CN104962610A - LAMP rapid detecting method for Clavibacter michiganensis subsp.nebraskensis(Cmn) - Google Patents
LAMP rapid detecting method for Clavibacter michiganensis subsp.nebraskensis(Cmn) Download PDFInfo
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Abstract
The invention relates to a LAMP rapid detecting method for Clavibacter michiganensis subsp.nebraskensis(Cmn). An LAMP primer is designed through the 16S-23S sequences of the Cmn issued by the GenBank, an LAMP detecting system for the Cmn is built, and reaction conditions are optimized. Genomes of the Cmn and bacterium suspension gradient diluents serve as templates to detect the sensitivity of the LAMP system; and results show that the detection sensitivity of the LAMP detecting system on the genomes and bacterium suspensions reach 50 fg and 3.8 CFU/mL respectively, and the detection sensitivity is 100 times higher than the common PCR sensitivity. Results show that the LAMP detecting system is simple and rapid, and is high in sensitivity and good in specificity, and the LAMP rapid detecting method has the huge potential on detection of the Cmn.
Description
Technical field
The present invention relates to agricultural and technical field of plant quarantine, be specifically related to state wilting germ LAMP method for quick in a kind of corn.
Background technology
State wilt disease in corn (Goss ' s Bacterial Wilt and Leaf Blight of Corn) be by holding peace clavibacter Nebraska subspecies (Clavibacter michiganensis subsp.nebraskensis, Cmn) a kind of serious bacterial disease caused, can cause corn yield rate of loss up to 50%.Since Late Cambrian in 1969 is in the middle part of the Nebraska State, this disease passes band by seed and carries out long-distance communications
[3], diffused to the many states of the U.S. and Canadian some areas at present, not yet found in China.Corn is in the sown area of China and output Jun Ju second place of the world, and China's major part corn-growing regions is applicable to generation and the propagation of Cmn, this germ is once import into, by serious threat China corn grain production safety, therefore state wilt disease in prevention and corntrol corn, to guaranteeing that Maize Production Sustainable development has very important meaning.
Cmn is Gram-positive, does not produce spore, and not solid acid, strict aerobic, is under the jurisdiction of Firmicutes (Firmicutes), heavy wall Gammaproteobacteria (Firmibacteria), rod type Bacillaceae (Clavibacter).The methods such as isolated culture, Serologic detection and molecular Biological Detection are mainly contained at present for its detection method, isolated culture and Serology test complicated operation, consuming time longer and sensitivity is lower is not suitable for rapid detection.Molecular biology for detection possesses quick, accurate and sensitive feature, but its testing process needs the equipment supports such as PCR instrument, cannot realize execute-in-place.
In corn, wilting germ Cmn in state can endanger dent corn, sweet corn, flint corn, quick-fried corn, Herba Setariae Viridis and sugarcane.Cmn can with seed dispersal (Biddle et al.1990), and China's major part corn-growing regions is applicable to disease and occurs.Germ once import into grows surely, easily break out and cause plant disease epidemic, and disease control difficulty is large.China's corn yield and cultivated area all occupy the second in the world, in national economy, occupy critical role.2011, only the annual import volume of fodder maize China reached millions of tons.Therefore, germ imports China into and the potential risk of diffusive transport is huge.At present, the domestic report that there is not yet Cmn detection in inward corn sample, the micro-Pathogen detection method of accurate Fast Practical has very important potential using value.
Summary of the invention
The object of the invention is in order to solve existing cannot realize state wilting germ in execute-in-place, quick, accurate, sensitive detection corn defect and provide a kind of and facilitate wilting germ LAMP method for quick in state in execute-in-place, quick, accurate, sensitive corn.
To achieve these goals, the present invention is by the following technical solutions:
State wilting germ LAMP method for quick in a kind of corn, comprises the steps:
A) design primer, primer sequence is:
F3 forward outer primer: 5 '-CGCTCATGGGTGGAACAT-3 ' (SEQ ID NO.1);
The reverse outer primer of B3: 5 '-GTTCTCAATATACGGGCGGT-3 ' (SEQ ID NO.2);
FIP forward inner primer: 5 '-CATGCCCTCGACACACCAGACTGATGTGTCGGGCTGCTA-3 ' (SEQ ID NO.3);
The reverse inner primer of BIP: 5 '-CGCTGTTGGGTCCTGAGGGAACGAAAGGACGACGGAAAG-3 ' (SEQ ID NO.4);
LF forward cyclisation primer: 5 '-ACCCCACAAGGAGGCGTA-3 ' (SEQ ID NO.5);
The reverse cyclisation primer of LB: 5 '-GCACCTTCGGGTGTGTCTG-3 ' (SEQ ID NO.6);
B) yeast culture and the preparation of DNA masterplate: the activation culture LB solid medium of thalline, put into 25 DEG C of constant incubators after coated plate and cultivate 24h, rear picking list bacterium colony enters LB nutrient solution 25 DEG C and cultivates 12h (OD600=0.8), collected by centrifugation thalline, TIANGEN bacterial genomes DNA extraction kit is utilized to extract bacterial genomes, ultraviolet spectrophotometer Nanodrop 2000 Detection and Extraction quality and concentration ,-20 DEG C are for subsequent use;
C) LAMP amplification: genome step b) obtained carries out LAMP amplification, and it is 61 DEG C, 62 DEG C, 63 DEG C, 64 DEG C or 65 DEG C that LAMP system arranges temperature of reaction gradient; Inner primer, cyclisation primer and outer primer ratio are set and are respectively 2:2:1 (10pmol:10pmol:5pmol), 4:4:1 (20pmol:20pmol:5pmol), 6:4:1 (30pmol:20pmol:5pmol) or 8:4:1 (40pmol:20pmol:5pmol); Arranging reaction times gradient is 15min, 30min, 45min or 60min;
D) analyze: expansion product step c) obtained carries out estimating with negative control or in 2.0% agarose gel electrophoresis analysis.
As preferably, step c) gradient ratio of inner primer, cyclisation primer and outer primer is 6:4:1 or 8:4:1, the amplified reaction time is 15min or 30min, LAMP amplified reaction temperature is 62 DEG C, 63 DEG C or 64 DEG C.
As preferably, step c) gradient ratio of inner primer, cyclisation primer and outer primer is 6:4:1, the amplified reaction time is 30min, LAMP amplified reaction temperature is 64 DEG C.
As preferably, described thalline is state wilting germ in corn.
The invention has the beneficial effects as follows: wilting germ (Clavibactermichiganensis subsp.nebraskensis in state in the corn that the present invention utilizes GenBank to announce, Cmn) 16S-23S sequences Design LAMP (loop-mediated isothermalamplification) primer, establish state wilting germ LAMP detection system in corn, and reaction conditions is optimized.LAMP 64 DEG C of reaction 30min under inner primer, cyclisation primer and outer primer are than the 25 μ L systems for 6:4:1 (30pmol:20pmol:5pmol) are optimum reaction condition, and LAMP primer only can go out Cmn gradient band by specific amplification from 7 kinds of bacterial strains for examination.With the sensitivity that wilting germ genome in state in corn and bacteria suspension gradient dilution liquid are masterplate detection LAMP system, result shows, LAMP detection system reaches 50fg and 3.8CFU/mL respectively for the sensitivity of genome and bacteria suspension, more highly sensitive than regular-PCR 100 times.Result shows, LAMP detection system is simple, quick, highly sensitive, specificity is good, and in corn, state wilting germ context of detection has a high potential.
Accompanying drawing explanation
Fig. 1 is LAMP specificity experiments electrophorogram of the present invention.
In figure, M:DL2000; 1: blank; 2-8 representative strain genome is respectively Cmn, Pss, Aaa, Aac, Xo, Pa, Pas.
Fig. 2 is Cmn thalline LAMP sensitivity experiments electrophorogram of the present invention.
Fig. 3 is Cmn thalline regular-PCR sensitivity experiments electrophorogram.
In figure, M:DL2000; 1: blank; 2 to represent Cmn thalline masterplate concentration be 3.6 × 10
6cFU/ml, 3-8 represent Cmn thalline masterplate concentration dilution multiple respectively, 10 times, and 10
2doubly, 10
3doubly, 10
4doubly, 10
5doubly, 10
6doubly.
Embodiment
Below by specific embodiment, technical scheme of the present invention is described in further detail.Should be appreciated that embodiments of the invention are not limited to the following examples, any pro forma accommodation make the present invention and/or change all will fall into scope.
Method in following embodiment, if no special instructions, is the ordinary method of this area.
Wilting germ (Clavibacter michiganensis subsp.nebraskensis in state in the bacterial strain corn used in the present invention, Cmn), P.stwartii subsp.stewartii (Pantoea stewartii subsp.Stewartii, Pss) is provided by Zhejiang Entry-Exit Inspection and Quarantine Bureau doctor Zhang Mingzhe; Oat acidovorax avenae (Acidovorax avenae subsp.Avenae, Aaa), Acidovorax Avenae Subsp (Acidovorax avenae subsp.citrull, Aac), Xanthomonas campestris (Xanthomonasoryzae, Xo) is provided by Zhejiang University doctor Li Bin; The general bacterium of pineapple (Pantoea ananatis, Pa), Chinese sorghum pseudomonas (Pseudomonas andropogonis (Smith) stapp, Pas) is preserved by complex art service centre of Zhoushan Entry-Exit Inspection and Quarantine Bureau laboratory.
LAMP method thymus nucleic acid amplification kit (Rong Yan), bacterial genomes DNA extraction kit (TIANGEN), DL 2000DNA Ladder Marker (TaKaRa), agarose Agarose H (the raw work in Shanghai), LB agar (Hangzhou microorganism reagent company limited).
Embodiment 1
State wilting germ LAMP method for quick in a kind of corn, comprises the steps:
A) design primer, primer sequence is:
F3 forward outer primer: 5 '-CGCTCATGGGTGGAACAT-3 ' (SEQ ID NO.1);
The reverse outer primer of B3: 5 '-GTTCTCAATATACGGGCGGT-3 ' (SEQ ID NO.2);
FIP forward inner primer: 5 '-CATGCCCTCGACACACCAGACTGATGTGTCGGGCTGCTA-3 ' (SEQ ID NO.3);
The reverse inner primer of BIP: 5 '-CGCTGTTGGGTCCTGAGGGAACGAAAGGACGACGGAAAG-3 ' (SEQ ID NO.4);
LF forward cyclisation primer: 5 '-ACCCCACAAGGAGGCGTA-3 ' (SEQ ID NO.5);
The reverse cyclisation primer of LB: 5 '-GCACCTTCGGGTGTGTCTG-3 ' (SEQ ID NO.6);
B) yeast culture and the preparation of DNA masterplate: the activation culture of state wilting germ LB solid medium in corn, put into 25 DEG C of constant incubators after coated plate and cultivate 24h, rear picking list bacterium colony enters LB nutrient solution 25 DEG C and cultivates 12h (OD600=0.8), collected by centrifugation thalline, TIANGEN bacterial genomes DNA extraction kit is utilized to extract bacterial genomes, ultraviolet spectrophotometer Nanodrop 2000 Detection and Extraction quality and concentration ,-20 DEG C are for subsequent use;
C) LAMP amplification: genome step b) obtained carries out LAMP amplification, and it is 64 DEG C that LAMP system arranges temperature of reaction gradient; Inner primer, cyclisation primer and outer primer ratio are set and are respectively 6:4:1 (30pmol:20pmol:5pmol); Arranging reaction times gradient is 30min;
D) analyze: expansion product step c) obtained is in 2.0% agarose gel electrophoresis analysis.
Embodiment 2
State wilting germ LAMP method for quick in a kind of corn, comprises the steps:
A) design primer, primer sequence is:
F3 forward outer primer: 5 '-CGCTCATGGGTGGAACAT-3 ' (SEQ ID NO.1);
The reverse outer primer of B3: 5 '-GTTCTCAATATACGGGCGGT-3 ' (SEQ ID NO.2);
FIP forward inner primer: 5 '-CATGCCCTCGACACACCAGACTGATGTGTCGGGCTGCTA-3 ' (SEQ ID NO.3);
The reverse inner primer of BIP: 5 '-CGCTGTTGGGTCCTGAGGGAACGAAAGGACGACGGAAAG-3 ' (SEQ ID NO.4);
LF forward cyclisation primer: 5 '-ACCCCACAAGGAGGCGTA-3 ' (SEQ ID NO.5);
The reverse cyclisation primer of LB: 5 '-GCACCTTCGGGTGTGTCTG-3 ' (SEQ ID NO.6);
B) yeast culture and the preparation of DNA masterplate: the activation culture of state wilting germ LB solid medium in corn, put into 25 DEG C of constant incubators after coated plate and cultivate 24h, rear picking list bacterium colony enters LB nutrient solution 25 DEG C and cultivates 12h (OD600=0.8), collected by centrifugation thalline, TIANGEN bacterial genomes DNA extraction kit is utilized to extract bacterial genomes, ultraviolet spectrophotometer Nanodrop 2000 Detection and Extraction quality and concentration ,-20 DEG C are for subsequent use;
C) LAMP amplification: genome step b) obtained carries out LAMP amplification, and it is 62 DEG C that LAMP system arranges temperature of reaction gradient; Inner primer, cyclisation primer and outer primer ratio are set and are respectively 8:4:1 (40pmol:20pmol:5pmol); Arranging reaction times gradient is 15min;
D) analyze: expansion product step c) obtained is in 2.0% agarose gel electrophoresis analysis.
Embodiment 3
State wilting germ LAMP method for quick in a kind of corn, comprises the steps:
A) design primer, primer sequence is:
F3 forward outer primer: 5 '-CGCTCATGGGTGGAACAT-3 ' (SEQ ID NO.1);
The reverse outer primer of B3: 5 '-GTTCTCAATATACGGGCGGT-3 ' (SEQ ID NO.2);
FIP forward inner primer: 5 '-CATGCCCTCGACACACCAGACTGATGTGTCGGGCTGCTA-3 ' (SEQ ID NO.3);
The reverse inner primer of BIP: 5 '-CGCTGTTGGGTCCTGAGGGAACGAAAGGACGACGGAAAG-3 ' (SEQ ID NO.4);
LF forward cyclisation primer: 5 '-ACCCCACAAGGAGGCGTA-3 ' (SEQ ID NO.5);
The reverse cyclisation primer of LB: 5 '-GCACCTTCGGGTGTGTCTG-3 ' (SEQ ID NO.6);
B) yeast culture and the preparation of DNA masterplate: the activation culture of state wilting germ LB solid medium in corn, put into 25 DEG C of constant incubators after coated plate and cultivate 24h, rear picking list bacterium colony enters LB nutrient solution 25 DEG C and cultivates 12h (OD600=0.8), collected by centrifugation thalline, TIANGEN bacterial genomes DNA extraction kit is utilized to extract bacterial genomes, ultraviolet spectrophotometer Nanodrop 2000 Detection and Extraction quality and concentration ,-20 DEG C are for subsequent use;
C) LAMP amplification: genome step b) obtained carries out LAMP amplification, and it is 63 DEG C that LAMP system arranges temperature of reaction gradient; Inner primer, cyclisation primer and outer primer ratio are set and are respectively 2:2:1 (10pmol:10pmol:5pmol); Arranging reaction times gradient is 45min;
D) analyze: expansion product step c) obtained is in 2.0% agarose gel electrophoresis analysis.
Specificity verification: utilize TIANGEN bacterial genomes to extract the DNA of state wilting germ, reference strains in test kit extraction corn, ultraviolet spectrophotometer Nanodrop 2000 verifies extraction effect, getting 2ul is that masterplate carries out LAMP amplified reaction and pcr amplification reaction respectively, the specificity of checking LAMP optimization system.Glue figure is run as can be seen from electrophoresis, the sample liquid gel imaging only carrying out LAMP amplification with state wilting germ in corn for masterplate runs out of gradient band clearly, and gradient band (Fig. 2) does not all appear in other LAMP amplifications being masterplate with reference strains DNA.Result shows, the LAMP detection method high specificity for state wilting germ in corn that the present invention sets up.
Reference strains is P.stwartii subsp.stewartii (Pantoea stewartii subsp.Stewartii, Pss), oat acidovorax avenae (Acidovorax avenae subsp.Avenae, Aaa), Acidovorax Avenae Subsp (Acidovorax avenaesubsp.citrull, Aac), Xanthomonas campestris (Xanthomonas oryzae, Xo), the general bacterium of pineapple (Pantoea ananatis, Pa), Chinese sorghum pseudomonas (Pseudomonas andropogonis (Smith) stapp, Pas).
Sensitivity technique: get state wilting germ bacteria suspension stoste in 100ul corn, carry out 10 times of gradient dilutions with distilled water, it is 3.6 × 10 that coated plate measures state wilting germ bacteria suspension concentration in corn
6cFU/ml, getting 1 μ L diluent is respectively that masterplate carries out LAMP amplification and regular-PCR amplified reaction, and checking LAMP optimization system is for the susceptibility of thalline.Can find out that LAMP detection system detection sensitivity can reach 3.6CFU/mL by LAMP result Fig. 2, and the detection sensitivity of Fig. 3 regular-PCR is 3.6 × 10
2cFU/mL.Contrast LAMP system and regular-PCR system gel imaging result show, highly sensitive 100 times of the remolding sensitivity regular-PCR of LAMP detection system.
The output of China's corn and cultivated area Jun Ju second place of the world, corn occupies consequence in China's grain-production.From 2010, there is corn net importation situation in China, 2012 to 2014, and within 3 years, corn import total amount is more than 1,000 ten thousand tons.The risk that in corn, state wilting germ imports China into import corn is very high.The combination of isolated culture, euzymelinked immunosorbent assay (ELISA), Standard PCR, TagMan-PCR, Chao Shi PCR, REP-PCR genetic fingerprint technology and two or more method is mainly contained at present for the quarantine method of state wilting germ in corn, various method has its relative merits, the weak point of isolated culture is complicated operation, consuming time longer.Euzymelinked immunosorbent assay (ELISA) is mainly limited high with its insufficient sensitivity, and specificity is directly related with Dispersal risk.Standard PCR and its detection sensitivity of Chao Shi PCR, TagMan-PCR, REP-PCR genetic fingerprint technology developed thus improve many, but its operating process needs special instrument and consuming time longer, cannot realize that basic unit health officer is on-the-spot, the requirement of rapid quarantine.And antibody prepared by its specificity affects.Standard PCR and fluorescence quantitative PCR detection improve the sensitivity of detection, but they all need special equipment, for mechanism's particularly basic unit's inspection and quarantine department that some experiment conditions are limited, are unfavorable for promoting.The present invention adopts loop-mediated isothermal amplification technique (LAMP), effectively can overcome the deficiency of above method, its reaction process only needs the support of the small-sized equipment of metal bath, and result do not need by the equipment such as electrophoresis apparatus can directly visual or add fluorescence dye uv irradiating observe, this effectively overcomes the restriction of experimental site; Result shows, LAMP reacts thalline detection sensitivity up to 3.6CFU, and Site Detection can directly drench to be got bacterium liquid and test, and it is not high that this just effectively makes up other molecular biology experiment thalline sensitivity, genomic dna need be extracted detect, decrease the complicacy of operation; LAMP detection reaction time shorten was by 30 minutes simultaneously, which greatly enhances the efficiency of detection.Certainly, LAMP technology also has some self problem.First, design of primers is complicated, LAMP reaction comprise 3 pairs of primers (inner primer, outer primer, cyclisation primer) and in a lot of situation cyclisation design of primers unsuccessful.Secondly, amplified production is not easily verified, a series of gradient band appears in LAMP amplification, unless at drone design a pair specific cleavage site, otherwise cannot verify that whether its extension increasing sequence is consistent with target sequence.
SEQUENCE LISTING
<110> Zhoushan complex art service centre of Entry-Exit Inspection and Quarantine Bureau
State wilting germ LAMP method for quick in <120> corn
<130> ZH10012
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213> artificial sequence
<400> 1
cgctcatggg tggaacat 18
<210> 2
<211> 20
<212> DNA
<213> artificial sequence
<400> 2
gttctcaata tacgggcggt 20
<210> 3
<211> 39
<212> DNA
<213> artificial sequence
<400> 3
catgccctcg acacaccaga ctgatgtgtc gggctgcta 39
<210> 4
<211> 39
<212> DNA
<213> artificial sequence
<400> 4
cgctgttggg tcctgaggga acgaaaggac gacggaaag 39
<210> 5
<211> 18
<212> DNA
<213> artificial sequence
<400> 5
accccacaag gaggcgta 18
<210> 6
<211> 19
<212> DNA
<213> artificial sequence
<400> 6
gcaccttcgg gtgtgtctg 19
Claims (4)
1. a state wilting germ LAMP method for quick in corn, is characterized in that, comprise the steps:
A) design primer, primer sequence is:
F3 forward outer primer: 5 '-CGCTCATGGGTGGAACAT-3 ' (SEQ ID NO.1);
The reverse outer primer of B3: 5 '-GTTCTCAATATACGGGCGGT-3 ' (SEQ ID NO.2);
FIP forward inner primer: 5 '-CATGCCCTCGACACACCAGACTGATGTGTCGGGCTGCTA-3 ' (SEQ ID NO.3);
The reverse inner primer of BIP: 5 '-CGCTGTTGGGTCCTGAGGGAACGAAAGGACGACGGAAAG-3 ' (SEQ ID NO.4);
LF forward cyclisation primer: 5 '-ACCCCACAAGGAGGCGTA-3 ' (SEQ ID NO.5);
The reverse cyclisation primer of LB: 5 '-GCACCTTCGGGTGTGTCTG-3 ' (SEQ ID NO.6);
B) yeast culture and the preparation of DNA masterplate: the activation culture LB solid medium of thalline, put into 25 DEG C of constant incubators after coated plate and cultivate 24h, rear picking list bacterium colony enters LB nutrient solution 25 DEG C and cultivates 12h (OD600=0.8), collected by centrifugation thalline, TIANGEN bacterial genomes DNA extraction kit is utilized to extract bacterial genomes, ultraviolet spectrophotometer Nanodrop 2000 Detection and Extraction quality and concentration ,-20 DEG C are for subsequent use;
C) LAMP amplification: genome step b) obtained carries out LAMP amplification, and it is 61 DEG C, 62 DEG C, 63 DEG C, 64 DEG C or 65 DEG C that LAMP system arranges temperature of reaction gradient; Inner primer, cyclisation primer and outer primer ratio are set and are respectively 2:2:1 (10pmol:10pmol:5pmol), 4:4:1 (20pmol:20pmol:5pmol), 6:4:1 (30pmol:20pmol:5pmol) or 8:4:1 (40pmol:20pmol:5pmol); Arranging reaction times gradient is 15min, 30min, 45min or 60min;
D) analyze: expansion product step c) obtained carries out estimating with negative control or in 2.0% agarose gel electrophoresis analysis.
2. state wilting germ LAMP method for quick in a kind of corn according to claim 1, it is characterized in that, step c) gradient ratio of inner primer, cyclisation primer and outer primer is 6:4:1 or 8:4:1, the amplified reaction time is 15min or 30min, LAMP amplified reaction temperature is 62 DEG C, 63 DEG C or 64 DEG C.
3. state wilting germ LAMP method for quick in a kind of corn according to claim 2, it is characterized in that, step c) gradient ratio of inner primer, cyclisation primer and outer primer is 6:4:1, the amplified reaction time is 30min, LAMP amplified reaction temperature is 64 DEG C.
4. state wilting germ LAMP method for quick in a kind of corn according to claim 1, is characterized in that, described thalline is state wilting germ in corn.
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| CN112662794A (en) * | 2021-01-20 | 2021-04-16 | 舟山海关综合技术服务中心 | Fluorescence recombinase mediated isothermal amplification detection kit for wilting bacteria in China in corn |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102816835A (en) * | 2012-05-04 | 2012-12-12 | 中华人民共和国上海出入境检验检疫局 | Clavibacter michiganensis subsp.nebraskensis(Cmn) PCR detection method, and detection primer |
| CN104372109A (en) * | 2014-11-12 | 2015-02-25 | 中华人民共和国北仑出入境检验检疫局 | Loop-mediated isothermal amplification detection primer group, loop-mediated isothermal amplification detection kit and loop-mediated isothermal amplification detection method for maize chlorotic dwarf virus |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102816835A (en) * | 2012-05-04 | 2012-12-12 | 中华人民共和国上海出入境检验检疫局 | Clavibacter michiganensis subsp.nebraskensis(Cmn) PCR detection method, and detection primer |
| CN104372109A (en) * | 2014-11-12 | 2015-02-25 | 中华人民共和国北仑出入境检验检疫局 | Loop-mediated isothermal amplification detection primer group, loop-mediated isothermal amplification detection kit and loop-mediated isothermal amplification detection method for maize chlorotic dwarf virus |
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| NOTOMI T ET.AL，: "loop-mediated isothermal amplification of DNA", 《NUCLEIC ACIDS RESEARCH》 * |
Cited By (1)
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| CN112662794A (en) * | 2021-01-20 | 2021-04-16 | 舟山海关综合技术服务中心 | Fluorescence recombinase mediated isothermal amplification detection kit for wilting bacteria in China in corn |
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