CN104593502B - 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, it is related to a kind of loop-mediated isothermal amplification (LAMP) primer combination of detection tack anthrax
Thing and its application.
Background technology
Soybean anthracnose is the important disease in the world each Semen sojae atricolor producing region, generally betides China northeast, North China, East China, west
The important Semen sojae atricolor producing region such as north, south China, it has been reported that all there are generation in 16 provinces such as Heilungkiang, Jilin, Liaoning[1].Soybean anthracnose from
Seedling stage to harvest time all can occur, and cotyledon, blastostyle, blade, petiole, cane, pod and seed all can be aggrieved.Pathogenic bacteria is on cotyledon
Scab is rounded, bronzing to pitchy, depression.On stem, scab is brown or red rust color, slice shape, is recessed and is cracked.Strain
On phase blade, scab takes place mostly in vein, and in polygon, bronzing is to pitchy.On stem, scab is similar to seedling stage.Disease on pod
Speckle ovalize or subcircular, edge often swells, and central part is recessed, and vermilion point or little when moist on each affected part speckle face
Stain[2,3].
In recent years, constantly expand with soybean acreage, occurring of soybean anthracnose is in expand, increase to endanger
Trend, all there is the generation of anthrax in national each Semen sojae atricolor producing region, and Soybean production is caused with very big threat.In order to stop soybean anthracnose
The continuous expansion of spread scope, makes soybean anthracnose be controlled, and needs it is quickly and accurately detected.
Soybean anthracnose can be infected by multiple anthrax and cause, and China has reported the anthrax causing soybean anthracnose so far
There are tack anthrax (c.truncatum), colletotrichum gloeosporioides Penz (c.gloeosporioide), Colletotrichum capsici
(c.capsici), C. dematium (c.dematium) and destruction anthrax (c.destructivum) etc.[4,5].Wherein, tack
Anthrax is one of most commonly seen pathogen.The Molecular Detection being therefore directed to tack anthrax We conducted a series of grinding
Study carefully.
Tack anthrax (c.truncatum) is the important phytopathogen of a class, and its geographical distribution and host range are equal
Very extensive.This bacterium Invisible element classification position is Deuteromycotina, Coelomycetess, Melanconiales, Melanconiaceae, colletotrichum, sexual
State is then under the jurisdiction of Ascomycotina, gang pyrenomycetes, Sphaerials, spherical shell section, small cluster shell genus.This bacterium in pda culture medium, cause of disease bacterium colony
Circle, neat in edge, mycelia is sparse, is close to cultivate basal growth, just for white, afterwards for light gray, in incubation, bacterium colony has
Fan becomes phenomenon, and fan change part mycelia is more flourishing, villiform.Aceravlus black stack projection, Dan Shengzhi is grown thickly, subcircular,
Size variation is very big, and many aceravluses join together sometimes；Bristle pitchy, 1～3 separation, long 41.76～194.12
μm；Conidiophore is colourless to filbert；Conidium is colourless, crescent, no separates, two blunt point, size (18.82～
26.47) × (3.53～5.29) μm；Appresorium (6.60～10.63 (8.21)) × (4.31～8.00 (6.47)) μm, oval
Shape, accidental Rhizoma Zingiberis Recens lobed[5].
The classification of traditional pathogen, identification are based primarily upon morphological characteristic, Pathogenicity etc., complex operation, time-consuming,
Sensitivity is low, be easily subject to artificially and the factors such as environment are disturbed[6]It is impossible to make diagnosis in disease incubation period and initial phase, very
Hardly possible is timely monitored and effective control to disease.
With the development of molecular biology, Protocols in Molecular Biology has progressively been applied in the research of tack anthrax.On
Century, the method for common pcr that grows up of the eighties was used successfully to detection tack anthrax[7], but common pcr is rotten
Therefore outmoded technology and have some defects, such as detection time is still long, general 6～8h, the identification to target gene section
Site is few, and detection specificity is low with sensitivity；The cumbersome grade of detection process is it is difficult to meet demand.
Loop-mediated isothermal amplification technique (loop-mediated isothermal amplification, lamp) is Japan
A kind of new nucleic acid amplification technologies that can invent of Rong Yan strain formula[8], because it is simple to operate, quick, specificity is high, low cost
The advantages of, become the new nucleic acid amplification technologies that can substitute common pcr.It is the 4 kinds of spies of 6 regions designs for target gene
Different primer, causes self-loopa strand replacement reaction, 60～65 DEG C of scope 60～70min in the presence of bst Large fragment polymerase
Interior, while a large amount of synthesis target dna, the magnesium pyrophosphate precipitation with by-product white produces[9].Due to lamp amplification
Process relies on identification 6 isolated areas of target sequence, so atopic is very strong, and amplification process is in constant temperature
Under carry out, common water-bath or the equipment having stable thermal source just can meet and reacts requirement, and testing cost substantially reduces.
The selection of target gene is one of key factor of lamp detection.The target gene that common pcr commonly uses has ribosome
Internal gene transcribed spacers (internal transcribed space, its)[10], but many scholars think that this target can not
Enough differentiation colletotrichum funguses accurate in the level planted.
Modulation rna polymerase ii large subunit (the large subunit of rna polymerase that the present invention selects
Ii, rpb1) it is the topmost function subunit determining eukaryote messenger rna (mrna) transcription initiation and extending, exist multiple
Post translational modification, such as phosphorylation, glycosylation and ubiquitination.Conventional research finds, the ubiquitination of rpb1 removes and just participating in it
Often outside the protein metabolism under physiological statuss, may additionally facilitate the degraded of the rpb1 being stuck in transcription on dna, and promote
Transcription when extensive dna damages couples reparation (transcription coupled repair, tcr).Therefore, rpb1's is general
Elementization is modified by the activated protein level of tight control rpb1 it is ensured that being normally carried out of transcription, and to dna injury repairing and
Ensure that stability, the fidelity of hereditary material are significant[11].
Known tack anthrax Molecular Detection target its can not meet the needs of lamp design of primers, existing based on general
Logical pcr is more backward to the molecular detection technology of this pathogenic bacteria, and it is time-consuming partially long, to target gene section recognition site to have detection
Few, false drop rate is higher, and detection specificity is low with sensitivity, the defect such as detection process is cumbersome.
Content of the invention
The purpose of the present invention is the above-mentioned deficiency for prior art, provides a kind of ring mediation of detection tack anthrax etc.
Warm amplimer compositionss.
It is a further object of the present invention to provide the application of this Primer composition.
It is yet another object of the invention to provide a kind of lamp test kit of detection tack anthrax.
The purpose of the present invention can be achieved through the following technical solutions:
The application in detection tack anthrax as detection target of rpb1 gene order shown in seq id no.1, excellent
It is selected in lamp and detect the application in tack anthrax.
A kind of Primer composition detecting tack anthrax for lamp, the positive outer primer shown in seq id no.2
Reverse outer primer rpb1-b3 shown in rpb1-f3, seq id no.3, the positive inner primer rpb1- shown in seq id no.4
Reverse inner primer rpb1-bip shown in fip, seq id no.5, the ring primer rpb1-lf composition shown in seq id no.6.
Application in detection tack anthrax for the Primer composition of the present invention.
Application in preparing tack anthrax detectable for the Primer composition of the present invention.
A kind of lamp test kit for detecting tack anthrax, comprises Primer composition of the present invention.
Described lamp test kit preferably comprises detection solution, and the detection solution described in every milliliter is prepared by the following method
Obtain: 0.8 μm of positive inner primer rpb1-fip, 0.8 μm of reverse inner primer rpb1-bip, 0.1 μm of forward direction outer primer rpb1-f3,
0.1 μm of reverse outer primer rpb1-b3,0.1 μm of ring primer rpb1-lf, 1.4mmdntps, 8mmmgso4, 0.8m glycine betaine, 0.8m
tris-hcl(ph8.8)、0.4mm kcl、0.4mm(nh4)2so4, 4%triton x-100,8u μ l-1bst dna
Polymerase, adds ultra-pure water to be prepared into 1ml detection solution.
Described test kit also contains dyestuff sybr green i.
A kind of method that lamp detects tack anthrax, including the detection solution taking described in 18 μ l, 3 μ l sterilizing deionizations
Water, adds 4 μ l dna solution to be checked, and cumulative volume carries out lamp reaction for 25 μ l；Response procedures are: 62 DEG C, 70min；Add after amplification
Enter dyestuff sybr green i as reaction indicator, perusal, with the color change of sybr green i and fluorescence power
As result judgement standard: under ordinary light, yellow green represents test positive, that is, with the presence of tack anthrax, orange expression inspection
Surveying result is feminine gender, that is, do not contain tack anthrax, or contained tack anthrax does not reach test limit.
The present invention describes in detail:
1st, the design of primer
The screening of the selection of modulation gene and primer is the key factor of lamp detection.First look for pertinent literature choosing
Chs (chitin synthetase), sod are taken2(Mn oxide dismutase), gs (glutamine synthetase), its (ribosomal gene
Transcribed spacer), the availability target such as ef1 α (translation elongation factor), rpb1 (rna polymerase ii large subunit).With target chs
As a example, search and download the chs sequence contained by tack anthrax and its close kind in ncbi website, then soft using bioedit
Part carries out sequence alignment, chooses one section of its distinctive sequence as target sequence (200 in the chs sequence of tack anthrax
～400bp), with primer4 Photographing On-line primer.Chosen according to primer length, primer free energy and gc content etc. and suitably draw
Thing is tested, and therefrom in screening kind, versatility is good, inter-species high specificity, and specificity among genus are strong, the high primer of sensitivity.Successively
Screen other targets, through analysis during sequence alignment, we find that rpb1 gene is highly conserved between different strains in planting, inter-species has
Notable difference, final choice rpb1 is as the target gene of detection tack anthrax.Again the many sets being designed with this target base are drawn
Thing carries out test of many times and checking, and it is good to select a set of versatility, high specificity, and sensitivity is high, can be quick, accurately detect tack
Anthrax primer.
2nd, primer screening and proof procedure
(1) tack anthrax lamp primer is detected by versatility experiment sieving
1) for the purpose of screening the lamp primer that can detect tack anthrax, carry out following experiment: select tack anthrax
Reference culture, and it is derived from different regions, the dna of the tack anthrax of different host as template, take 4 μ l dna solution, plus
Enter 18 μ l difference the prepared reactant liquors of lamp primer that go out of drone designs and carry out lamp reaction, and 3 μ l sterile deionized waters are carried out
Lamp reacts, and response procedures are: 62 DEG C of 70min；Observing response pipe color change after reaction, detects, containing tack under ordinary light
The sample of anthrax strains as yellow green under ordinary light, and negative control is orange under ordinary light.If reaction can make after terminating owning
Sample containing tack anthrax is changed into yellow green, then this primer meets versatility requirement of experiment, can proceed with next step sieve
Choosing experiment；If all samples containing tack anthrax can not be made to be changed into yellow green, this primer does not meet requirement of experiment, gives up
Abandon.
(2) pass through specific test selective mechanisms tack anthrax lamp primer
1) for the purpose of screening the lamp primer that can specifically detect tack anthrax, carry out following experiment: select tack
Anthrax reference culture, close with it kind of colletotrichum gloeosporioides Penz and sharp spore anthrax, and other pathogenic fungi (purples spot of soybean
Bacterium, aspergillus oryzae, alternaric bacteria, Semen sojae atricolor charcoal rot bacterium, epicoccum nigrum, southern stem canker of soybean, Semen sojae atricolor north stem canker
Pathogenic bacteria, soybean Phomopsis seed decay pathogen, brown stem rot bacterium, phytophthora sojae kaufmann＆gerdemann, sharp spore reaping hook pathogenic bacteria, the flat umbilicuss of Semen Maydiss are compacted
Spore) dna as template, take 4 μ l dna solution, add 18 μ l through 1) the lamp primer institute that goes out of the different drone designs that filter out
Prepare reactant liquor and 3 μ l sterile deionized waters carry out lamp reaction, response procedures are: 62 DEG C of 70min；Observing response pipe after reaction
Color change, detects under ordinary light, the sample containing tack anthrax strains as yellow green under ordinary light, and contains the sample of other kinds
Product and negative control are orange under ordinary light.If after reaction terminates, except tack anthrax is yellow green, 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) pass through sensitivity test selective mechanisms tack anthrax lamp primer
For the purpose of screening the lamp primer that can delicately detect tack anthrax, carry out following experiment: select tack
Anthrax reference culture, standard tack anthrax bacteria strain dna is used depc water afterwards with spectrophotometric determination concentration (1ng/ μ l)
Carry out 10 doubling dilutions, -70 DEG C preserve as template.Take each concentration dna diluent 4 μ l after 10 doubling dilutions respectively as mould
Plate, adds 18 μ l through 2) the prepared reactant liquor of lamp primer that goes out of the different drone designs that filter out and 3 μ l sterile deionized waters
Carry out lamp reaction, response procedures are: 62 DEG C of 70min；Observing response pipe color change after reaction, detects under ordinary light, negative
It is orange to impinging upon under ordinary light, record is changed into the concentration of yellowish green sample, choose detection sensitivity highest primer.
Through series of experiments, the present invention finally adopts a set of versatility in the primer designing with rpb1 for target gene
Good, high specificity, sensitivity is high, can be quick, accurately detects the primer of tack anthrax, by nvitrogen (Shanghai) company
Synthesis, its sequence information is as follows:
Table 1
3. the bacterial strain of participating in the experiment that this institute of bacterial strain of participating in the experiment uses is as shown in table 2:
Table 2
Beneficial effect:
Inventor finds this gene order different strains in colletotrichum kind when rpb1 gene order is compared
Between highly conserved, there is abundant change in inter-species, can be used as the candidate targets gene of this pathogenic bacteria Molecular Detection.The present invention analyzes
Tack anthrax rpb1 gene and difference in sequence for other anthrax, design and have screened four specific lamp primers
With a ring primer, establish the lamp system of detection tack anthrax on this basis.The present invention solves in prior art
Cycle length needed for the biological detection method of tack anthrax, waste time and energy, loaded down with trivial details, poor specificity problem and pcr detection skill
Art needs thermal cycler instrument it is impossible to the problem of quick detection tack anthrax, detection cycle short (only needing 70min), high specificity,
Susceptiveness is high, can perusal testing result.
Compared with prior art, its advantage and good effect show the present invention:
(1) practicality is good.Common pcr reaction carries out gel electrophoresiss and with seeing under ultraviolet light after ethidium bromide staining to product
Examine dish and can differentiate result, this not only increases detection required time and detection operation link, be also easy to cause product to spread, become real
Test important sources of chamber contamination；And ethidium bromide (eb) has severe toxicity, can accumulate carcinogenic；Additionally, ultraviolet light also can be to reality
The personnel of testing cause a certain degree of injury.And lamp reaction only need to be carried out in thermostat water bath, reaction is passed through after terminating
The color change of sybr greeni just with the naked eye can directly observe judged result under ordinary light, and the present invention is simple to operate, is taken
Between short (completing one-time detection only needs 2h), pollution little, there is higher practicality.
(2) constant-temperature amplification.Unlike pcr method has to thermal cycle, thus break away from the dependence to thermal cycler instrument, as long as
There is stable thermal source such as thermostat water bath lamp reaction just can complete the instrument and equipment it is not necessary to costliness, so being easy to basic unit
Agricultural production unit is applied.Why lamp can react under constant thermal source is because with the addition of in lamp reactant liquor
Glycine betaine, makes double-strand dna be in the dynamic equilibrium unwind, and realizes amplification in the presence of bst dna polymerase.
(3) accuracy is high.Time-consuming, sensitivity is low for traditional tack anthrax detection technique, it is artificial to be easily subject to and environment etc.
The interference of factors；And the present invention chooses the specific lamp primer of the distinctive one section of rpb1 sequential design of tack anthrax,
Identify 6 isolated areas on target sequence by 4 primer specificity, identify 2 independent zones of target sequence with respect to pcr primer
For domain, specificity and sensitivity are higher.
(4) it is obviously improved technical merit.Based on target gene rpb1 design and the high specific primer screening and lamp skill
Art combines the tack anthrax rapid molecular detection system of foundation, overcame the defect existing for de-correlation technique, and shortened
Detection required time, simplifies operating process, improves specificity and the sensitivity of detection, be that technical merit is upgraded.
Brief description
Fig. 1 naked-eye observation result, ep pipe in left side is in yellow green, be expressed as the detection of tack anthrax positive (+), right side ep manages
In orange, be expressed as the detection of tack anthrax negative (-).
The versatility based on the lamp technology for detection tack anthrax of target gene rpb1 for the Fig. 2
Wherein, No. 1 is tack anthrax bacteria reference culture (positive control), and 2～No. 8 is to adopt on the difference host of different regions
The tack anthrax bacteria bacterial strain that sample is separated to, No. 9 is water (negative control).2～No. 8 hosts and area see that this specification table 2 carries
For bacterial strain information of participating in the experiment.Semen sojae atricolor from Jiangsu Jiangpu farm, Semen arachidis hypogaeae, Fructus Capsici and Jiangsu Taixing, Xuzhou, Jiangsu Rhizoma Zingiberis Recens
The tack anthrax being separated on the Semen sojae atricolor on ground such as weir, Jiangsu Jiangyan City, Hubei Wuhan, Anhui Suzhou, Anhui Long Kang farm totally 70
Strain, has been involved in verifying, result shows to be all positive reaction (yellow green), illustrates this technology to from different hosts and area
The detection of tack anthrax is all applicable.Fig. 2 only have chosen 8 plants of representative bacterial strains and lists.
The result of lamp technology for detection colletotrichum gloeosporioides Penz, sharp spore anthrax and other funguses based on target gene rpb1 for the Fig. 3
Wherein, No. 1 is tack anthrax bacteria reference culture (positive control), and 2～No. 7 is glue spore anthrax bacteria
(colletotrichum gloeosporioide) different strains, No. 8, No. 9 is point spore anthrax bacteria
(colletotrichum.acutatum) different strains, No. 10 is Cercospora kikuchii (cercospora kikuchii), 11
Number be aspergillus oryzae (aspergillus oryzae), No. 12 be alternaric bacteria (alternaria alternata), No. 13 are
Semen sojae atricolor charcoal rot bacterium (macrophomina phaseolina), No. 14 is epicoccum nigrum (epicoccum nigrum), and No. 15 are
Southern stem canker of soybean (diaporthe phaseolorum var.caulivora), No. 16 is Semen sojae atricolor north stem canker
Pathogenic bacteria (diaporthe phaseolorumvar.meridionalis), No. 17 is soybean Phomopsis seed decay pathogen
(phomopsis longicolla), No. 18 is brown stem rot bacterium (phialophora sojae), and No. 19 is soybean phytophthora
Pathogenic bacteria (phytophthora sojae), No. 20 sharp spore reaping hook pathogenic bacterias (fusarium oxysporum), the flat umbilicuss of No. 21 Semen Maydiss
Compacted spore (bipolaris maydis), No. 22 is water (negative control).What this specification table 2 provided seen in pathogen host and area
Participate in the experiment bacterial strain information.Result shows, all colletotrichum gloeosporioides Penzs supplying examination, sharp spore anthrax and other pathogenic fungi are all in orange
Negative reaction, only No. 1 tack anthrax bacteria reference culture is in yellowish green positive reaction.Show the rpb1- that the present invention sets up
Lamp technology can distinguish target pathogenic bacteria (tack anthrax) and non-targeted pathogen (other anthrax and other pathogen).
The sensitivity based on the lamp technology for detection tack anthrax bacteria of target gene rpb1 for the Fig. 4
Lamp expands variable concentrations tack anthrax bacteria genome dna；No. 1 is tack anthrax bacteria reference culture genome
(positive control), 2～9 be 25 μ l reaction systems in contain respectively 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg,
The lamp amplification of 10fg tack anthrax bacteria dna, No. 10 is negative control.Lamp amplified reaction can specifically identify flat
Head anthrax bacteria, picture represents that sensitivity can reach 100pg/ μ l.
The tack anthrax that the lamp technology for detection based on target gene rpb1 for the Fig. 5 is fallen ill in soybean plant strain
Wherein No. 1 is tack anthrax bacteria reference culture genome (positive control), and 2～7 is inoculation tack anthrax bacteria
Soybean plant strain genome after 6 days for the bacterial strain, 8 is the blank soybean plant strain genome after 6 days for the pda culture medium of inoculation, and 9 is aquesterilisa
Replace dna to expand as negative control.Result shows, the soybean plant strain (2～No. 7 pipes) of inoculation tack anthrax is under ordinary light
Yellowish green positive reaction, effect and direct detection tack anthrax dna (No. 1 pipe) do not have difference, and inoculate blank pda culture
Soybean plant strain after 6 days for the base (No. 8 pipes) and negative control aquesterilisa (No. 9 pipes) then in ordinary light in orange negative reaction, explanation
The rpb1-lamp technology that the present invention sets up can specifically detect tack anthrax from the soybean plant strain of inoculation morbidity, can
For field quick detection.
The sensitivity in actual applications of the lamp technology based on target gene rpb1 for the Fig. 6
No. 1 is Semen sojae atricolor tack anthrax bacteria reference culture genome (positive control), and 2～No. 6 is in every 50g Semen sojae atricolor sample
Add the dna of the Semen sojae atricolor sample extraction of different spore amounts, 2～No. 6 spore additions are followed successively by 10000,1000,100,50,10
Individual.No. 7, No. 8 are respectively the dna of Semen sojae atricolor sample extraction adding 0 spore and aquesterilisa replacement dna amplification as negative right
According to.Result shows, the sensitivity of the rpb1-lamp technology for detection tack anthrax that the present invention sets up is that 10 spore/50g are big
Bean.
Fig. 7 collects in the lamp technology for detection market based on target gene rpb1 the tack anthrax in Semen sojae atricolor
Extract dna to from Liaoning, Anhui, Jiangsu, Hubei, Shandong, Heilungkiang, the Semen sojae atricolor of zhejiang and other places and soybean pod residuum
Carry out lamp detection.Wherein, No. 1 is tack anthrax bacteria reference culture genome (positive control), and 2～No. 11 are respectively derived from
Dalian, Fuyang, Xuzhou, Hubei Wuhan, Shandong Weifang, Shandong Dezhou, Shandong Zibo, Heilungkiang Mudanjiang,
The dna that Zhejiang Jiaxing, the regional soybean seed in 10, Jiangsu Taizhou are extracted.No. 12 is negative control.Result shows, from the Liao Dynasty
Peaceful Dalian, Fuyang, Xuzhou, Hubei Wuhan, Shandong Weifang, the local Semen sojae atricolor in 6, Shandong Dezhou and soybean pod residuum dna
In yellowish green positive reaction, illustrate that it carries tack anthrax.
Specific embodiment
Main agents and instrument that following examples use: bst dna polymerase (new england biolabs),
betaine、mgs04(sigma)；Dntps, eppendorf common pcr amplification instrument, eppendorf biophotometer light splitting
Photometer, upper Nereid grand experimental apparatus factory dk-8d type electrical heating thermostat water bath.
A kind of lamp detection kit for detecting tack anthrax of embodiment 1
Test kit reaction system: detection solution and dyestuff sybr green i.
Detection solution manufacturing method is as follows:
0.8 μm of positive inner primer rpb1-fip, 0.8 μm of reverse inner primer rpb1-bip, 0.1 μm of positive outer primer rpb1-
F3,0.1 μm of reverse outer primer rpb1-b3,0.1 μm of ring primer rpb1-lf, 1.4mmdntps, 8mmmgso4, 0.8m glycine betaine,
0.8m tris-hcl(ph8.8)、0.4mm kcl、0.4mm(nh4)2so4, 4%triton x-100,8u μ l-1bst dna
Polymerase, adds ultra-pure water to be prepared into 1ml detection solution, the pot-life is 1 year.
Embodiment 2 test kit versatility of the present invention is investigated
Select the tack anthrax reference culture shown in table 2, and the tack anthrax from different regions, different host
Dna as template, take 4 μ l dna solution, add 18 μ l detection solution and 3 μ l sterile deionized waters to carry out lamp reaction, instead
The program is answered to be: 62 DEG C of 70min；Add 0.25 μ l dyestuff sybr green i after amplification as reaction indicator, observe after reaction
Reaction tube color change, detects under ordinary light, the sample from different hosts and the tack anthrax in area all becomes under ordinary light
For yellow green, negative control is orange under ordinary light.Illustrate this technology to the tack anthrax from different hosts and area
Detection is all applicable.Fig. 2 only have chosen 8 plants of representative bacterial strains and lists.
Embodiment 3
Select tack anthrax reference culture, close with it kind of colletotrichum gloeosporioides Penz and sharp spore anthrax, and other cause of diseases
Funguses (Cercospora kikuchii, aspergillus oryzae, alternaric bacteria, Semen sojae atricolor charcoal rot bacterium, epicoccum nigrum, southern stem canker of soybean,
Semen sojae atricolor north stem canker, soybean Phomopsis seed decay pathogen, brown stem rot bacterium, phytophthora sojae kaufmann＆gerdemann, sharp spore reaping hook disease
Bacterium, Bipolaris maydis) dna as template, take 4 μ l dna solution, add 18 μ l detection solution and 3 μ l sterilizing deionization
Water carries out lamp reaction, and response procedures are: 62 DEG C of 70min；0.25 μ l dyestuff sybr green i is added as reaction after amplification
Indicator, observing response pipe color change after reaction, detects under ordinary light, the sample containing tack anthrax strains under ordinary light
For yellow green, and containing the sample of other kinds and negative control is orange under ordinary light.Result shows, all glue spore anthraxs for examination
, all in orange negative reaction, only No. 1 tack anthrax bacteria reference culture is in yellow green for bacterium, sharp spore anthrax and other pathogen
Positive reaction.Show that the rpb1-lamp technology that the present invention sets up can distinguish target pathogenic bacteria (tack anthrax) and non-mesh
Secondary disease opportunistic pathogen (other anthrax and other pathogen) (Fig. 3).
Embodiment 4
Select tack anthrax reference culture, by standard tack anthrax bacteria strain dna spectrophotometric determination concentration (1 μ
G/ μ l) after carry out 10 doubling dilutions with depc water, -70 DEG C preserve as template.Take each concentration dna after 10 doubling dilutions respectively
Diluent 4 μ l, as template, adds 18 μ l detection solution and 3 μ l sterile deionized waters to carry out lamp reaction, response procedures are: 62
℃70min；0.25 μ l dyestuff sybr green i is added as reaction indicator, after reaction, observing response pipe color becomes after amplification
Change.Result such as Fig. 4, represent that sensitivity of the present invention can reach 100pg/ μ l.
Embodiment 5 is based on the tack in the lamp technology for detection artificial vaccination morbidity group Semen sojae atricolor diseased tissuess of target gene rpb1
Anthrax
With plant genes group dna extracts kit () extract 6 plants of inoculation tack anthrax bacterias after 6 days
Soybean plant strain dna, as template be used for lamp expand, using tack anthrax bacteria reference culture dna as the positive right
According to healthy plant dna and aquesterilisa replacement dna expands as negative control.Take 4 μ ldna solution, add 18 μ l embodiment 1 institute
The detection solution stated and 3 μ l sterile deionized waters carry out lamp reaction, and response procedures are: 62 DEG C, 70min.Add after amplification
0.25 μ l dyestuff sybr green i as reaction indicator, observing response pipe color change after reaction, under ordinary light detect, knot
Fruit is as shown in figure 5, display this method can become under ordinary light from the soybean plant strain (2～No. 7 pipes) of inoculation tack anthrax bacteria
Yellow green, can specifically detect that tack anthrax bacteria, effect and direct detection tack anthrax bacteria dna (No. 1 pipe) do not have
There is difference, and inoculate soybean plant strain (No. 8 pipes) after 6 days for the blank pda culture medium, negative control aquesterilisa (No. 9 pipes) then normal
It is orange it is seen that this method can be used for Fields detection under light.
The tack charcoal fallen ill in soybean plant strain diseased tissuess in the lamp technology for detection field based on target gene rpb1 for the embodiment 6
Cellulitis bacterium
Gather from Jiangsu Jiangpu farm, Xuzhou, Hubei Wuhan, Fuyang, Anhui Suzhou, Anhui Long Kang farm
Choose typical incidence tissue in Semen sojae atricolor sample, cleaning is dried, with plant genes group dna extracts kit () extract its genome.It is used for lamp as template to expand, tack anthrax bacteria reference culture dna is made
For positive control, aquesterilisa replacement dna amplification is as negative control.Take 4 μ ldna solution, add the above-mentioned detection solution of 18 μ l and 3
μ l sterile deionized water carries out lamp reaction, and response procedures are: 62 DEG C, 70min.Dyestuff sybr is added after amplified reaction terminates
Green i is as indicator.If reactant liquor color is changed into yellow green represents test positive, contain in examination soy bean plant tissue
Tack anthrax；If the orange invariant representation of color keep is detected as feminine gender, do not contain tack anthrax in examination soy bean plant tissue.
1. testing result is as follows:
Nanjing and Xuzhou: 61 parts of samples, detect 15 positives；
Hubei Wuhan: 50 parts of samples, detect 33 positives；
Anhui Suzhou and Huaiyuan: 43 parts of samples, detect 13 positives.
2. the Semen sojae atricolor diseased tissuess material of pair test positive carries out pathogenicbacteria separation, and the pathogen isolating ground is through morphology
Observe and its sequencing compares, be tack anthrax, prove that detection positive findingses are correctly reliable further.
The sensitivity in actual applications of the lamp technology based on target gene rpb1 for the embodiment 7
Choose same kind, areal, the no Semen sojae atricolor of tack anthrax and soybean pod residuum sample, enter according to the following steps
OK:
1) randomization
Semen sojae atricolor and soybean pod residue sample rock 5 points of samplings of mixing in plate, choose 6 parts of sample next step standby.
2) washing of sample
6 parts of Semen sojae atricolor Semen sojae atricolor and soybean pod residuum are respectively weighed 50g, pour in 250ml triangular flask, add the sterilized water of 100ml,
Deca 20% tween 3-5 drips, and is then respectively adding the conidium of 10000,1000,100,50,10,0 tack anthrax, envelope
Live bottleneck, be placed in 220rpm, 30min on shaking table, concussion washing.
3) collection of filtrate
Mixed liquor after concussion washing 200 mesh steel are sieved through filter, and the Soybeanresidue being sieved with a small amount of aseptic water washing steel,
Filtrate collection is in former triangular flask.
4) be collected by centrifugation precipitation,
The filtrate collected is attached in 100ml centrifuge tube, is centrifuged by several times, 6500rpm, 10min, abandon supernatant, it is heavy to collect
Form sediment.
5) precipitate is processed
Put the precipitate in 37 DEG C of calorstats and dry.
6) extraction of the total genome of precipitate
Due to containing a large amount of soil constituents in precipitate, so experiment is using the soil microorganism dna of mobio company of the U.S.
Strength extracts kitDna isolation kit is extracted, and method is as follows:
The precipitate 0.25g taking drying is added in powerbead pipe, adds 60 μ l c1, and whirlpool shakes 10min；Room temperature
10000g, 30s are centrifuged.In Aspirate supernatant 400～500 μ l to 2ml collection tube, add 250 μ in centrifuge tube
L c2, whirlpool shakes 5s, is placed in 4 DEG C, 5min；Room temperature is centrifuged 10000g, 1min；Draw supernatant 600 μ l to new 2ml
In collection tube；Add 200 μ l c3 in centrifuge tube, after whirlpool concussion 5s, be placed in 4 DEG C of 5min；Room temperature is centrifuged
10000g, 1min；Draw in supernatant 750ul to new 2ml collection tube；Xiang Guanzhong adds 1.2ml c4, whirlpool
Concussion 5s；Mixed liquor is transferred in spin fittle, 10000g, 1min are centrifuged, and abandon filtrate；Add 500ul in filter post
C5,10000g, 30s, abandon filtrate；Filter post idle running centrifugation, 10000g, 1min；Filter post is positioned over new 2ml collection
In tube, 37 DEG C of constant temperature dry；Add 100ul c6 in filter post, place 2min, 10000g, 1min are centrifuged.Gained filtrate is
The genome extracting, places standby.
7) extract Semen sojae atricolor tack anthrax lamp detection in genome
Tack anthrax lamp detects: takes 4uldna solution, adds 18 μ l mentioned reagent box solution and 3 μ l sterilizing deionization
Water carries out lamp reaction.
Reaction condition: 62 DEG C, 70min.
Amplified production detects: adds dyestuff sybr green i after amplified reaction terminates as indicator.If reactant liquor
Color is changed into yellow green and represents test positive, contains tack anthrax in examination Semen sojae atricolor；If the orange invariant representation of color keep
It is detected as feminine gender, do not contain tack anthrax in examination Semen sojae atricolor.Result shows, the rpb1-lamp technology that the present invention sets up is for big
In bean and soybean pod residuum sample, the detection sensitivity of tack anthrax is 10 spore/50g Semen sojae atricolor (Fig. 6).
The tack anthrax in Semen sojae atricolor is collected in the lamp technology for detection market based on target gene rpb1 for the embodiment 8
From Dalian, Fuyang, Xuzhou, Hubei Wuhan, Shandong Weifang, Shandong Dezhou, Shandong Zibo, black dragon
River Mudanjiang, Zhejiang Jiaxing, Jiangsu Taizhou collect 10 parts of soybean seed, 10 portions of Semen sojae atricolor and soybean pod residuum are respectively weighed 50g,
Pour in 250ml triangular flask, add the sterilized water of 100ml, Deca 20% tween 3-5 drips, and seals bottleneck, is placed on shaking table
220rpm, 30min, concussion washing.Then it is repeated in 3～6 steps in example 3, extract genome.
Extract tack anthrax lamp detection in genome
Tack anthrax lamp detects: takes 4 μ ldna solution, adds 18 μ l mentioned reagent box solution and 3 μ l sterilizing deionization
Water carries out lamp reaction.
Reaction condition: 62 DEG C, 70min.
Amplified production detects: adds dyestuff sybr green i after amplified reaction terminates as indicator.If reactant liquor
Color is changed into yellow green and represents test positive, contains tack anthrax in examination Semen sojae atricolor；If the orange invariant representation of color keep
It is detected as feminine gender, do not contain tack anthrax in examination Semen sojae atricolor.Result shows, from Dalian, Fuyang, Xuzhou,
Hubei Wuhan, Shandong Weifang, the local Semen sojae atricolor in 6, Shandong Dezhou and soybean pod residuum dna are in yellowish green positive reaction, and it is described
Carry tack anthrax (Fig. 7).
List of references
1. He Yongmei, Luo Guangyao (2012). the identification of soybean anthracnose and preventing and treating. pesticide market information 19:44.
2. kingdom's honor (2009). " Taiwan 75 " eats soybean pod anthrax process for comprehensively treating research and extension raw. Zhejiang
University's master thesis.
3. Sun Zhi peak, Lou Binggan, kingdom is flourish to wait (2008). soybean pod anthrax bacteria biological characteristicses. Zhejiang Agriculture
Report 20 (6): 432-436.
4. building soldier does, Chen Wujian, woods hairpin etc. (2009). a kind of new soybean pod anthrax symptom type and its cause of disease mirror
Fixed. plant protection journal 36 (05): 229-233.
5. Xiao Jiewen, Liu Yuelian, Ran Junxiang etc. (2011). the isolation identification research of anthrax in Brazil Soybeans. Chinese agriculture
Learn circular 27 (05): 333-337.
6.daniells j,davis d,peterson r,et al.(1995).goldfinger:not as
resistant to sigatoka/yellow sigatoka as first thought.infomusa 4(1):6.
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.
11. Li Hui (20086) hect family ubiquitin ligase wwp2 ubiquitination rna polymerase ii large subunit rpb1
[d]. Postgraduate School, Chinese Academy of Sciences (Shanghai life science institute)
Claims (7)
1. a kind of detect the Primer composition of tack anthrax it is characterised in that for lamp shown in seq id no.2 just
To outer primer rpb1-f3, the reverse outer primer rpb1-b3 shown in seq id no.3, just inwardly drawing shown in seq id no.4
Reverse inner primer rpb1-bip shown in thing rpb1-fip, seq id no.5, the ring primer rpb1-lf shown in seq id no.6
Composition.
2. application in detection tack anthrax for the Primer composition described in claim 1.
3. application in preparing tack anthrax detectable for the Primer composition described in claim 1.
4. a kind of lamp test kit for detecting tack anthrax is it is characterised in that comprise the primer sets described in claim 1
Compound.
5. lamp test kit according to claim 4 is it is characterised in that comprise to detect solution, the detection described in every milliliter is molten
Liquid is prepared by the following method and obtains: 0.8 μm of positive inner primer rpb1-fip, 0.8 μm of reverse inner primer rpb1-bip, 0.1
μm positive outer primer rpb1-f3,0.1 μm of reverse outer primer rpb1-b3,0.1 μm of ring primer rpb1-lf, 1.4 mmdntps, 8
mmmgso4, 0.8 m glycine betaine, 0.8 m tris-hcl (ph8.8), 0.4 mm kcl, 0.4 mm (nh4)2so4、4%
triton x-100、8u•μl-1Bst dna polymerase, adds ultra-pure water to be prepared into 1 ml detection solution.
6. the lamp test kit according to claim 4 or 5 is it is characterised in that described test kit also contains dyestuff sybr
green i.
7. a kind of lamp detects the method for tack anthrax it is characterised in that including taking the inspection described in 18 μ l claim 5
Survey solution, 3 μ l sterile deionized waters, add 4 μ l dna solution to be checked, cumulative volume carries out lamp reaction for 25 μ l；Reaction interval
Sequence is: 62 DEG C, 70 min；Add dyestuff sybr green i as reaction indicator after amplification, perusal, with sybr
The color change of green i and fluorescence power are as result judgement standard: under ordinary light, yellow green represents test positive, that is,
With the presence of tack anthrax, orange expression testing result is feminine gender, that is, do not contain tack anthrax, or contained tack anthrax is not
Reach test limit.
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| CN109609683A (en) * | 2019-01-25 | 2019-04-12 | 福建省农业科学院果树研究所 | A kind of LAMP detection primer detecting colletotrichum gloeosporioides Penz in olive tissue |
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