CN104593502A - Loop-mediated isothermal amplification primer composition capable of detecting colletotrichum truncatum and application thereof - Google Patents
Loop-mediated isothermal amplification primer composition capable of detecting colletotrichum truncatum and application thereof Download PDFInfo
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Abstract
The invention discloses a loop-mediated isothermal amplification primer composition capable of detecting colletotrichum truncatum and application thereof. The primer composition comprises a forward outer primer Rpb1-F3 as shown in SEQ ID NO.2, a reverse outer primer Rpb1-B3 as shown in SEQ ID NO.3, a forward inner primer Rpb1-FIP as shown in SEQ ID NO.4, a reverse inner primer Rpb1-BIP as shown in SEQ ID NO.5 and a loop primer Rpb1-LF as shown in SEQ ID NO.6. By the primer composition, the problems that the biological detection method requires long period, wastes time and energy and is tedious and poor in specificity and a thermal cycling instrument is needed and colletotrichum truncatum cannot be rapidly detected by virtue of the PCR detection technology in the prior art are solved and the primer composition has the advantages of short detection period (only 70 minutes), strong specificity and high sensitivity, and the detection results can be visually observed.
Description
Technical field
The invention belongs to field of biological detection, relate to a kind of the loop-mediated isothermal amplification (LAMP) primer composition and the application thereof that detect tack anthrax-bacilus.
Background technology
Soybean anthracnose is the important disease in each soybean producing region, the world, generally betides the important soybean producing regions such as China northeast, North China, East China, northwest, south China, and according to reports, all there are generation in 16 provinces such as Heilungkiang, Jilin, Liaoning
[1].Soybean anthracnose all can occur from seedling stage to harvesting time, and cotyledon, blastostyle, blade, petiole, cane, pod and seed all can be injured.Germ scab on cotyledon is rounded, and sorrel is to chocolate, depression.On stem, scab is brown or red rust look, slice shape, depression and be full of cracks.On Adult plant blade, scab mainly betides vein, and in polygon, sorrel is to chocolate.On stem, scab is similar to seedling stage.Scab ovalize or subcircular on pod, edge often swells, and central part caves in, and time moist, vermilion red point or pore appears in each affected part spot face
[2,3].
In recent years, along with soybean acreage constantly expands, the trend of generation in the harm that expands, increases the weight of of soybean anthracnose, all there is the generation of anthrax in national each soybean producing region, causes very large threat to Soybean production.In order to stop the continuous expansion of soybean anthracnose spread scope, soybean anthracnose being controlled, needing to detect quickly and accurately it.
Soybean anthracnose can be infected by multiple anthrax-bacilus and cause, and China has reported and caused the anthrax-bacilus of soybean anthracnose have tack anthrax-bacilus (C.truncatum), colletotrichum gloeosporioides Penz (C.gloeosporioide), Colletotrichum capsici (C.capsici), C. dematium (C.dematium) and destroy anthrax-bacilus (C.destructivum) etc. so far
[4,5].Wherein, tack anthrax-bacilus is one of pathogenic bacteria the most common.Therefore the Molecular Detection for tack anthrax-bacilus We conducted a series of research.
Tack anthrax-bacilus (C.truncatum) is the important phytopathogen of a class, and its regional distribution and host range are all very extensive.This bacterium Invisible element classification position is Deuteromycotina, Coelomycetes, Melanconiales, Melanconiaceae, colletotrichum, and Perfect stage is then under the jurisdiction of Ascomycotina, gang pyrenomycetes, Sphaerials, spherical shell section, small cluster shell genus.This bacterium is on PDA substratum, and cause of disease bacterium colony is circular, neat in edge, and mycelia is more sparse, is close to substratum growth, is just white, is light grey afterwards, and in culturing process, bacterium colony has fan realization to resemble, and it is more flourishing that fan becomes part mycelia, fine hair shape.Acervulus black stack projection, Dan Shengzhi is grown thickly, subcircular, and size variation is very large, and many acervuluses join together sometimes; Bristle chocolate, 1 ~ 3 separation, long 41.76 ~ 194.12 μm; Conidiophore is colourless to filbert; Conidium is colourless, crescent, without separating, and the blunt point in two, size (18.82 ~ 26.47) × (3.53 ~ 5.29) μm; Appressorium (6.60 ~ 10.63 (8.21)) × (4.31 ~ 8.00 (6.47)) μm, oval, accidental ginger lobe shape
[5].
The classification of tradition pathogenic bacteria, qualification are mainly based on morphological characteristic, Pathogenicity etc., and complex operation, length consuming time, sensitivity is low, the interference that is subject to the factors such as artificial and environment
[6], diagnosis can not be made in disease latent period and initial phase, be difficult to monitor timely disease and effectively control.
Along with molecular biological development, Protocols in Molecular Biology is progressively applied in the research of tack anthrax-bacilus.The method of the regular-PCR that last century, the eighties grew up has been used successfully to and has detected tack anthrax-bacilus
[7], but regular-PCR is rotten therefore outmoded technology and the some defects of tool, and such as detection time is still long, general 6 ~ 8h, few to the recognition site of target gene section, detection specificity and sensitivity on the low side; Testing process is more loaded down with trivial details, is difficult to satisfy the demands.
Loop-mediated isothermal amplification technique (Loop-mediated isothermal amplification, LAMP) is a kind of new nucleic acid amplification technologies that the Rong Yan strain formula of Japan can be invented
[8], 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, self-circulation strand replacement reaction is caused under the effect of Bst Large fragment polymerase, in 60 ~ 65 DEG C of scope 60 ~ 70min, while a large amount of synthesis target dna, being attended by by product---the magnesium pyrophosphate precipitation of white produces
[9].Target sequence 6 isolated areas are identified because LAMP amplification procedure relies on, so atopic is very strong, and amplification process is carried 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.
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)
[10], but many scholars think that this target can not distinguish colletotrichum fungi accurately in the level of planting.
The large subunit of modulation rna plymerase ii (the large subunit of RNA polymerase II that the present invention selects, Rpb1) be the topmost function subunit determining eukaryote messenger RNA(mRNA) (mRNA) transcription initiation and extension, there is multiple posttranslational modification, as phosphorylation, glycosylation and ubiquitination.Research in the past finds, the ubiquitination of Rpb1 is except the protein metabolism under its normal physiological condition of participation, also can promote the degraded being stuck in the Rpb1 on DNA in transcription, and transcribing when promoting extensive DNA damage is coupled reparation (Transcription Coupled Repair, TCR).Therefore, the ubiquitination of Rpb1, by the activated protein level of tight control Rpb1, ensure that normally carrying out of transcribing, and to DNA damage reparation with ensure that the stability of genetic material, fidelity are significant
[11].
Known tack anthrax-bacilus Molecular Detection target ITS can not meet the needs of LAMP primer design, existing based on regular-PCR to the molecular detection technology of this germ comparatively backwardness, and it is consuming time partially long to have detection, few to target gene section recognition site, false drop rate is higher, detection specificity and sensitivity on the low side, the defects such as testing process is more loaded down with trivial details.
Summary of the invention
The object of the invention is the above-mentioned deficiency for prior art, a kind of loop-mediated isothermal amplification (LAMP) primer composition detecting tack anthrax-bacilus is provided.
Another object of the present invention is to provide the application of this Primer composition.
Another object of the present invention is to provide a kind of LAMP kit detecting tack anthrax-bacilus.
Object of the present invention realizes by following technical scheme:
Rpb1 gene order shown in SEQ ID NO.1 is detecting the application in tack anthrax-bacilus as detecting target, preferably detects the application in tack anthrax-bacilus at LAMP.
A kind of Primer composition detecting tack anthrax-bacilus for LAMP, by the forward outer primer Rpb1-F3 shown in SEQ ID NO.2, reverse outer primer Rpb1-B3 shown in SEQ ID NO.3, forward inner primer Rpb1-FIP shown in SEQ ID NO.4, the ring primer Rpb1-LF shown in reverse inner primer Rpb1-BIP, SEQ ID NO.6 shown in SEQ ID NO.5 forms.
Primer composition of the present invention is detecting the application in tack anthrax-bacilus.
Primer composition of the present invention is preparing the application in tack anthrax-bacilus detection reagent.
For detecting a LAMP kit for tack anthrax-bacilus, comprise Primer composition of the present invention.
Described LAMP kit preferably comprises detection solution, and the detection solution described in every milliliter prepares by the following method: 0.8 μM of forward inner primer Rpb1-FIP, 0.8 μM of reverse inner primer Rpb1-BIP, 0.1 μM of forward outer primer Rpb1-F3,0.1 μM of reverse outer primer Rpb1-B3,0.1 μM of ring primer Rpb1-LF, 1.4mMdNTPs, 8mMMgSO
4, 0.8M trimethyl-glycine, 0.8M Tris-HCl (pH8.8), 0.4mM KCl, 0.4mM (NH4)
2sO
4, 4%Triton X-100,8U μ L
-1bst DNA polymerase, adds ultrapure water and is prepared into 1mL detection solution.
Described test kit is also containing dyestuff SYBR Green I.
LAMP detects a method for tack anthrax-bacilus, and comprise the detection solution got described in 18 μ L, 3 μ L sterilizing deionized waters, add 4 μ L DNA solution to be checked, and cumulative volume is that 25 μ L carry out LAMP reaction; Response procedures is: 62 DEG C, 70min; Dyestuff SYBR Green I is added as reaction indicator after amplification, visual inspection, strong and weak as result criterion with the colour-change of SYBR Green I and fluorescence: under ordinary light, yellow-green colour represents test positive, namely tack anthrax-bacilus is had to exist, orange expression detected result is negative, and namely not containing tack anthrax-bacilus, or contained tack anthrax-bacilus does not reach detectability.
The present invention describes in detail:
1, the design of primer
Choosing of modulation gene is the key factor that LAMP detects with the screening of primer.First search pertinent literature and have chosen CHS (chitin synthetase), SOD
2the operability target such as (Mn oxide dismutase), GS (glutamine synthetase), ITS (Internal Transcribed Spacer), EF1 α (translation elongation factor), RPB1 (the large subunit of rna plymerase ii).For target CHS, search in NCBI website and download the CHS sequence contained by tack anthrax-bacilus and close kind thereof, then BIOEDIT software is used to carry out sequence alignment, one section of its distinctive sequence is chosen as target sequence (200 ~ 400bp), with Primer4 Photographing On-line primer in the CHS sequence of tack anthrax-bacilus.Choose suitable primer according to primer length, primer free energy and GC content etc. to test, therefrom screening kind of interior versatility is good, and high specificity between kind, specificity among genus is strong, highly sensitive primer.Screen other targets successively, we analyze and find Rpb1 gene high conservative between different strains in kind when sequence alignment, and tool notable difference between kind, the final Rpb1 that selects is as the target gene of detection tack anthrax-bacilus.Again test of many times and checking are carried out to the many covers primer designed with this target base, select a set of versatility good, high specificity, highly sensitive, fast, can accurately detect tack anthrax-bacilus primer.
2, primer screening and proof procedure
(1) tack anthrax-bacilus LAMP primer is detected by versatility experiment sieving
1) to screen for the purpose of the LAMP primer that can detect tack anthrax-bacilus, carry out following experiment: select tack anthrax-bacilus reference culture, and from the DNA of the tack anthrax-bacilus of different areas, different host as template, get 4 μ l DNA solutions, add the reaction solution that LAMP primer is prepared that the different drone designs of 18 μ L go out and carry out LAMP reaction, carry out LAMP reaction with 3 μ L sterilizing deionized waters, response procedures is: 62 DEG C of 70min; Observing response pipe colour-change after reaction, detects under ordinary light, and the sample containing tack anthrax-bacilus strains as yellow-green colour under ordinary light, and negative control is orange under ordinary light.If all samples containing tack anthrax-bacilus can be made after reaction terminates to become yellow-green colour, then this primer meets versatility requirement of experiment, can proceed next step screening experiment; If all samples containing tack anthrax-bacilus can not be made to become yellow-green colour, then this primer does not meet requirement of experiment, discarded.
(2) by specific test selective mechanisms tack anthrax-bacilus LAMP primer
1) to screen for the purpose of the LAMP primer that can detect tack anthrax-bacilus specifically, carry out following experiment: select tack anthrax-bacilus reference culture, close with it kind of colletotrichum gloeosporioides Penz and sharp spore anthrax-bacilus, and other pathogenic fungi (Cercospora kikuchiis, aspergillus oryzae, alternaric bacteria, soybean charcoal rot bacterium, epicoccum nigrum, southern stem canker of soybean, soybean north stem canker, soybean Phomopsis seed decay pathogen, brown stem rot bacterium, phytophthora sojae kaufmann&gerdemann, point spore reaping hook germ, Bipolaris maydis) DNA as template, get 4 μ l DNA solutions, adding 18 μ L through 1) reaction solution that LAMP primer is prepared that goes out of the different drone designs that filter out and 3 μ L sterilizing deionized waters carry out LAMP reaction, response procedures is: 62 DEG C of 70min, observing response pipe colour-change after reaction, detects under ordinary light, and the sample containing tack anthrax-bacilus strain as yellow-green colour under ordinary light, and contains the sample of other kinds and negative control is orange under ordinary light.If after reaction terminates, except tack anthrax-bacilus is yellow-green colour, other samples are orange, then this primer meets specific requirement of experiment, can carry out next step screening experiment; Otherwise this primer does not meet requirement of experiment, discarded.
(3) by sensitivity test selective mechanisms tack anthrax-bacilus LAMP primer
To screen for the purpose of the LAMP primer that can detect tack anthrax-bacilus delicately, carry out following experiment: select tack anthrax-bacilus reference culture, standard tack anthrax-bacilus bacterial strain DNA spectrophotometric determination concentration (1ng/ μ l) is carried out 10 doubling dilutions with DEPC water afterwards, preserves as template for-70 DEG C.Get each concentration DNA diluent 4 μ l after 10 doubling dilutions respectively as template, adding 18 μ L through 2) reaction solution that LAMP primer is prepared that goes out of the different drone designs that filter out and 3 μ L sterilizing deionized waters carry out LAMP reaction, and response procedures is: 62 DEG C of 70min; Observing response pipe colour-change after reaction, detect under ordinary light, negative control is orange under ordinary light, and record becomes the concentration of yellowish green sample, chooses the primer that detection sensitivity is the highest.
Through series of experiments, the present invention finally adopts a set of versatility in the primer designed for target gene with Rpb1 good, high specificity, highly sensitive, fast, can accurately detect the primer of tack anthrax-bacilus, synthesized by nvitrogen (Shanghai) company, its sequence information is as follows:
Table 1
3. participate in the experiment this institute of bacterial strain use bacterial strain of participating in the experiment as shown in table 2:
Table 2
Beneficial effect:
To finding this gene order high conservative between different strains in Colletotrichum kind during the comparison of Rpb1 gene order, there is abundant change between kind, can be used as the candidate targets gene of this germ Molecular Detection in contriver.The present invention analyzes tack anthrax-bacilus Rpb1 gene and the difference of other anthrax-bacilus in sequence, designs and has screened four specific LAMP primer and a ring primer, establishes the LAMP system detecting tack anthrax-bacilus on this basis.Cycle needed for the biological detection method that the invention solves tack anthrax-bacilus in prior art is grown, waste time and energy, the problem of loaded down with trivial details, poor specificity and PCR detection technique need thermal cycler instrument, cannot the problem of rapid detection tack anthrax-bacilus, sense cycle short (only needing 70min), high specificity, susceptibility be high, can visual inspection detected result.
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 can be differentiated result with observing dish under UV-light after ethidium bromide staining, and this not only increases detection required time and detects operation link, also easily causes product to spread, becomes an important sources of laboratory pollution; And ethidium bromide (EB) has severe toxicity, can accumulate carcinogenic; In addition, UV-light also can cause injury to a certain degree to experimenter.And LAMP reaction only need be carried out in thermostat water bath, after reaction terminates by the colour-change of SYBR GreenI just can under ordinary light with the naked eye direct observe and decide result, the present invention is simple to operate, required time short (complete one-time detection and only need 2h), pollute little, there is stronger practicality.
(2) constant-temperature amplification.Must thermal cycling unlike PCR method, so just break away from the dependence to thermal cycler instrument, as long as there is stable thermal source just can complete as thermostat water bath LAMP reaction, do not needed expensive plant and instrument, apply so be convenient to basic agriculture production unit.Why LAMP can react under constant thermal source is because with the addition of trimethyl-glycine in LAMP reaction solution, in running balance double-stranded DNA being in unwind, under the effect of Bst archaeal dna polymerase, realizes amplification.
(3) accuracy is high.The interference that traditional tack anthrax-bacilus detection technique length consuming time, sensitivity is low, be subject to the factors such as artificial and environment; And the present invention chooses the distinctive one section of specific LAMP primer of Rpb1 sequences Design of tack anthrax-bacilus, by 6 isolated areas on 4 primer specificity identification target sequences, for 2 isolated areas of PCR primer identification target sequence, specificity and sensitivity higher.
(4) significantly upgrade technologies.The tack anthrax-bacilus rapid molecular detection system set up that to combine with LAMP technology with the high specific primer screened is designed based on target gene Rpb1, overcame the defect existing for de-correlation technique, shorten detection required time, simplify operating process, improve specificity and the sensitivity of detection, is that state of the art is upgraded.
Accompanying drawing explanation
Fig. 1 naked-eye observation result, left side EP manages in yellow-green colour, and be expressed as tack anthrax-bacilus and detect positive (+), right side EP manages in orange, is expressed as tack anthrax-bacilus and detects negative (-).
Fig. 2 is based on the versatility of the LAMP technology detection tack anthrax-bacilus of target gene Rpb1
Wherein, No. 1 is tack anthrax bacteria reference culture (positive control), and 2 ~ No. 8 tack anthrax bacteria bacterial strains being the different host's up-sampling in different areas and being separated to, No. 9 is water (negative control).The bacterial strain information of participating in the experiment that this specification sheets table 2 provides is seen in 2 ~ No. 8 hosts and area.Tack anthrax-bacilus totally 70 strains that soybean from the soybean on farm, Jiangpu, Jiangsu, peanut, the ground such as capsicum and Taixing, Jiangsu, Xuzhou, Jiangsu Jiangyan City, Jiangsu Jiangyan City, Wuhan, Hubei, Suzhou, Anhui, Long Kang farm, Anhui is separated to, all take part in checking, result display is all positive reaction (yellow-green colour), illustrates that the detection of this technology to the tack anthrax-bacilus from different host and area is all applicable.Fig. 2 only have chosen the representative bacterial strain of 8 strain and lists.
Fig. 3 detects the result of colletotrichum gloeosporioides Penz, sharp spore anthrax-bacilus and other fungies based on the LAMP technology of target gene Rpb1
Wherein, No. 1 is tack anthrax bacteria reference culture (positive control), 2 ~ No. 7 is glue spore anthrax bacteria (Colletotrichum gloeosporioide) different strains, No. 8, No. 9 is sharp spore anthrax bacteria (Colletotrichum.acutatum) different strains, No. 10 is Cercospora kikuchii (Cercospora kikuchii), No. 11 is aspergillus oryzae (Aspergillus oryzae), No. 12 is alternaric bacteria (Alternaria alternata), No. 13 is soybean charcoal rot bacterium (Macrophomina phaseolina), No. 14 is epicoccum nigrum (Epicoccum nigrum), No. 15 is southern stem canker of soybean (Diaporthe phaseolorum var.caulivora), No. 16 is soybean north stem canker (Diaporthe phaseolorumvar.meridionalis), No. 17 is soybean Phomopsis seed decay pathogen (Phomopsis longicolla), No. 18 is brown stem rot bacterium (Phialophora sojae), No. 19 is phytophthora sojae kaufmann&gerdemann (Phytophthora sojae), No. 20 sharp spore reaping hook germs (Fusarium oxysporum), No. 21 Bipolaris maydis (Bipolaris maydis), No. 22 is water (negative control).The bacterial strain information of participating in the experiment that this specification sheets table 2 provides is seen in pathogenic bacteria host and area.Result shows, and all colletotrichum gloeosporioides Penzs for examination, sharp spore anthrax-bacilus and other pathogenic fungies are all in orange negative reaction, and only No. 1 tack anthrax bacteria reference culture is yellowish green positive reaction.Show that the Rpb1-LAMP technology that the present invention sets up can distinguish target pathogenic bacteria (tack anthrax-bacilus) and non-targeted pathogenic bacteria (other anthrax-bacilus and other pathogenic bacterias).
Fig. 4 is based on the sensitivity of the LAMP technology detection tack anthrax bacteria of target gene Rpb1
LAMP amplification different concns tack anthrax bacteria genomic dna; No. 1 is tack anthrax bacteria reference culture genome (positive control), 2 ~ 9 is the LAMP amplification respectively containing 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg tack anthrax bacteria DNA in the reaction system of 25 μ L, and No. 10 is negative control.LAMP amplified reaction can identify tack anthrax bacteria specifically, and picture represents that sensitivity can reach 100pg/ μ L.
Fig. 5 detects the tack anthrax-bacilus of falling ill in soybean plant strain based on the LAMP technology of target gene Rpb1
Wherein No. 1 is tack anthrax bacteria reference culture genome (positive control), 2 ~ 7 is the soybean plant strain genome of inoculation tack anthrax bacteria bacterial strain after 6 days, 8 is the soybean plant strain genome of the blank PDA substratum of inoculation after 6 days, and 9 for aqua sterilisa replacement DNA cloning is as negative control.Result shows, the soybean plant strain (2 ~ No. 7 pipes) of inoculation tack anthrax-bacilus is yellowish green positive reaction under ordinary light, effect and direct-detection tack anthrax-bacilus DNA (No. 1 pipe) do not have difference, inoculate the soybean plant strain of blank PDA substratum after 6 days (No. 8 pipes) and negative control aqua sterilisa (No. 9 pipes) then in the negative reaction that ordinary light is orange, illustrate that the Rpb1-LAMP technology that the present invention sets up can detect tack anthrax-bacilus specifically from the soybean plant strain of inoculation morbidity, may be used for field rapid detection.
Fig. 6 is based on the LAMP technology sensitivity in actual applications of target gene Rpb1
No. 1 is soybean tack anthrax bacteria reference culture genome (positive control), 2 ~ No. 6 is the DNA adding the soybean sample extraction of different spore amount in every 50g soybean sample, and 2 ~ No. 6 spore additions are followed successively by 10000,1000,100,50,10.No. 7, No. 8 DNA being respectively the soybean sample extraction of interpolation 0 spore and aqua sterilisa replace DNA cloning as negative control.Result shows, and the sensitivity of the Rpb1-LAMP technology for detection tack anthrax-bacilus that the present invention sets up is 10 spores/50g soybean.
Fig. 7 is based on the tack anthrax-bacilus in the LAMP technology detection market collection soybean of target gene Rpb1
LAMP detection is carried out to extracting DNA from the soybean of Liaoning, Anhui, Jiangsu, Hubei, Shandong, Heilungkiang, zhejiang and other places and the residual body of beanpod.Wherein, No. 1 is tack anthrax bacteria reference culture genome (positive control), and 2 ~ No. 11 are respectively the DNA extracted from the soybean seeds that Dalian, Fuyang, Xuzhou, Wuhan, Hubei, Weifang, Shandong, Dezhou, Shandong, Shandong Zibo, Mudanjiang, Heilungkiang, Zhejiang Jiaxing, 10, Taizhou, Jiangsu are regional.No. 12 is negative control.Result shows, and the soybean local from Dalian, Fuyang, Xuzhou, Wuhan, Hubei, Weifang, Shandong, 6, Dezhou, Shandong and beanpod residual body DNA are yellowish green positive reaction, illustrate that it carries tack anthrax-bacilus.
Embodiment
The main agents that following examples use and instrument: Bst archaeal dna polymerase (New England Biolabs), Betaine, MgS0
4(Sigma); DNTPs, eppendorf regular-PCR amplification instrument, eppendorf Biophotometer spectrophotometer, upper Nereid grand laboratory apparatus factory DK-8D type electrically heated thermostat water bath.
Embodiment 1 one kinds is for detecting the LAMP detection kit of tack anthrax-bacilus
Test kit reaction system: detect solution and dyestuff SYBR Green I.
Detection solution manufacturing method is as follows:
0.8 μM of forward inner primer Rpb1-FIP, 0.8 μM of reverse inner primer Rpb1-BIP, 0.1 μM of forward outer primer Rpb1-F3,0.1 μM of reverse outer primer Rpb1-B3,0.1 μM of ring primer Rpb1-LF, 1.4mMdNTPs, 8mMMgSO
4, 0.8M trimethyl-glycine, 0.8M Tris-HCl (pH8.8), 0.4mM KCl, 0.4mM (NH4)
2sO
4, 4%Triton X-100,8U μ L
-1bst DNA polymerase, add ultrapure water and be prepared into 1mL detection solution, the storage life is 1 year.
Embodiment 2 test kit versatility of the present invention is investigated
Tack anthrax-bacilus reference culture shown in option table 2, and from the DNA of the tack anthrax-bacilus of different areas, different host as template, get 4 μ l DNA solutions, add 18 μ L and detect solution and 3 μ L sterilizing deionized waters and carry out LAMP reaction, response procedures is: 62 DEG C of 70min; 0.25 μ L dyestuff SYBR Green I is added as reaction indicator after amplification, observing response pipe colour-change after reaction, detect under ordinary light, the sample from different host and regional tack anthrax-bacilus all becomes yellow-green colour under ordinary light, and negative control is orange under ordinary light.Illustrate that the detection of this technology to the tack anthrax-bacilus from different host and area is all applicable.Fig. 2 only have chosen the representative bacterial strain of 8 strain and lists.
Embodiment 3
Select tack anthrax-bacilus reference culture, close with it kind of colletotrichum gloeosporioides Penz and sharp spore anthrax-bacilus, and other pathogenic fungi (Cercospora kikuchiis, aspergillus oryzae, alternaric bacteria, soybean charcoal rot bacterium, epicoccum nigrum, southern stem canker of soybean, soybean north stem canker, soybean Phomopsis seed decay pathogen, brown stem rot bacterium, phytophthora sojae kaufmann&gerdemann, point spore reaping hook germ, Bipolaris maydis) DNA as template, get 4 μ l DNA solutions, add 18 μ L to detect solution and 3 μ L sterilizing deionized waters and carry out LAMP reaction, response procedures is: 62 DEG C of 70min, 0.25 μ L dyestuff SYBR Green I is added as reaction indicator after amplification, observing response pipe colour-change after reaction, detect under ordinary light, the sample containing tack anthrax-bacilus strains as yellow-green colour under ordinary light, and is orange under ordinary light containing the sample of other kinds and negative control.Result shows, and all colletotrichum gloeosporioides Penzs for examination, sharp spore anthrax-bacilus and other pathogenic bacterias are all in orange negative reaction, and only No. 1 tack anthrax bacteria reference culture is yellowish green positive reaction.Show that the Rpb1-LAMP technology that the present invention sets up can distinguish target pathogenic bacteria (tack anthrax-bacilus) and non-targeted pathogenic bacteria (other anthrax-bacilus and other pathogenic bacterias) (Fig. 3).
Embodiment 4
Select tack anthrax-bacilus reference culture, standard tack anthrax-bacilus bacterial strain DNA spectrophotometric determination concentration (1 μ g/ μ l) is carried out 10 doubling dilutions with DEPC water afterwards, preserve as template for-70 DEG C.Get each concentration DNA diluent 4 μ l after 10 doubling dilutions respectively as template, add 18 μ L and detect solution and 3 μ L sterilizing deionized waters and carry out LAMP reaction, response procedures is: 62 DEG C of 70min; 0.25 μ L dyestuff SYBR Green I is added as reaction indicator, observing response pipe colour-change after reaction after amplification.Result, as Fig. 4, represents that sensitivity of the present invention can reach 100pg/ μ L.
Embodiment 5 is based on the tack anthrax-bacilus in the LAMP technology detection artificial inoculation morbidity group soybean diseased tissues of target gene Rpb1
With plant genes group DNA extraction kit (
) extract the DNA that the soybean plant strain of tack anthrax bacterias after 6 days is inoculated in 6 strains, it can be used as template to increase for LAMP, using tack anthrax bacteria reference culture DNA as positive control, healthy plant DNA and aqua sterilisa replace DNA cloning as negative control.Get 4 μ LDNA solution, add detection solution described in 18 μ L embodiments 1 and 3 μ L sterilizing deionized waters carry out LAMP reaction, response procedures is: 62 DEG C, 70min.0.25 μ L dyestuff SYBR Green I is added as reaction indicator after amplification, observing response pipe colour-change after reaction, detect under ordinary light, result as shown in Figure 5, display present method can become yellow-green colour from the soybean plant strain (2 ~ No. 7 pipes) of inoculation tack anthrax bacteria under ordinary light, tack anthrax bacteria can be detected specifically, effect and direct-detection tack anthrax bacteria DNA (No. 1 pipe) do not have difference, and inoculate the soybean plant strain of blank PDA substratum after 6 days (No. 8 pipes), negative control aqua sterilisa (No. 9 pipes) is then orange under ordinary light, visible present method may be used for Fields detection.
Embodiment 6 detects the tack anthrax-bacilus of falling ill in soybean plant strain diseased tissues in field based on the LAMP technology of target gene Rpb1
From farm, Jiangpu, Jiangsu, Xuzhou, Wuhan, Hubei, Fuyang, Suzhou, Anhui, Long Kang farm, Anhui gather soybean sample choose typical incidence tissue, cleaning is dried, with plant genes group DNA extraction kit (
) extract its genome.It can be used as template to increase for LAMP, using tack anthrax bacteria reference culture DNA as positive control, aqua sterilisa replaces DNA cloning as negative control.Get 4 μ LDNA solution, add the above-mentioned detection solution of 18 μ L and 3 μ L sterilizing deionized waters carry out LAMP reaction, response procedures is: 62 DEG C, 70min.Dyestuff SYBR Green I is added as indicator after amplified reaction terminates.If reaction solution color becomes yellow-green colour represent test positive, contain tack anthrax-bacilus in examination soy bean plant tissue; If color keeps orange invariant representation to be detected as feminine gender, do not contain tack anthrax-bacilus in examination soy bean plant tissue.
1. detected result is as follows:
Nanjing and Xuzhou: 61 increments originally, detect 15 positives;
Wuhan, Hubei: 50 increments originally, detect 33 positives;
Suzhou, Anhui and Huaiyuan: 43 increments originally, detect 13 positives.
2. the soybean diseased tissues material of pair test positive carries out pathogenicbacteria separation, and the pathogenic bacteria of isolating ground, through morphological observation and ITS order-checking comparison, is tack anthrax-bacilus, proves to detect positive findings further correctly reliable.
Embodiment 7 is based on the LAMP technology sensitivity in actual applications of target gene Rpb1
Choose same kind, areal, without the soybean of tack anthrax-bacilus and the residual sample body of beanpod, carry out according to the following steps:
1) random sampling
Soybean and beanpod residue sample rock mixing 5 sampling in plate, choose 6 increments this next step for subsequent use.
2) washing of sample
6 parts of soybean soybean and the residual body of beanpod are respectively taken 50g, pour in 250ml triangular flask, add the sterilized water of 100ml, drip 20% tween 3-5 to drip, then add the conidium of 10000,1000,100,50,10,0 tack anthrax-bacilus respectively, seal bottleneck, be placed in 220rpm on shaking table, 30min, concussion washing.
3) collection of filtrate
The mixed solution sieved filter of 200 order steel after concussion washing, and the Soybeanresidue sieved with a small amount of aseptic water washing steel, filtrate collection is in former triangular flask.
4) centrifugal collecting precipitation,
The filtrate of collecting installed in 100ml centrifuge tube, gradation is centrifugal, and 6500rpm, 10min abandon supernatant, collecting precipitation.
5) throw out process
Throw out is placed in 37 DEG C of thermostat containers to dry.
6) the total genomic extraction of throw out
Owing to containing a large amount of soil constituent in throw out, so experiment adopts the soil microbial DNA brute force of MOBIO company of the U.S. to extract test kit
dNA Isolation Kit extracts, and method is as follows:
The throw out 0.25g getting oven dry joins in PowerBead pipe, adds 60 μ l C1, whirlpool concussion 10min; Room temperature 10000g, 30s are centrifugal.In Aspirate supernatant 400 ~ 500 μ l to 2ml Collection Tube, add 250 μ l C2 in centrifuge tube, whirlpool concussion 5s, is placed in 4 DEG C, 5min; Room temperature centrifugal 10000g, 1min; Draw supernatant 600 μ l in new 2ml Collection Tube; In centrifuge tube, add 200 μ l C3, after whirlpool concussion 5s, be placed in 4 DEG C of 5min; Room temperature centrifugal 10000g, 1min; Draw supernatant 750ul in new 2ml Collection Tube; Xiang Guanzhong adds 1.2ml C4, whirlpool concussion 5s; Be transferred to by mixed solution in Spin Fittle, 10000g, 1min are centrifugal, abandon filtrate; In filter post, add 500ul C5,10000g, 30s, abandon filtrate; The idle running of filter post is centrifugal, 10000g, 1min; Be positioned in new 2ml Collection Tube by filter post, 37 DEG C of constant temperature dry; Add 100ul C6 in filter post, place 2min, 10000g, 1min is centrifugal.Gained filtrate is the genome of extraction, places for subsequent use.
7) extract soybean tack anthrax-bacilus LAMP in genome to detect
Tack anthrax-bacilus LAMP detects: get 4uLDNA solution, adds 18 μ L mentioned reagent box solution and 3 μ L sterilizing deionized waters carry out LAMP reaction.
Reaction conditions: 62 DEG C, 70min.
Amplified production detects: after amplified reaction terminates, add dyestuff SYBR Green I as indicator.If reaction solution color becomes yellow-green colour represent test positive, contain tack anthrax-bacilus in examination soybean; If color keeps orange invariant representation to be detected as feminine gender, do not contain tack anthrax-bacilus in examination soybean.Result shows, and the Rpb1-LAMP technology that the present invention sets up is 10 spores/50g soybean (Fig. 6) for the detection sensitivity of tack anthrax-bacilus in soybean and the residual sample body of beanpod.
Embodiment 8 is based on the tack anthrax-bacilus in the LAMP technology detection market collection soybean of target gene Rpb1
From Dalian, Fuyang, Xuzhou, Wuhan, Hubei, Weifang, Shandong, Dezhou, Shandong, Shandong Zibo, Mudanjiang, Heilungkiang, Zhejiang Jiaxing, Taizhou, Jiangsu collect soybean seeds 10 parts, 10 portions of soybean and the residual body of beanpod are respectively taken 50g, pour in 250ml triangular flask, add the sterilized water of 100ml, drip 20% tween 3-5 to drip, seal bottleneck, be placed in 220rpm on shaking table, 30min, concussion washing.Then repeat 3 ~ 6 steps in example 3 successively, extract genome.
Extract tack anthrax-bacilus LAMP in genome to detect
Tack anthrax-bacilus LAMP detects: get 4 μ LDNA solution, adds 18 μ L mentioned reagent box solution and 3 μ L sterilizing deionized waters carry out LAMP reaction.
Reaction conditions: 62 DEG C, 70min.
Amplified production detects: after amplified reaction terminates, add dyestuff SYBR Green I as indicator.If reaction solution color becomes yellow-green colour represent test positive, contain tack anthrax-bacilus in examination soybean; If color keeps orange invariant representation to be detected as feminine gender, do not contain tack anthrax-bacilus in examination soybean.Result shows, and the soybean local from Dalian, Fuyang, Xuzhou, Wuhan, Hubei, Weifang, Shandong, 6, Dezhou, Shandong and beanpod residual body DNA are yellowish green positive reaction, illustrate that it carries tack anthrax-bacilus (Fig. 7).
Reference
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3. Sun Zhi peak, Lou Binggan, kingdom's honor waits (2008). soybean pod anthrax bacteria biological characteristics. and Zhejiang Agriculture journal 20 (6): 432-436.
4. building soldier does, Chen Wujian, woods hairpin etc. (2009). a kind of new soybean pod anthrax symptom type and Pathogen identification thereof. and plant protection journal 36 (05): 229-233.
5. Xiao Jiewen, Liu Yuelian, Ran Junxiang etc. (2011). the isolation identification research of anthrax-bacilus in Brazil Soybeans. Chinese agronomy circular 27 (05): 333-337.
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7.L.S.Chen.Chu.C.D.LiuR.S.Chen,J.G.Tsay(2006).PCR‐based Detection and Differentiation of Anthracnose Pathogens,Colletotrichum gloeosporioides and C.truncatum,from Vegetable Soybean in Taiwan.Journal of Phytopathology 154(11‐12):654-662.
8.Notomi,T.,Okayama,H.,Masubuchi,H.,Yonekawa,T.,Watanabe,K.,Amino,N.,and Hase,T.(2000).Loop-mediated isothermal amplification of DNA.Nucleic Acids Research 28:e63-e63.
9.Yasuyoshi Mori,Kentaro Nagamine,Norihiro Tomita,and Tsugunori Notomi(2001).Detection of Loop-Mediated Isothermal Amplification Reaction by Turbidity Derived from Magnesium Pyrophosphate Formation.Biochemical and Biophysical Research Communications289:150-154.
10.White,T.J.,Bruns,T.,Lee,S.,and Taylor,J.(1990).Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics.Pages 315-322in:PCR Protocols:A Guide to Methods and Applications.M.A.Innis,D.H.Gelfand,J.J.Sninsky,and T.J.White,eds.Academic Press,San Diego,CA.
The large subunit Rpb1 [D] of 11. Li Hui (20086) HECT family ubiquitin ligase Wwp2 ubiquitination rna plymerase ii. Postgraduate School, Chinese Academy of Sciences (Shanghai life science institute)
Claims (9)
- Rpb1 gene order shown in 1.SEQ ID NO.1 is detecting the application in tack anthrax-bacilus as detecting target.
- 2. application according to claim 1, is characterized in that the Rpb1 gene order shown in SEQ ID NO.1 is as detecting the application of target in LAMP detection tack anthrax-bacilus.
- 3. one kind is detected the Primer composition of tack anthrax-bacilus for LAMP, it is characterized in that by the forward outer primer Rpb1-F3 shown in SEQ ID NO.2, reverse outer primer Rpb1-B3 shown in SEQ ID NO.3, forward inner primer Rpb1-FIP shown in SEQ ID NO.4, the ring primer Rpb1-LF shown in reverse inner primer Rpb1-BIP, SEQ ID NO.6 shown in SEQ ID NO.5 forms.
- 4. Primer composition according to claim 3 is detecting the application in tack anthrax-bacilus.
- 5. Primer composition according to claim 3 is preparing the application in tack anthrax-bacilus detection reagent.
- 6., for detecting a LAMP kit for tack anthrax-bacilus, it is characterized in that comprising Primer composition according to claim 3.
- 7. LAMP kit according to claim 6, it is characterized in that comprising detection solution, the detection solution described in every milliliter prepares by the following method: 0.8 μM of forward inner primer Rpb1-FIP, 0.8 μM of reverse inner primer Rpb1-BIP, 0.1 μM of forward outer primer Rpb1-F3,0.1 μM of reverse outer primer Rpb1-B3,0.1 μM of ring primer Rpb1-LF, 1.4mMdNTPs, 8mMMgSO 4, 0.8M trimethyl-glycine, 0.8M Tris-HCl (pH8.8), 0.4mM KCl, 0.4mM (NH4) 2sO 4, 4%TritonX-100,8U μ L -1bst DNA polymerase, adds ultrapure water and is prepared into 1mL detection solution.
- 8. the LAMP kit according to claim 6 or 7, is characterized in that described test kit also containing dyestuff SYBR GreenI.
- 9. LAMP detects a method for tack anthrax-bacilus, and it is characterized in that comprising and get 18 μ L detection solution according to claim 7,3 μ L sterilizing deionized waters, add 4 μ L DNA solution to be checked, and cumulative volume is that 25 μ L carry out LAMP reaction; Response procedures is: 62 DEG C, 70min; Dyestuff SYBR Green I is added as reaction indicator after amplification, visual inspection, strong and weak as result criterion with the colour-change of SYBR GreenI and fluorescence: under ordinary light, yellow-green colour represents test positive, namely tack anthrax-bacilus is had to exist, orange expression detected result is negative, and namely not containing tack anthrax-bacilus, or contained tack anthrax-bacilus does not reach detectability.
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