CN104962610B - Wilting germ LAMP quick determination methods in state in a kind of corn - Google Patents
Wilting germ LAMP quick determination methods in state in a kind of corn Download PDFInfo
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Abstract
The present invention relates to wilting germ LAMP quick determination methods in state in a kind of corn, state wilting germ 16S 23S sequences Design LAMP primers in the corn announced using GenBank, wilting germ LAMP detection architectures in state in corn are established, and reaction condition is optimized.The sensitivity of LAMP systems is detected using wilting germ genome in state in corn and bacteria suspension gradient dilution liquid as masterplate, as a result show, LAMP detection architectures have respectively reached 50fg and 3.8CFU/mL for the sensitivity of genome and bacteria suspension, than 100 times of regular-PCR high sensitivity.As a result show, LAMP detection architectures are simple, quick, high sensitivity, specificity are good, and state wilting germ context of detection has a high potential in corn.
Description
Technical field
The present invention relates to agricultural and technical field of plant quarantine, and in particular to state wilting germ LAMP is quick in a kind of corn
Detection method.
Background technology
State wilt disease (Goss ' s Bacterial Wilt and Leaf Blight of Corn) is by holding peace in corn
Clavibacter Nebraska subspecies (Clavibacter michiganensis subsp.nebraskensis, Cmn) cause
A kind of serious bacterial disease, corn yield loss late can be caused to be up to 50%.Interior cloth was found in first since 1969
In the middle part of the California of Lars, the disease passes band by seed and carries out long-distance communications[3], the more states in the U.S. and Canadian portion have been diffused at present
Region-by-region, not yet found in China.Sown area and yield Jun Ju second place of the world of the corn in China, and China is most of
Corn-growing regions are adapted to Cmn generation and propagation, and once the germ is incoming, by serious threat China corn grain production safety,
Therefore prevent and control state wilt disease in corn, to ensuring that Maize Production sustainable development has particularly important meaning.
Cmn is Gram-positive, does not produce spore, not solid acid, strict aerobic, is under the jurisdiction of Firmicutes (Firmicutes),
Heavy wall Gammaproteobacteria (Firmibacteria), rod type Bacillus (Clavibacter).Mainly there is separation currently for its detection method
The methods of cultivation, Serologic detection and molecular Biological Detection, isolated culture and Serology test complex operation, consumption
When the relatively low unsuitable quick detection of longer and sensitivity.Molecular biology for detection possesses the characteristics of quick, accurate and sensitive,
But its detection process needs the equipment such as PCR instrument to support, can not realize execute-in-place.
Wilting germ Cmn in state can endanger dent corn, corn, flint corn, quick-fried corn, green bristlegrass and sweet in corn
Sugarcane.Cmn can be with seed dispersal (Biddle et al.1990), and China major part corn-growing regions are adapted to disease.Disease
Bacterium easily breaks out once incoming colonize and causes plant disease epidemic, and disease control difficulty is big.China's corn yield and cultivated area are equal
The second in the world is occupied, critical role is occupied in national economy.2011, only the annual import volume in fodder maize China was up to millions of
Ton.Therefore, germ is passed to China and the potential risk of diffusive transport is huge.At present, it is domestic there is not yet Cmn in the corn sample that enters the territory
The report of detection, the micro Pathogen detection method of accurate Fast Practical have highly important potential using value.
The content of the invention
It is an object of the invention in order to solve it is existing can not realize execute-in-place, it is quick, accurate, sensitively detect corn
The defects of interior state wilting germ and provide it is a kind of facilitate execute-in-place, state wilting germ LAMP in quick, accurate, sensitive corn
Quick determination method.
To achieve these goals, the present invention uses following technical scheme：
State wilting germ LAMP quick determination methods, comprise the following steps in a kind of corn：
A) primer is designed, primer sequence is：
F3 forward direction outer primers：5’-CGCTCATGGGTGGAACAT-3’(SEQ ID NO.1)；
The reverse outer primers of B3：5’-GTTCTCAATATACGGGCGGT-3’(SEQ ID NO.2)；
FIP forward direction inner primers：5’-CATGCCCTCGACACACCAGACTGATGTGTCGGGCTGCTA-3’(SEQ ID
NO.3)；
The reverse inner primers of BIP：5’-CGCTGTTGGGTCCTGAGGGAACGAAAGGACGACGGAAAG-3’(SEQ ID
NO.4)；
The positive cyclisation primers of LF：5’-ACCCCACAAGGAGGCGTA-3’(SEQ ID NO.5)；
LB is reversely cyclized primer：5’-GCACCTTCGGGTGTGTCTG-3’(SEQ ID NO.6)；
B) prepared by thalline culture and DNA masterplates：The activation culture of thalline LB solid mediums, 25 DEG C of perseverances are put into after coated plate
24h is cultivated in warm incubator, rear picking single bacterium falls into 25 DEG C of culture 12h (OD600=0.8) of LB nutrient solutions, thalline is collected by centrifugation,
Bacterial genomes, ultraviolet specrophotometer Nanodrop 2000 are extracted using TIANGEN bacterial genomes DNA extraction kit
Detection and Extraction quality and concentration, -20 DEG C standby；
C) LAMP is expanded：The genome that step b) is obtained carries out LAMP amplifications, and LAMP systems setting reaction temperature gradient is
61 DEG C, 62 DEG C, 63 DEG C, 64 DEG C or 65 DEG C；It is respectively 2 to set inner primer, cyclisation primer and outer primer ratio：2：1(10pmol：
10pmol：5pmol), 4：4：1(20pmol：20pmol：5pmol), 6：4：1(30pmol：20pmol：5pmol) or 8：4：1
(40pmol：20pmol：5pmol)；It is 15min, 30min, 45min or 60min to set reaction time gradient；
D) analyze：The extension product that step c) is obtained is estimated with negative control or in 2.0% agarose gel electrophoresis
Analysis.
Preferably, the gradient ratio of step c) inner primers, cyclisation primer and outer primer is 6：4：1 or 8：4：1, amplified reaction
Time is 15min or 30min, LAMP amplified reaction temperature are 62 DEG C, 63 DEG C or 64 DEG C.
Preferably, the gradient ratio of step c) inner primers, cyclisation primer and outer primer is 6：4：1, the amplified reaction time is
30min, LAMP amplified reaction temperature are 64 DEG C.
Preferably, the thalline is state wilting germ in corn.
The beneficial effects of the invention are as follows：The present invention utilizes state wilting germ in the corn of GenBank announcements
(Clavibacter michiganensis subsp.nebraskensis, Cmn) 16S-23S sequences Design LAMP (loop-
Mediated isothermal amplification) primer, wilting germ LAMP detection architectures in state in corn are established, and
Reaction condition is optimized.LAMP is 6 in inner primer, cyclisation primer and outer primer ratio：4：1(30pmol：20pmol：
The lower 64 DEG C of reactions 30min of 25 μ L systems 5pmol) is optimum reaction condition, and LAMP primer is only capable of from 7 kinds of bacterial strains for examination
Specific amplification goes out Cmn gradient bands.Examined using wilting germ genome in state in corn and bacteria suspension gradient dilution liquid as masterplate
The sensitivity of LAMP systems is surveyed, is as a result shown, LAMP detection architectures respectively reach for the sensitivity of genome and bacteria suspension
50fg and 3.8CFU/mL, than 100 times of regular-PCR high sensitivity.As a result show, LAMP detection architectures are simple, quick, sensitivity
High, specific good, state wilting germ context of detection has a high potential in corn.
Brief description of the drawings
Fig. 1 is LAMP specificity experiments electrophoretogram of the present invention.
In figure, M：DL2000；1：Blank control；2-8 representative strain genomes are respectively Cmn, Pss, Aaa, Aac, Xo,
Pa, Pas.
Fig. 2 is Cmn thalline LAMP sensitivity experiments electrophoretograms of the present invention.
Fig. 3 is Cmn thalline regular-PCR sensitivity experiments electrophoretograms.
In figure, M：DL2000；1：Blank control；2 represent Cmn thalline masterplate concentration as 3.6 × 106CFU/ml, 3-8 distinguish
Represent Cmn thalline masterplate concentration dilution multiple as, 10 times, 102Times, 103Times, 104Times, 105Times, 106Times.
Embodiment
Below by specific embodiment, technical scheme is described in further detail.It should be appreciated that this hair
Bright embodiment is not limited to the following examples, and any formal accommodation and/or change made to the present invention all will
Fall into the scope of the present invention.
Method in following embodiments, it is the conventional method of this area unless otherwise instructed.
Wilting germ (Clavibacter michiganensis in state in the bacterial strain corn used in the present invention
Subsp.nebraskensis, Cmn), P.stwartii subsp.stewartii (Pantoea stewartii subsp.Stewartii,
Pss) provided by Zhejiang Entry-Exit Inspection and Quarantine Bureau doctor Zhang Mingzhe；Oat acidovorax avenae (Acidovorax avenae
Subsp.Avenae, Aaa), it is Acidovorax Avenae Subsp (Acidovorax avenae subsp.citrull, Aac), yellow
Monad (Xanthomonas oryzae, Xo) is provided by Zhejiang University doctor Li Bin；General bacterium (the Pantoea of pineapple
Ananatis, Pa), sorghum pseudomonad (Pseudomonas andropogonis (Smith) stapp, Pas) comes in and goes out by Zhoushan
Complex art service centre of border inspection and quarantine bureau laboratory preserves.
LAMP method DNA amplification kits (Rong Yan), bacterial genomes DNA extraction kit (TIANGEN),
DL 2000DNA Ladder Marker (TaKaRa), agarose Agarose H (Shanghai life work), LB agar (Hangzhou microorganism
Reagent Co., Ltd).
Embodiment 1
State wilting germ LAMP quick determination methods, comprise the following steps in a kind of corn：
A) primer is designed, primer sequence is：
F3 forward direction outer primers：5’-CGCTCATGGGTGGAACAT-3’(SEQ ID NO.1)；
The reverse outer primers of B3：5’-GTTCTCAATATACGGGCGGT-3’(SEQ ID NO.2)；
FIP forward direction inner primers：5’-CATGCCCTCGACACACCAGACTGATGTGTCGGGCTGCTA-3’(SEQ ID
NO.3)；
The reverse inner primers of BIP：5’-CGCTGTTGGGTCCTGAGGGAACGAAAGGACGACGGAAAG-3’(SEQ ID
NO.4)；
The positive cyclisation primers of LF：5’-ACCCCACAAGGAGGCGTA-3’(SEQ ID NO.5)；
LB is reversely cyclized primer：5’-GCACCTTCGGGTGTGTCTG-3’(SEQ ID NO.6)；
B) prepared by thalline culture and DNA masterplates：The activation culture of state wilting germ LB solid mediums, coated plate in corn
After be put into 25 DEG C of constant incubators and cultivate 24h, rear picking single bacterium falls into 25 DEG C of culture 12h (OD600=0.8) of LB nutrient solutions,
Thalline is collected by centrifugation, bacterial genomes, ultraviolet specrophotometer are extracted using TIANGEN bacterial genomes DNA extraction kit
The Detection and Extraction quality of Nanodrop 2000 and concentration, -20 DEG C standby；
C) LAMP is expanded：The genome that step b) is obtained carries out LAMP amplifications, and LAMP systems setting reaction temperature gradient is
64℃；It is respectively 6 to set inner primer, cyclisation primer and outer primer ratio：4：1(30pmol：20pmol：5pmol)；Reaction is set
Time gradient is 30min；
D) analyze：The extension product that step c) is obtained is analyzed in 2.0% agarose gel electrophoresis.
Embodiment 2
State wilting germ LAMP quick determination methods, comprise the following steps in a kind of corn：
A) primer is designed, primer sequence is：
F3 forward direction outer primers：5’-CGCTCATGGGTGGAACAT-3’(SEQ ID NO.1)；
The reverse outer primers of B3：5’-GTTCTCAATATACGGGCGGT-3’(SEQ ID NO.2)；
FIP forward direction inner primers：5’-CATGCCCTCGACACACCAGACTGATGTGTCGGGCTGCTA-3’(SEQ ID
NO.3)；
The reverse inner primers of BIP：5’-CGCTGTTGGGTCCTGAGGGAACGAAAGGACGACGGAAAG-3’(SEQ ID
NO.4)；
The positive cyclisation primers of LF：5’-ACCCCACAAGGAGGCGTA-3’(SEQ ID NO.5)；
LB is reversely cyclized primer：5’-GCACCTTCGGGTGTGTCTG-3’(SEQ ID NO.6)；
B) prepared by thalline culture and DNA masterplates：The activation culture of state wilting germ LB solid mediums, coated plate in corn
After be put into 25 DEG C of constant incubators and cultivate 24h, rear picking single bacterium falls into 25 DEG C of culture 12h (OD600=0.8) of LB nutrient solutions,
Thalline is collected by centrifugation, bacterial genomes, ultraviolet specrophotometer are extracted using TIANGEN bacterial genomes DNA extraction kit
The Detection and Extraction quality of Nanodrop 2000 and concentration, -20 DEG C standby；
C) LAMP is expanded：The genome that step b) is obtained carries out LAMP amplifications, and LAMP systems setting reaction temperature gradient is
62℃；It is respectively 8 to set inner primer, cyclisation primer and outer primer ratio：4：1(40pmol：20pmol：5pmol)；Reaction is set
Time gradient is 15min；
D) analyze：The extension product that step c) is obtained is analyzed in 2.0% agarose gel electrophoresis.
Embodiment 3
State wilting germ LAMP quick determination methods, comprise the following steps in a kind of corn：
A) primer is designed, primer sequence is：
F3 forward direction outer primers：5’-CGCTCATGGGTGGAACAT-3’(SEQ ID NO.1)；
The reverse outer primers of B3：5’-GTTCTCAATATACGGGCGGT-3’(SEQ ID NO.2)；
FIP forward direction inner primers：5’-CATGCCCTCGACACACCAGACTGATGTGTCGGGCTGCTA-3’(SEQ ID
NO.3)；
The reverse inner primers of BIP：5’-CGCTGTTGGGTCCTGAGGGAACGAAAGGACGACGGAAAG-3’(SEQ ID
NO.4)；
The positive cyclisation primers of LF：5’-ACCCCACAAGGAGGCGTA-3’(SEQ ID NO.5)；
LB is reversely cyclized primer：5’-GCACCTTCGGGTGTGTCTG-3’(SEQ ID NO.6)；
B) prepared by thalline culture and DNA masterplates：The activation culture of state wilting germ LB solid mediums, coated plate in corn
After be put into 25 DEG C of constant incubators and cultivate 24h, rear picking single bacterium falls into 25 DEG C of culture 12h (OD600=0.8) of LB nutrient solutions,
Thalline is collected by centrifugation, bacterial genomes, ultraviolet specrophotometer are extracted using TIANGEN bacterial genomes DNA extraction kit
The Detection and Extraction quality of Nanodrop 2000 and concentration, -20 DEG C standby；
C) LAMP is expanded：The genome that step b) is obtained carries out LAMP amplifications, and LAMP systems setting reaction temperature gradient is
63℃；It is respectively 2 to set inner primer, cyclisation primer and outer primer ratio：2：1(10pmol：10pmol：5pmol)；Reaction is set
Time gradient is 45min；
D) analyze：The extension product that step c) is obtained is analyzed in 2.0% agarose gel electrophoresis.
Specificity verification:Utilize state wilting germ, reference bacterium in TIANGEN bacterial genomes extracts kit extraction corn
The DNA of strain, ultraviolet specrophotometer Nanodrop 2000 verify extraction effect, take 2ul to carry out LAMP amplifications respectively instead for masterplate
Should and pcr amplification reaction, verify LAMP optimization systems specificity.Glue figure is run from electrophoresis to can be seen that only with state in corn
Wilting germ is that the sample liquid gel imaging of masterplate progress LAMP amplifications runs out of clearly gradient band, and other are with reference strains
DNA is that the LAMP amplifications of masterplate do not occur gradient band (Fig. 2).As a result show, what the present invention established is directed in corn
The LAMP detection method high specificity of state wilting germ.
Reference strains be P.stwartii subsp.stewartii (Pantoea stewartii subsp.Stewartii, Pss),
Oat acidovorax avenae (Acidovorax avenae subsp.Avenae, Aaa), Acidovorax Avenae Subsp (Acidovorax
Avenae subsp.citrull, Aac), Xanthomonas campestris (Xanthomonas oryzae, Xo), the general bacterium (Pantoea of pineapple
Ananatis, Pa), sorghum pseudomonad (Pseudomonas andropogonis (Smith) stapp, Pas).
Sensitivity technique：Wilting germ bacteria suspension stoste in state in 100ul corns is taken, it is dilute to carry out 10 times of gradients with distilled water
Release, state wilting germ bacteria suspension concentration is 3.6 × 10 in coated plate measure corn6CFU/ml, 1 μ L dilutions are taken to enter for masterplate respectively
Row LAMP is expanded and regular-PCR amplified reaction, and checking LAMP optimization systems are directed to the sensitivity of thalline.Can by LAMP result figures 2
To find out that LAMP detection architectures detection sensitivity can reach 3.6CFU/mL, and the detection sensitivity of Fig. 3 regular-PCRs be 3.6 ×
102CFU/mL.Contrast LAMP systems and regular-PCR system gel imaging result show that the remolding sensitivity of LAMP detection architectures is general
Logical PCR 100 times of high sensitivity.
The yield and cultivated area Jun Ju second places of the world, corn of China's corn occupy important in China's grain-production
Status.From 2010, there is corn net importation situation in China, and 2012 to 2014, corn import total amount exceeded within 3 years
10000000 tons.State wilting germ is very high with the risk in the incoming China of import corn in corn.Currently for state wilt disease in corn
The quarantine method of bacterium mainly has isolated culture, ELISA, Standard PCR, TagMan-PCR, Chao Shi PCR, REP-PCR bases
Because of the combination of fingerprint technique and two or more method, various methods have its advantage and disadvantage, the deficiency of isolated culture
Part is complex operation, takes longer.ELISA is mainly limited not high enough with its sensitivity and specific anti-with preparation
Body is directly related.Standard PCR and Chao Shi PCR thus developed, TagMan-PCR, REP-PCR genetic fingerprints technology its
Detection sensitivity improves many, but its operating process needs special instrument and time-consuming longer, can not realize that basic unit quarantines people
Member scene, the requirement of rapid quarantine.And its specificity is influenceed by the antibody prepared.Standard PCR and fluorescence quantitative PCR detection
The sensitivity of detection is improved, but they are required for special equipment, for the limited mechanism of some experiment conditions particularly base
Layer inspection and quarantine department, is unfavorable for promoting.The present invention uses loop-mediated isothermal amplification technique (LAMP), can effectively overcome the above
The deficiency of method, its course of reaction only needs the support of the small-sized equipment of metal bath, and result need not pass through the equipment such as electrophoresis apparatus
Directly visually or the observation of fluorescent dye ultraviolet irradiation can be added, this effectively overcomes the limitation of experimental site；As a result show,
LAMP reactions thalline detection sensitivity is up to 3.6CFU, and Site Detection, which can directly drench, takes bacterium solution to be tested, and this is with regard to effectively more
It is not high to mend other molecular biology experiment thalline sensitivity, genomic DNA need to be extracted and detected, reduce the complexity of operation
Property；The LAMP detection reaction time shortens to 30 minutes simultaneously, and which greatly enhances the efficiency of detection.Certainly, LAMP technology also has
Some itself the problem of.First, design of primers is complicated, and LAMP reactions include 3 pairs of primers (inner primer, outer primer, cyclisation primer)
And it is unsuccessful to be cyclized design of primers in many cases.Secondly, amplified production is not easy to verify, a series of gradients occur in LAMP amplifications
Band, unless in a pair of specific cleavage sites of drone design, otherwise can not verify its extension increasing sequence and target sequence whether one
Cause.
SEQUENCE LISTING
<110>Complex art service centre of Zhoushan Entry-Exit Inspection and Quarantine Bureau
<120>Wilting germ LAMP quick determination methods in state in a kind of 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 (3)
1. wilting germ LAMP quick determination methods in state in a kind of corn, it is characterised in that comprise the following steps：
a）Primer is designed, primer sequence is：
SEQ ID NO.1：F3 forward direction outer primers：5’-CGCTCATGGGTGGAACAT-3’；
SEQ ID NO.2：The reverse outer primers of B3：5’-GTTCTCAATATACGGGCGGT-3’；
SEQ ID NO.3：FIP forward direction inner primers：5’-CATGCCCTCGACACACCAGACTGATGTGTCGGGCTGCTA-3’；
SEQ ID NO.4：The reverse inner primers of BIP：5’-CGCTGTTGGGTCCTGAGGGAACGAAAGGACGACGGAAAG-3’；
SEQ ID NO.5：The positive cyclisation primers of LF：5’-ACCCCACAAGGAGGCGTA-3’ ；
SEQ ID NO.6：LB is reversely cyclized primer：5’-GCACCTTCGGGTGTGTCTG-3’ ；
b）It is prepared by thalline culture and DNA masterplates：The activation culture of thalline LB solid mediums, 25 DEG C of constant temperature trainings are put into after coated plate
Support and 24 h are cultivated in case, rear picking single bacterium falls into 25 DEG C of culture 12h of LB nutrient solutions to OD600=0.8, and thalline is collected by centrifugation, utilizes
TIANGEN bacterial genomes DNA extraction kit extracts bacterial genomes, and ultraviolet specrophotometer Nanodrop 2000 is detected
Quality and concentration are extracted, -20 DEG C standby；
c）LAMP is expanded：Step b）Obtained genome carries out LAMP amplifications, and it is 62 that LAMP systems, which set reaction temperature gradient,
DEG C, 63 DEG C or 64 DEG C；It is respectively 6 to set inner primer, cyclisation primer and outer primer ratio：4：1 or 8：4：1；Reaction time is set
Gradient is 15 min or 30 min；
d）Analysis：Step c）Obtained extension product is estimated with negative control or analyzed in 2.0% agarose gel electrophoresis.
2. wilting germ LAMP quick determination methods in state in a kind of corn according to claim 1, it is characterised in that step
c）The gradient ratio of inner primer, cyclisation primer and outer primer is 6：4：1, the amplified reaction time is 30min, LAMP amplified reaction temperature
For 64 DEG C.
3. wilting germ LAMP quick determination methods in state in a kind of corn according to claim 1, it is characterised in that step
c）Inner primer, cyclisation primer and outer primer are respectively 30 pmol：20 pmol：5 pmol or 40 pmol：20 pmol：5
pmol。
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Citations (2)
| 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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2015
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Patent Citations (2)
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| loop-mediated isothermal amplification of DNA;Notomi t et.al，;《Nucleic acids research》;20020615;e63 * |
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