, and (16,20,21,22) are the important diseases that has a strong impact on China northeast and Huang-Huai-Hai Soybean production, particularly serious at Sanjiang Plain in Heilongjiang Province.
Soybean Fusarium can be infected and be caused by multiple Fusariumsp, and the Fusariumsp that China has reported so far has Fusarium oxysporum (F.oxysporum), Fusarium solani (F.solani), scouring rush's Fusariumsp (F.equiseti), Fusarium graminearum (F.graninearum), oat Fusariumsp (F.aveneum), F.semitectum bacterium (F.semitectum) etc.(19) wherein for the Molecular Detection of soybean scouring rush Fusariumsp, we have carried out a series of research.
Traditional fungi strain authentication method, mainly taking morphology as foundation, is observed colonial morphology and individual cells microscopic features by after cultivating, and strain identification is arrived to guiding principle, order, section, genus and species.But the morphological specificity complexity of fungi, and some mushroom morphological specificity and physiological and biochemical property unstable along with the variation of environment, therefore, in traditional classification of fungi, often cause the inconsistent of categorizing system.Simultaneously low, the interference (2) that is subject to the factors such as artificial and environment of traditional classification authentication method length consuming time, sensitivity, can not make diagnosis in disease latent period and initial phase, is difficult to disease to monitor timely and effectively control.
Since the eighties in 20th century, along with the fast development of Protocols in Molecular Biology, the appearance of different kinds of molecules Biological assay, makes molecular biology play the part of more and more important role (3,5,9,15) in strain identification and classification of organisms research.Also make identifying more accurate, reliable in basic qualification taking morphological specificity and physiological and biochemical index of bacterial classification simultaneously.At present there is PCR for detection of the method for soybean scouring rush Fusariumsp, Real-time PCR, molecule marker etc., (8,10) although these methods in specificity and sensitivity, be greatly improved, but need to rely on accurate temperature cycling device, testing process complexity, can not meet the demand of rapid detection.
Loop-mediated isothermal amplification technique (Loop-mediated isothermal amplification, LAMP) be a kind of new nucleic acid amplification technologies (12) that Japanese Rong Yan strain formula can be invented, because it is simple to operate, quick, specificity is high, low cost and other advantages, become the new nucleic acid amplification technologies that can substitute regular-PCR.It is 6 species specific primers of zone design 4 for target gene, under the effect of Bst large fragment polysaccharase, cause self-circulation strand replacement reaction, in 60~65 DEG C of scope 60min, when synthesizing target dna in a large number, being attended by by product---the magnesium pyrophosphate precipitation of white produces.Because LAMP amplification procedure relies on 6 isolated areas of identification target sequence, so atopic is very strong, and amplification process is to carry out under constant temperature, ortho-water bath or have the equipment of stable thermal source just can meet to react requirement, testing cost reduces greatly.
In addition, common PCR reaction is carried out gel electrophoresis to product and is easy to cause product diffusion, and this is a main source of laboratory pollution; And ethidium bromide (EB) has huge poison, can accumulate carcinogenic; Long-term observation ultraviolet lamp also can cause injury to a certain degree to experimenter.And LAMP reaction only need be carried out in thermostat water bath, after reaction finishes, by adding SYBR Green I to observe just direct judged result of color and change in fluorescence, greatly reduce the injury to experimenter, and increased the using value in field.
Summary of the invention
Object of the present invention for the required cycle of biological detection method of soybean scouring rush Fusariumsp in prior art grow, waste time and energy, problem and PCR detection technique loaded down with trivial details, poor specificity need thermal cycling instrument, problem that cannot rapid detection soybean scouring rush Fusariumsp, provides the LAMP primer sets compound that detects soybean scouring rush Fusariumsp.
Another object of the present invention is to provide a kind of method that detects soybean scouring rush Fusariumsp.
Object of the present invention can be achieved through the following technical solutions:
Soybean scouring rush Fusariumsp CYP51c gene order shown in SEQ ID NO.1 detects the application in soybean scouring rush Fusariumsp as target at LAMP.
For detection of the LAMP primer sets compound of soybean scouring rush Fusariumsp, formed by the reverse outer primer B3 shown in the reverse inner primer BIP shown in the forward inner primer FIP shown in SEQ ID NO.2, SEQ ID NO.3, the forward outer primer F3 shown in SEQ ID NO.4, SEQ ID NO.5, the ring primer LB shown in the ring primer LF shown in SEQ ID NO.6, SEQ ID NO.7.
Primer sets compound of the present invention is in the application detecting in soybean scouring rush Fusariumsp.
The application of primer sets compound of the present invention in the LAMP detection reagent of preparation soybean scouring rush Fusariumsp.
Detect a LAMP test kit for soybean scouring rush Fusariumsp, contain primer sets compound of the present invention.
The preferred inclusion test solution of described test kit and dyestuff SYBR Green I; Wherein said detection solution is by 32mM forward inner primer FIP, the reverse inner primer BIP of 32mM, 8mM forward outer primer F3, the reverse outer primer B3 of 8mM, 8mM ring primer LF, 8mM ring primer LB, 56mM dNTPs, 0.8M Tris-HCl (pH8.8), 0.4mM KCl, 0.4mM (NH4)
2sO
4, 0.24mM MgSO
4, 4%Triton X-100, Bst DNA polymerase320 unit, add ultrapure water to prepare.
Detect a method for soybean scouring rush Fusariumsp, get microbial DNA to be checked, taking this DNA as template, utilize described primer sets compound to carry out LAMP; After amplified reaction finishes, add dyestuff SYBR Green I, under daylight, estimate or observe fluorescence in 245nm length ultraviolet rayed; With the colour-change of SYBR Green I and fluorescence power as result criterion: under daylight, range estimation is yellow-green colour and represents that detected result is positive, there is soybean scouring rush Fusariumsp, under daylight, range estimation is yellow and represents that detected result is negative, does not have soybean scouring rush Fusariumsp; Under UV-light, the green fluorescence of sending out strong represents test positive, has soybean scouring rush Fusariumsp, does not have fluorescence to represent to detect negative, does not have soybean scouring rush Fusariumsp (Fig. 1).
Wherein, LAMP response procedures is preferably: 62 DEG C, and 60min.
Beneficial effect
The selection of target gene is one of important factor of LAMP detection.The target gene that regular-PCR is conventional has Internal Transcribed Spacer (Internal transcribed space, ITS), but the differentiation fusarium fungi that this target can not be very clear and definite.The sterol 14 α-demethylase of CYP51 genes encoding is the Cytochrome P450 family member who distributes the widest, is the key enzyme in biosterin building-up process.In Human genome and plant pathogenic fungi, find the CYP51 gene of multiple copies, as contained two kinds of copies CYP51A, CYP51B in aspergillus tubigensis, rice blast fungus etc.Also there is in addition the third copy CYP51C, be found to be present in specifically in Fusariumsp kind gene.Contriver is in earlier stage also to find in the research of regular-PCR technology for detection soybean Fusarium oxysporum, CYP51C gene order high conservative between different strains in Fusarium kind, between kind, having abundant variation, is to compare rDNA-ITS, the better Molecular Detection target of β-tubulin sequence.The present invention has analyzed soybean scouring rush Fusariumsp CYP51C gene and the difference of other Fusariumsps in sequence, select wherein 6 specific regions, design four specific LAMP primers and two ring primers, successfully set up on this basis the LAMP system that detects soybean scouring rush Fusariumsp.
Compared with prior art, its advantage and positively effect show in the present invention:
(1) practicality is good.Common PCR reaction is carried out gel electrophoresis to product and is easy to cause product diffusion, and this is a main source of laboratory pollution; And ethidium bromide (EB) has huge poison, can accumulate carcinogenic; Long-term observation ultraviolet lamp also can cause injury to a certain degree to experimenter.And LAMP reaction only need be carried out in thermostat water bath, just direct judged result of the color by SYBR Green I after reaction finishes and change in fluorescence, thus increase its using value in field.
(2) constant-temperature amplification.Must thermal cycling unlike PCR method, so just break away from the dependence to thermal cycling instrument, as long as there is stable thermal source LAMP reaction just can occur, expand greatly the scope that LAMP uses, why LAMP can react under constant thermal source is because added trimethyl-glycine in LAMP reaction solution, make in the running balance of double-stranded DNA in unwinding, under the effect of BstDNA polysaccharase, realize amplification.
(3) accuracy is high.Because traditional soybean scouring rush Fusariumsp detection technique is just determined Quarantine Objects according to morphological specificity, the interference that authentication method length consuming time, sensitivity is low, be subject to the factors such as artificial and environment; And the present invention is according to the CYP51C sequence of soybean scouring rush Fusariumsp, very conservative in the genome of this sequence in soybean scouring rush Fusariumsp, the CYP51C sequence of the CYP51C sequence of soybean scouring rush Fusariumsp and other Fusariumsps is compared, choose the specific LAMP primer of the distinctive one section of CYP51C sequences Design of soybean scouring rush Fusariumsp.LAMP reaction is by 6 isolated areas on 4 primer specificity identification target sequences, and for 2 isolated areas of PCR primer identification target sequence, specificity and sensitivity are all higher.
Figure of description
Fig. 1 LAMP detects soybean scouring rush's Fusariumsp positive findings and negative findings schematic diagram.Wherein, A figure is visual observation figure, and B figure is observed result under UV-light; In every photos, the EP on the left side manages positive result, and the EP on the right manages negative result.
Fig. 2 LAMP detects the specificity of soybean scouring rush Fusariumsp
By the bacterial strain of 8 other kinds of fusarium is amounted to 180 bacterial strains, and and 11 other belong to pathogenic bacteria bacterial strains and amount to 76 bacterial strains and carried out LAMP amplification, specificity LAMP reaction can only produce yellowish green colour-change and produce green fluorescence (1-3: soybean scouring rush Fusariumsp bacterial strain in the F.eauiseti bacterial strain for examination; 4: layer goes out Fusariumsp; 5: Fusarium graminearum; 6: Fusarium oxysporum; 7: Fusarium solani; 8: avenge rotten Fusariumsp; 9: oat Fusariumsp; 10: Fusarium moniliforme; 11: yellow Fusariumsp; 12: aspergillus oryzae; 13: alternaric bacteria; 14: glue born of the same parents anthrax-bacilus; 15: tack anthrax-bacilus; 16: soybean rest fungus; 17: soybean is intended the swollen maize ear rot bacterium of stem point; 18: soyabean phytophthora; 19: soybean charcoal rot bacterium; 20: rice blast fungus; 21: oil bottle mould; 22: ball Tuber Melanosporum; 23-24: negative control.) upper two rows are for visual observation, lower two rows are observed result under UV-light.
Fig. 3 LAMP detects the sensitivity of soybean scouring rush Fusariumsp
LAMP amplification soybean scouring rush Fusariumsp different concns genomic dna; In the reaction system of 25 μ L, contain respectively the amplification of 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg DNA.
Color and fluorescence judge that LAMP detects the sensitivity colour developing figure of soybean scouring rush Fusariumsp bacterium.Positive reaction presents yellow-green colour and has strong green fluorescence, and negative control is yellow, not aobvious fluorescence.Result shows that the sensitivity of LAMP reaction reaches 10pg.First row is visual observation, and second row is observed result under UV-light.
Fig. 4 LAMP detects the soybean scouring rush Fusariumsp in disease plant
Get the soybean plants being infected by scouring rush's Fusariumsp and organize, extract genome, carry out LAMP amplification.Color and fluorescence judge that LAMP reaction can detect the scouring rush's Fusariumsp infecting in plant.(1-4: disease plant; 5: scouring rush's Fusariumsp genome; 6: healthy plant; 7: negative control.) first row is visual observation, second row is observed result under UV-light.
Embodiment
Embodiment 1 field gathers in plant detects soybean scouring rush Fusariumsp
Soybean scouring rush Fusariumsp detection kit, comprises following composition:
Four Auele Specific Primer FIP, BIP, F3, B3 and two ring primer LF, the LB of soybean scouring rush Fusariumsp LAMP Molecular Detection:
F3 (forward outer primer): GCGTACCCGGTACCGAAT (18bp, SEQ ID NO.4);
B3 (oppositely outer primer): GGACTGGTGACAGACTTGTT (20bp, SEQ ID NO.5);
FIP (forward inner primer) is (F1C+F2):
GGAGGGTCGAGGGAAGAACTCT-TAGTGCCTCCGTCCCATAC(41bp,SEQ?ID?NO.2);
BIP (oppositely inner primer) is (B1C+B2):
TGGGATCCTCATCGCTGGGA-CCGAAGCCATAATCCACAGT(40bp,SEQ?ID?NO.3)。
LF (ring primer): TGCCAGGAGATGCAAGAAGT (20bp, SEQ ID NO.6)
LB (ring primer): CGAGCCTCTTGAGAAGAACGCC (22bp, SEQ ID NO.7)
Test kit reaction system
1mL detects solution and comprises: 32mM forward inner primer FIP, the reverse inner primer BIP of 32mM, 8mM forward outer primer F3, the reverse outer primer B3 of 8mM, 8mM ring primer LF, 8mM ring primer LB, 56mM dNTPs, 0.8M Tris-HCl (pH 8.8), 0.4mM KCl, 0.4mM (NH4)
2sO
4, 0.24mM MgSO
4, 4%Triton X-100, Bst DNA polymerase320 unit, add ultrapure water to be prepared into 1mL and detect solution; Dyestuff SYBR Green I25 μ L.Storage life is 1 year.
Embodiment 2
In order to verify the specificity of LAMP method, select soybean scouring rush Fusariumsp bacterial strain, (layer goes out Fusariumsp to the Fusariumsp bacterial strain not of the same race with scouring rush's sickle spore; Fusarium graminearum; Fusarium oxysporum; Fusarium solani; Avenge rotten Fusariumsp; Oat Fusariumsp; Fusarium moniliforme; Yellow Fusariumsp), and the bacterial strain (aspergillus oryzae not belonging to together with soybean point sickle spore; Alternaric bacteria; Glue born of the same parents anthrax-bacilus; Tack anthrax-bacilus; Soybean rest fungus; Soybean is intended the swollen maize ear rot bacterium of stem point; Soyabean phytophthora; Soybean charcoal rot bacterium; Rice blast fungus; Oil bottle mould; Ball Tuber Melanosporum) DNA as template, get 4 μ l DNA solutions, utilize the detection kit of embodiment 1, carry out LAMP reaction, LAMP response procedures: 62 DEG C, 60min.After amplified reaction finishes, add dyestuff SYBR Green I, under daylight, estimate or observe fluorescence in 245nm length ultraviolet rayed; Result shows while removing to increase the DNA profiling of soybean scouring rush Fusariumsp with LAMP primer, produces yellowish green colour-change and generation green fluorescence; And other bacterial strain is the same with negative control, there is no colour-change or strong green fluorescence (Fig. 2).
Embodiment 3
In order to determine the sensitivity of LAMP detection method, using spectrophotometric determination concentration DNA for of the soybean scouring rush Fusariumsp bacterial strain extracting (1 μ g/ μ l) afterwards with DEPC water carry out 10 doubling dilution , ?70 DEG C of preservations as template.Get respectively each concentration DNA diluent 4 μ L after 10 doubling dilutions as template, by the method for embodiment 2, carry out LAMP reaction and result observation.Result shows that LAMP method can detect that concentration is the DNA (Fig. 3) of the soybean scouring rush Fusariumsp of 10pg
Embodiment 4 detects soybean scouring rush Fusariumsp from the inward soybean of customs carries disease germs soil sample:
Above-mentioned soybean scouring rush Fusariumsp detection kit, for detection of the method for soybean scouring rush Fusariumsp, comprising:
1) enrichment of oospore in soil:
Get 20 grams of pedotheques to be checked, grind, successively adopt the 200 larger grogs in eye mesh screen place to go, then filter through 400,500,800 eye mesh screens, repeatedly rinse with 3 premium on currency simultaneously, from 800 mesh sieve online collection oospore, use 1ml aqueous suspension.Because oospore can not see through 800 eye mesh screens, processing can reach the effect that makes oospore enrichment like this.
2) from micro-oospore, extract DNA:
The oospore suspending with sterilized water is transferred in the centrifuge tube of 1.5mL, under 12000r.min-1 rotating speed centrifugal 5 minutes, pouring liquids;
Add 50 μ L CTAB buffer, grind, then add 500 μ L CTAB buffer, water-bath 30 minutes;
Add the extracting of equal-volume chloroform, at 12000rmin
-1under rotating speed centrifugal 10 minutes, draw supernatant;
Add the 3M NaAc of 1/10 volume, 2 times of volumes without water-ice ethanol, precipitation at room temperature 30 minutes, 12000rmin
-1under rotating speed centrifugal 10 minutes, fall dry liquids;
Add 1mL70% (V/V) washing with alcohol, 12000rmin
-1under rotating speed centrifugal 10 minutes, fall dry liquids, dry to alcohol-free taste;
Add 10 μ L aseptic double-distilled waters and dissolve, for the template of LAMP amplification.
3) soybean scouring rush Fusariumsp LAMP detects, and comprising:
(1) LAMP of soybean scouring rush Fusariumsp detects: get 4 μ L DNA solutions, add 18 μ L test kit solution and 3 μ L sterilizing deionized waters, cumulative volume is 25 μ L;
(2) response procedures is: 62 DEG C, and 60min;
(3) detection of amplified production: after amplification, add 0.25 μ L dyestuff SYBR Green I as reaction indicator, visual inspection, and 245nm length ultraviolet rayed is observed fluorescence.With the colour-change of SYBR Green I and fluorescence power as result criterion.Under daylight, yellow-green colour represents test positive, has soybean scouring rush Fusariumsp, and yellow expression detected result is negative; Under UV-light, strong green fluorescence represents test positive, has soybean scouring rush Fusariumsp, does not have fluorescence to represent to detect negative.
Embodiment 5 identifies soybean scouring rush Fusariumsp from morbidity soyabean tissue
With after 70% alcohol disinfecting, adopt improved NaOH method to extract DNA at the soybean leaves being infected by pathogen or rhizome position.Get the plant tissue of one section of neopathy, every milligram of tissue adds 10 μ L 0.5mol/L NaOH, is transferred in the centrifuge tube of 1.5mL 12000rmin in mortar after fully grinding
-1centrifugal 5min under rotating speed, gets 5 μ L supernatant liquors and adds 495 μ L0.1mmol/L Tris (pH8.0), mixes rear absorption 4uL DNA solution, by the method for embodiment 2, carries out LAMP reaction, and with scouring rush's Fusariumsp genome, healthy plant; And blank feminine gender is for contrast, the results are shown in Figure 4, visible disease plant pipe is estimated as yellow-green colour under daylight, produces green fluorescence under UV-light, identical with the color of soybean scouring rush Fusariumsp genome pipe; Prove that the cause of disease detecting is soybean scouring rush Fusariumsp; Under daylight, range estimation is for yellow for healthy plant and blank negative control, and ultraviolet is administered without fluorescence.
Claims (8)
- Soybean scouring rush Fusariumsp CYP51c gene order shown in 1.SEQ ID NO.1 detects the application in soybean scouring rush Fusariumsp as target at LAMP.
- 2. for detection of the LAMP primer sets compound of soybean scouring rush Fusariumsp, it is characterized in that being formed by the reverse outer primer B3 shown in the reverse inner primer BIP shown in the forward inner primer FIP shown in SEQ ID NO.2, SEQ ID NO.3, the forward outer primer F3 shown in SEQ ID NO.4, SEQ ID NO.5, the ring primer LB shown in the ring primer LF shown in SEQ ID NO.6, SEQ ID NO.7.
- 3. primer sets compound claimed in claim 2 is in the application detecting in soybean scouring rush Fusariumsp.
- 4. the application of primer sets compound claimed in claim 2 in the LAMP detection reagent of preparation soybean scouring rush Fusariumsp.
- 5. detect a LAMP test kit for soybean scouring rush Fusariumsp, it is characterized in that containing primer sets compound claimed in claim 2.
- 6. the LAMP test kit of detection soybean scouring rush Fusariumsp according to claim 5, is characterized in that described test kit inclusion test solution and dyestuff SYBR Green I; Wherein said detection solution by: 32mM forward inner primer FIP, the reverse inner primer BIP of 32mM, 8mM forward outer primer F3, the reverse outer primer B3 of 8mM, 8mM ring primer LF, 8mM ring primer LB, 56mM dNTPs, 0.8M Tris ?HCl (pH8.8), 0.4mM KCl, 0.4mM (NH4) 2sO 4, 0.24mM MgSO 4, 4%Triton X ?100, Bst DNA polymerase320 unit/mL, add ultrapure water to prepare.
- 7. detect a method for soybean scouring rush Fusariumsp, it is characterized in that getting microbial DNA to be checked, taking this DNA as template, utilize the primer sets compound described in claim 2 to carry out LAMP; After amplified reaction finishes, add dyestuff SYBR Green I, under daylight, estimate or observe fluorescence in 245nm length ultraviolet rayed; With the colour-change of SYBR Green I and fluorescence power as result criterion: under daylight, range estimation is yellow-green colour and represents that detected result is positive, there is soybean scouring rush Fusariumsp, under daylight, range estimation is yellow and represents that detected result is negative, does not have soybean scouring rush Fusariumsp; Under UV-light, the green fluorescence of sending out strong represents test positive, has soybean scouring rush Fusariumsp, does not have fluorescence to represent to detect negative, does not have soybean scouring rush Fusariumsp.
- 8. the method for detection soybean scouring rush Fusariumsp according to claim 7, is characterized in that LAMP response procedures is: 62 DEG C, and 60min.
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CN105256049A (en) * | 2015-11-09 | 2016-01-20 | 南京农业大学 | Loop-mediated isothermal amplification primer combination for detecting yellow fusarium solani and application of loop-mediated isothermal amplification primer combination |
CN105256049B (en) * | 2015-11-09 | 2019-01-25 | 南京农业大学 | It is a kind of detect yellow Fusariumsp loop-mediated isothermal amplification (LAMP) primer composition and its application |
CN108315469A (en) * | 2018-04-09 | 2018-07-24 | 中国农业科学院农产品加工研究所 | Primer composition and kit of the pathogenic sickle-like bacteria of ring mediated isothermal amplification method detection and application thereof |
CN114045358A (en) * | 2021-10-15 | 2022-02-15 | 南京农业大学 | Primer composition for detecting twelve potato disease pathogenic bacteria based on loop-mediated isothermal amplification technology and detection method |
CN114045358B (en) * | 2021-10-15 | 2023-08-18 | 南京农业大学 | Primer composition for detecting twelve potato disease pathogenic bacteria based on loop-mediated isothermal amplification technology and detection method |
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