CN103740806B - A kind of method of No. 4 microspecies in the detection by quantitative banana blight bacteria torrid zone from soil - Google Patents
A kind of method of No. 4 microspecies in the detection by quantitative banana blight bacteria torrid zone from soil Download PDFInfo
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Abstract
The invention discloses a kind of method of No. 4 microspecies in the detection by quantitative banana blight bacteria torrid zone from soil, the method step is: carry out pre-treatment to test sample, extracts the DNA of test sample; The preparation of Auele Specific Primer; Fluorescence nucleic acid constant-temperature amplification detection technique RealAmp reacts; The structure of RealAmp typical curve; Result judges.The method of a kind of No. 4 microspecies in the detection by quantitative banana blight bacteria torrid zone from soil of the present invention overcomes the deficiency of gene amplification method in the past, and have high specific, highly sensitive, detection by quantitative lower limit is 4.3 × 10
-4ng/ μ l, easy to operate, used time is short, only need 2-3h, not vulnerable to pollution thing impact and can Site Detection pedotheque, do not need costliness and the temperature cycling device of precision, only need can reach the thermostat of 63 DEG C, analyze the method judging reaction product very simple, only need visual inspection yet, the content of No. 4 microspecies in the banana blight bacteria torrid zone in soil can be differentiated again quantitatively, be suitable for applying widely.
Description
Technical field
The invention belongs to No. 4 microspecies detection fields in the banana blight bacteria torrid zone, particularly relate to a kind of method of No. 4 microspecies in the detection by quantitative banana blight bacteria torrid zone from soil.
Background technology
Banana is Musaceae banana, is the tropical and subtropical zone important cash crop in area and food crop.Banana blight is the destructive disease of banana industry, seriously governs China's banana industry and develops in a healthy way.
Pathogen Causing Banana Fusarium Wilt is FusariumoxysporumSchlevht.f.sp.cubense (E.F.Smith) Snyd. & Hans., former name FusariumcubenseE.F.Smith, belong to imperfect fungi, knurl seat Zoopagales, Fusarium, Chinese is Fusarium oxysporum Cuba specialized form.This bacterium is divided into 3 microspecies.In China, be mainly No. 1 microspecies and No. 4 microspecies.No. 1 microspecies occur with a long history, can solve the harm of No. 1 microspecies by replanting Cavendish class banana.No. 4 microspecies imported China in 1996, the safety of serious harm China banana industry.No. 4 microspecies are subdivided into again No. 4, subtropics microspecies and the torrid zone No. 4 microspecies, in China's mainly No. 4 microspecies in the torrid zone.
The propagation harm of banana blight divides long-distance communications and closely propagates, and at a distance based on the allocation and transportation of pathogenic bacteria with tissue culture seedlings of bananas secondary seedling, closely propagating is that pathogenic bacteria spreads with soil-borne in any of several broadleaf plants garden.In production, in the urgent need to soil surface characters Fast Detection Technique, be used for detecting tissue cultured seedling secondary seedling soil, newly open up Banana soil and whether carry disease germs and the quick diagnosis of doubtful diseased plant.Therefore, the important guarantee that microspecies quick, stable detection method in a set of Pathogen Causing Banana Fusarium Wilt No. 4, torrid zone is China's banana safety in production is set up.
In recent years, the method of PCR-based has been used successfully to No. 4 microspecies in the detection Pathogen Causing Banana Fusarium Wilt torrid zone to have report to confirm, PCR method on the operating time and detect specificity and sensitivity in comparatively ordinary method improve a lot, but, PCR method must have accurate temperature cycling device, and its detection time also still long (2 ~ 3 hours), and reaction process is easy to the impact of contaminated thing, thus makes this method can not meet the needs of actual detection.Therefore, need exploitation one highly sensitive and willing method, the detection method of PCR can be replaced to a certain extent, and, common PCR method can only the having or nothing of qualitative detection pathogenic bacteria, can not the number of detection by quantitative pathogenic bacteria, more can not specify in soil have how many pathogenic bacterias? therefore, the newest fruits of biotech development is applied to Pathogen Causing Banana Fusarium Wilt No. 4, torrid zone microspecies to detect, significant.
Japanese scholars Notomi equals within 2000, to develop ring mediated isothermal nucleic acid amplification reaction (loop-mediatedisothermalamplification first, LAMP), this technology relies on six special primers and a kind of archaeal dna polymerase with strand displacement characteristic, quick under isothermal conditions, efficiently, high special, amplified target sequence with sensitivity, this technology first utilizes two inner primers and two outer primers, stem cyclic DNA structure is formed by strand displacement effect, then under the effect of inner primer by a large amount of amplified target sequence of automated cycle strand replacement reaction that ring mediates, there is high specificity, susceptibility is high, the feature such as simple and easy to do.Real-time fluorescence nucleic acid constant-temperature amplification detection technique (real-timefluorescenceloop-mediatedisothermalamplificatio nassay, be called for short RealAmp) be a kind of novel nucleic acids detection technique that the nucleic acid constant-temperature amplification technology of a new generation and real-time fluorescence detection technique are combined, can detection by quantitative pathogen, there is the advantages such as highly sensitive, high specific, low stain, stable reaction.In addition, SYBRGreenI is one of detection method that current LAMP product is the sensitiveest, but SYBRGreenI's adds the reaction efficiency that may suppress LAMP in RealAmp, therefore, in actually operating, generally adopt SYTO-9 fluorescence dye to carry out RealAmp reaction, and using a kind of auxiliary judgment measure of SYBRGreenI dyed color judged result as detection by quantitative.
The method is normally carried out as follows: step one, carry out pre-treatment to tested sample, the DNA of the tested sample of rapid extraction; The preparation of step 2, Auele Specific Primer: after determining the target gene of sample to be measured, obtains Auele Specific Primer 2 right, inner primer and each a pair of outer primer; Step 3, real-time fluorescence nucleic acid constant-temperature amplification (RealAmp) react: mix through the sample of pre-treatment, primer, reaction buffer with BstDNA polysaccharase, within 1.5 hours, carry out endless chain replacement(metathesis)reaction 63 DEG C of insulations; Step 4, drawing standard curve: the relation between the template concentrations that typical curve dilutes according to difference and corresponding Tt value builds, X-axis represents the logarithmic value of initial template concentration, and Y-axis represents that different concns template amplification reaches the threshold value time used (Tt).Step 5, analysis judge reaction product result.
The people such as Dita pass through the difference analyzed between the transcribed spacer of Pathogen Causing Banana Fusarium Wilt 20 Different Nutrition affinity types, devise the Auele Specific Primer of the pcr amplification of No. 4 microspecies in the torrid zone to FocTR4-F(5 '-CACGTTTAAGGTGCCATGAGAG-3 ')/FocTR4-R(5 '-CGCACGCCAGGACTGCCTCGTGA-3 '), amplified fragments size is 463bp.The research contents that the people such as Dita carry out, the method by means of only PCR detects No. 4 microspecies in the Pathogen Causing Banana Fusarium Wilt torrid zone qualitatively, is not detect from soil quantitatively.The people such as Li use loop-mediated isothermal amplification (LAMP) to have detected banana blight bacteria No. 4 microspecies (Fusariumoxysporiumf.sp.cubenserace4) in China by the method for SCAR, but not No. 4 microspecies in the detection by quantitative banana blight bacteria torrid zone from soil.
Use the PCR detection technique that the people such as Dita set up, need to extract the sample gene group DNA of higher degree, used time longer, can not Site Detection pedotheque, more can not detect pathogenic bacteria quantitatively from soil, and need the expensive and temperature cycling device of precision, and must could to the positive or negative differentiating amplified production by electrophoresis apparatus, cost is high, not easy to operate, need the professional with certain state of the art to carry out testing.The people such as Li have detected banana blight bacteria No. 4 microspecies of China by loop-mediated isothermal amplification (LAMP), and from soil, do not detect No. 4 microspecies in the banana blight bacteria torrid zone, do not detect the pathogenic bacteria content in soil quantitatively, the content of pathogenic bacteria in soil can not be specified yet.
Summary of the invention
The object of the present invention is to provide a kind of method of No. 4 microspecies in the detection by quantitative banana blight bacteria torrid zone from soil, being intended to solve normal PCR detection technique, to need to extract the sample gene group DNA of higher degree, used time longer, can not Site Detection pedotheque, pathogenic bacteria can not be detected quantitatively from soil, and need costliness and the temperature cycling device of precision, cost is high, not easy-operating problem.
The present invention is achieved in that a kind of method of No. 4 microspecies in the detection by quantitative banana blight bacteria torrid zone from soil, the method for No. 4 microspecies in the detection by quantitative banana blight bacteria torrid zone should comprise the following steps from soil:
Step one, pre-treatment is carried out to test sample, extract the DNA of test sample;
The preparation of step 2, Auele Specific Primer;
Step 3, fluorescence nucleic acid constant-temperature amplification detection technique RealAmp react;
The structure of step 4, RealAmp typical curve;
Step 5, result judge.
Further, carry out pre-treatment to test sample, the concrete grammar extracting the DNA of test sample is as follows:
The DNA nucleic acid extraction of test sample: add 300 μ lDNA extracting solutions in 0.1g detected sample, after grinding to form pasty state, after 95 DEG C of water bath with thermostatic control 10min, 25 DEG C, under 10000rpm centrifugal 2 minutes, gets 50 μ l supernatant liquors as detection template DNA;
DNA extraction liquid comprises: 100mMTris-HClpH7.4,1MKC1,10mMEDTA and 2%w/vPVPP.
Further, the preparation of Auele Specific Primer designs the LAMP detection primer of No. 4 microspecies in the banana blight bacteria torrid zone with reference to the 463bp fragment that banana blight bacteria No. 4, the torrid zone microspecies specific detection primers that Dita designs obtain, obtain RealAmp Auele Specific Primer 2 right, inner primer and each a pair of outer primer;
The primer mixed solution be made up of two pairs of primers, two pairs of primers are respectively: outer primer FocTR4-F3 i.e. 5 '-CACGTTTAAGGTGCCATGAGAG-3 ', outer primer FocTR4-B3 i.e. 5 '-CGCACGCCAGGACTGCCTCGTGA-3 ', inner primer FocTR4-FIP i.e. 5 '-ATTCAAGCCGGATTGACGGATTGGATATGTAGAGAATGTGGTGG-3 ', inner primer FocTR4-BIP i.e. 5 '-GGGAGCCAAGAAGAAGCAGGACCTTCGATTCTTGTATC-3 ', the concentration of described outer primer FocTR4-F3 and described outer primer FocTR4-B3 is respectively 5pmol/ μ l; The concentration of described inner primer FocTR4-FIP and described inner primer FocTR4-BIP is 40pmol/ μ l.
Further, step 3 fluorescence nucleic acid constant-temperature amplification detection technique (RealAmp) reacts specific as follows:
Mix through the sample of pre-treatment, primer, reaction buffer with BstDNA polysaccharase, carry out endless chain replacement(metathesis)reaction at 63 DEG C of insulation 90min;
RealAmp25 μ L reaction system: the nucleic acid DNA of 1 μ L extracting is as template, 2.5 μ L10 × BstDNApolymersebuffer, 1.4mMdNTPs, 0.8mM trimethyl-glycine, 8mMMgSO4, the outer primer of 0.2 μM, inner primer, the 1 μ LBstDNApolymerase of 1.6 μMs, 0.2 μM of SYTO-9 fluorescence dye, moisturizing to 25 μ L, and then the paraffin oil system such as adding covers whole reaction solution;
RealAmp reaction is carried out on ESE-QuantTubeScanner, 80 DEG C of reaction 10min termination reactions after 63 DEG C of reaction 90min;
RealAmp reaction before in reaction tubes, cover the SYBRGreenI fluorescence dye added as 1 μ L1:10 doubly dilutes, question response terminate rear brief centrifugation SYBRGreenI is added in LAMP reaction solution carry out colour developing observation; When positive control reaction is set, replace by the oranges and tangerines genome DNA infecting HLB; When negative control reaction is set, replace with 100mMTris-HClpH8.0 and 50mMEDTA, the reaction tubes containing the reaction soln prepared is placed in 63 DEG C of reaction 90min; Reaction terminates rear display screen display amplification curve, and X-axis represents proliferation time, Y-axis display fluorescent value.
Further, it is characterized in that, the construction process of step 4 RealAmp typical curve is as follows:
Adopt No. 4 microspecies Fusariumoxysporiumf.sp.cubenseTripicalrace4 in the banana blight bacteria torrid zone of Dita design, FocTR4 specific detection primer FocTR4-F5 '-CACGTTTAAGGTGCCATGAGAG-3 '/FocTR4-R5 '-CGCACGCCAGGACTGCCTCGTGA-3 ', amplified fragments size is 463bp; Clone the plasmid after this sequence to pMD18-T carrier order-checking correctly, called after pMD18-T-TR4, for building typical curve.
Further, the PCR detection reaction system method of No. 4 microspecies in the banana blight bacteria torrid zone is as follows:
2.5 μ L10 × PCRbuffer, 2 μ LdNTPs, 0.25 μ LTaq polysaccharase, each 1 μ L of primers F ocTR4-F/FocTR4-R, 1 μ LDNA, moisturizing to 25 μ L;
The condition of PCR reaction: 94 DEG C of denaturation 3min, 94 DEG C of sex change 30s, 57 DEG C of annealing 30s, 72 DEG C extend 60s, carry out 35 circulations; 72 DEG C extend 7min;
After reaction terminates, get 5 μ L reaction product, EB dyeing observations after 1% agarose gel electrophoresis;
Pcr amplification band is cut after glue reclaims connection pMD-18-T carrier and is checked order correctly, by this plasmid called after pMD18-T-TR4, after 10 times of gradient dilutions, build the typical curve of RealAmp of No. 4 microspecies in the banana blight bacteria torrid zone as template thus for assessment of the detection sensitivity of RealAmp.
Further, result judges employing two kinds of method judged results, and a kind of is the judgement of detection by quantitative result, and directly after ESE-QuantTubeScanner reaction terminates, instrument is automatically according to typical curve display quantitative result; In addition, reaction terminates centrifugally to be mixed in reaction solution by the SYBRGreenI covered in reaction tubes afterwards, and adopt covered colour developing to carry out judged result, if reaction solution color is orange, represent that result be negative, if reaction solution color be green, expression result is the positive.
Real-time fluorescence nucleic acid constant-temperature amplification detection technique (Real-timefluorescenceloop-meidatedisothermalamplificatio nassay of the present invention, be called for short RealAmp) be a kind of novel nucleic acids detection technique that the nucleic acid constant-temperature amplification technology of a new generation and real-time fluorescence detection technique are combined, can detection by quantitative pathogen, overcome the deficiency of first-generation LAMP detection method in the past, there is highly sensitive, high specific, low stain, the advantages such as stable reaction, adopt covered (closed-tubedetectiontechnique) not vulnerable to pollution thing impact and can Site Detection pedotheque, analyze and judge that the method for reaction product is very simple, be suitable for applying widely.
The method of a kind of No. 4 microspecies in the detection by quantitative banana blight bacteria torrid zone from soil of the present invention overcomes the deficiency of gene amplification method in the past, and have high specific, highly sensitive, detection by quantitative lower limit is 4.3 × 10
-4ng/ μ l, easy to operate, used time is short, only need 2-3h, not vulnerable to pollution thing impact and can Site Detection pedotheque, do not need costliness and the temperature cycling device of precision, only need can reach the thermostat of 63 DEG C, analyze the method judging reaction product very simple, only need visual inspection yet, the content of No. 4 microspecies in the banana blight bacteria torrid zone in soil can be differentiated again quantitatively, be suitable for applying widely.
Accompanying drawing explanation
Fig. 1 is the method flow diagram of No. 4 microspecies in the detection by quantitative banana blight bacteria torrid zone from soil provided by the invention.
Fig. 2 is No. 4 microspecies LAMP detection primer design diagrams in the Pathogen Causing Banana Fusarium Wilt torrid zone that the embodiment of the present invention provides.
Fig. 3 is the specificity analyses of No. 4 microspecies RealAmp detections in the Pathogen Causing Banana Fusarium Wilt torrid zone that the embodiment of the present invention provides;
(A) with Pathogen Causing Banana Fusarium Wilt (Fusariumoxysporumf.sp.cubense (Foc)) No. 1 microspecies (race1), No. 4, subtropics microspecies (SubtropicalRace4, ST4), No. 4 microspecies (TropicalRace4 in the torrid zone, TR4), melon didymella bryoniae (Mycosphaerellamelonis), cucumber fusarium axysporum (Fusariumoxysporumf.sp.cucumerium), the DNA of withered germ of water-melon (Fusariumoxysporumf.sp.niveum) and soil, water is the specificity (respectively the 1-8 of reference numeral) that contrast is used for testing LAMP, 2% agarose gel electrophoresis detects the product of each PCR pipe, only template is that FocTR4 genomic dna shows gradient band, all the other are all without gradient band, " M " MarkerDL5000,
(B) use the specific PCR of No. 4 microspecies in the Pathogen Causing Banana Fusarium Wilt torrid zone to detect primer, the 1-8 in amplification " A ", only template is that FocTR4 genomic dna shows single band, and all the other are all without band; " M " MarkerDL2000;
(C) color reaction that RealAmp detects shows, only template is the colour developing of FocTR4 genomic dna is green (positive), and all the other are orange (feminine gender);
(D) amplification curve after ESE-QuantTubeScanner instrument carries out RealAmp amplified reaction only has 1, the PCR pipe that what corresponding numbering was corresponding is is template with FocTR4 genomic dna.
Fig. 4 is sensitivity and the typical curve of No. 4 microspecies RealAmp detections in the Pathogen Causing Banana Fusarium Wilt torrid zone that the embodiment of the present invention provides;
(A) to comprise pMD-18-T-TR4 plasmid DNA between RealAmp amplification region for template, according to 10 times of gradient series dilution methods by pMD-18-T-TR4 plasmid DNA from 4.3 × 10
1ng/ μ l is diluted to 4.3 × 10 successively
-5ng/ μ l, take sterilized water as negative control (respectively the 1-8 of reference numeral), 2% agarose gel electrophoresis detects the product of each PCR pipe, and pMD-18-T-TR4 plasmid DNA concentration is 4.3 × 10
1ng/ μ l ~ 4.3 × 10
-4ng/ μ l shows gradient band, and concentration is 4.3 × 10
-4the gradient band of ng/ μ l is more weak, and all the other are all without gradient band " M " MarkerDL2000;
(B) color reaction that RealAmp detects shows, with pMD-18-T-TR4 plasmid DNA concentration for 4.3 × 10
1ng/ μ l ~ 4.3 × 10
-4shown in green (positive) of ng/ μ l, all the other are orange (feminine gender);
(C) amplification curve after ESE-QuantTubeScanner instrument carries out RealAmp amplified reaction has 6, and what corresponding numbering was corresponding is for 4.3 × 10 with pMD-18-T-TR4 plasmid DNA concentration
1ng/ μ l ~ 4.3 × 10
-4the PCR pipe of ng/ μ l;
(D) typical curve built there is linear relationship between the starting template DNA of different concns and corresponding Tt value.
Fig. 5 is the Field information of No. 4 microspecies RealAmp detections in the Pathogen Causing Banana Fusarium Wilt torrid zone that the embodiment of the present invention provides;
(A) ESE-QuantTubeScanner instrument carries out the amplification curve after RealAmp amplified reaction, with concentration for 8.6 × 10
-4ng plasmid DNA is positive control, and sterilized water is negative contrast;
(B) content of No. 4 microspecies in the banana blight bacteria torrid zone in every gram of soil calculating of establishing criteria Curves.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Fig. 1 shows the method flow of No. 4 microspecies in the detection by quantitative banana blight bacteria torrid zone from soil provided by the invention.For convenience of explanation, illustrate only part related to the present invention.
Below in conjunction with drawings and the specific embodiments, application principle of the present invention is further described.
Step one, pre-treatment is carried out to test sample, the DNA of rapid extraction test sample:
The DNA nucleic acid extraction of test sample: add 300 μ lDNA extracting solutions in 0.1g detected sample, after grinding to form pasty state, after 95 DEG C of water bath with thermostatic control 10min, 25 DEG C, under 10000rpm centrifugal 2 minutes, gets 50 μ l supernatant liquors as detection template DNA.
DNA extraction liquid comprises: 100mMTris-HCl(pH7.4), 1MKC1,10mMEDTA and 2%(w/v) PVPP.
The preparation of step 2, Auele Specific Primer: with reference to No. 4 microspecies (Fusariumoxysporiumf.sp.cubenseTripicalrace4 in the banana blight bacteria torrid zone of people's designs such as Dita, FocTR4) the 463bp fragment that specific detection primer obtains designs the LAMP detection primer of No. 4 microspecies in the banana blight bacteria torrid zone, obtain RealAmp Auele Specific Primer 2 right, inner primer and each a pair of outer primer.The fragment of specific 463bp contains the sequence of whole RealAmp.
The primer mixed solution be made up of two pairs of primers, two pairs of primers are respectively: outer primer FocTR4-F3(5 '-CACGTTTAAGGTGCCATGAGAG-3 '), outer primer FocTR4-B3(5 '-CGCACGCCAGGACTGCCTCGTGA-3 '), inner primer FocTR4-FIP(5 '-ATTCAAGCCGGATTGACGGATTGGATATGTAGAGAATGTGGTGG-3 '), inner primer FocTR4-BIP(5 '-GGGAGCCAAGAAGAAGCAGGACCTTCGATTCTTGTATC-3 '), the concentration of described outer primer FocTR4-F3 and described outer primer FocTR4-B3 is respectively 5pmol/ μ l, the concentration of described inner primer FocTR4-FIP and described inner primer FocTR4-BIP is 40pmol/ μ l.
The specific detection primer of No. 4 microspecies in the table 1 Pathogen Causing Banana Fusarium Wilt torrid zone
Step 3, fluorescence nucleic acid constant-temperature amplification detection technique (RealAmp) are reacted
Mix through the sample of pre-treatment, primer, reaction buffer with BstDNA polysaccharase, carry out endless chain replacement(metathesis)reaction at 63 DEG C of insulation 90min.RealAmp is at the enterprising line operate of ESE-QuantTubeScanner, ESE-QuantTubeScanner is that the RealAmp of a thermal cycling and fluoroscopic examination and one increases instrument, there are 8 bottoming holes, instrument self directly can show detected result with display screen, can also be connected on computer by conputer controlled.The amplification curve shown on a display screen after amplification terminates has several numerical value:
Tt value (thresholdtime, Tt) refers to the time that fluorescent signal in each reaction tubes experiences when arriving the thresholding of setting, with the Ct value comparing class in fluorescent PCR seemingly.
Threshold value (threshold) refers to the fluorescent signal value of setting.Fluorescent signal value refers to the fluorescence signal intensity that reaction tubes Instrumental measures, and represents with milli-volts (mV).
RealAmp25 μ L reaction system: the nucleic acid DNA of 1 μ L extracting is as template, 2.5 μ L10 × BstDNApolymersebuffer, 1.4mMdNTPs, 0.8mM trimethyl-glycine, 8mMMgSO4, the outer primer (B3 and F3) of 0.2 μM, inner primer (FIP and BIP), the 1 μ LBstDNApolymerase(8U/ μ L of 1.6 μMs), 0.2 μM of SYTO-9 fluorescence dye, moisturizing to 25 μ L, and then the paraffin oil system such as adding covers whole reaction solution and prevents volatilization.RealAmp reaction is carried out on ESE-QuantTubeScanner (ESEGmbh, Stockach, Germany), 80 DEG C of reaction 10min termination reactions after 63 DEG C of reaction 90min.RealAmp reaction before in reaction tubes, cover the SYBRGreenI fluorescence dye added as 1 μ L1:10 doubly dilutes, question response terminate rear brief centrifugation SYBRGreenI is added in LAMP reaction solution carry out colour developing observation, avoid detection Aerosol Pollution of uncapping.When positive control reaction is set, replace by the oranges and tangerines genome DNA infecting HLB; When arranging negative control reaction, with TE(100mMTris-HCl(pH8.0), 50mMEDTA) replace, the reaction tubes containing the reaction soln prepared is placed in 63 DEG C of reaction 90min.Reaction terminates rear display screen display amplification curve, and X-axis represents proliferation time, Y-axis display fluorescent value.
The structure of step 4, RealAmp typical curve
In order to the detection sensitivity of accurate assessment RealAmp also builds typical curve thus, adopt No. 4 microspecies (Fusariumoxysporiumf.sp.cubenseTripicalrace4 in the banana blight bacteria torrid zone of people's designs such as Dita, FocTR4) specific detection primer FocTR4-F(5 '-CACGTTTAAGGTGCCATGAGAG-3 ')/FocTR4-R(5 '-CGCACGCCAGGACTGCCTCGTGA-3 '), amplified fragments size is 463bp.The fragment of specific 463bp contains the sequence of whole RealAmp, and therefore, clone the plasmid after this sequence to pMD18-T carrier order-checking correctly, called after pMD18-T-TR4, for building typical curve.
The method of the PCR detection reaction system reference Dita of No. 4 microspecies in the banana blight bacteria torrid zone etc.: 2.5 μ L10 × PCRbuffer(Mg
2+), 2 μ LdNTPs(2.5mMeach), 0.25 μ LTaq polysaccharase (5U/ μ L), each 1 μ L(10pM of primers F ocTR4-F/FocTR4-R), 1 μ LDNA, moisturizing to 25 μ L; The condition of PCR reaction: 94 DEG C of denaturation 3min, 94 DEG C of sex change 30s, 57 DEG C of annealing 30s, 72 DEG C extend 60s, carry out 35 circulations; 72 DEG C extend 7min.After reaction terminates, get 5 μ L reaction product, EB dyeing observations after 1% agarose gel electrophoresis.Pcr amplification band is cut after glue reclaims connection pMD-18-T carrier and is checked order correctly, by this plasmid called after pMD18-T-TR4(10ng/ μ L), build the typical curve of RealAmp of No. 4 microspecies in the banana blight bacteria torrid zone as template thus for assessment of the detection sensitivity of RealAmp after 10 times of gradient dilutions.Relation between the template pMD18-T-TR4 plasmid concentration that typical curve dilutes according to difference and corresponding Tt value builds, and X-axis represents the logarithmic value of initial template concentration, and Y-axis represents that different concns template amplification reaches the threshold value time used (Tt).
Step 5, result judge
This experiment employing two kinds of method judged results, a kind of is the judgement of detection by quantitative result, and directly after ESE-QuantTubeScanner reaction terminates, instrument is automatically according to typical curve display quantitative result.In addition, in order to the fast results adapting to field judges, reaction terminates rear hand and to get rid of or the SYBRGreenI covered in reaction tubes is mixed in reaction solution by brief centrifugation, covered colour developing is adopted to carry out judged result, if reaction solution color is orange, represent that result is negative, if reaction solution color is green, represent that result is positive.Covered detection can avoid the crossed contamination between sample, and can as the auxiliary judgment mode outside detection by quantitative result.
Structure and the Field information analysis of specificity, susceptibility and the typical curve that RealAmp detects is carried out according to above-mentioned steps.
The specificity analyses that RealAmp detects:
With Pathogen Causing Banana Fusarium Wilt (Fusariumoxysporumf.sp.cubense (Foc)) No. 1 microspecies (race1), No. 4, subtropics microspecies (SubtropicalRace4, ST4), No. 4 microspecies (TropicalRace4 in the torrid zone, TR4), melon didymella bryoniae (Mycosphaerellamelonis), cucumber fusarium axysporum (Fusariumoxysporumf.sp.cucumerium), the DNA of withered germ of water-melon (Fusariumoxysporumf.sp.niveum) and healthy Banana soil, negative control tests the specificity of LAMP, it is that the colour developing of FocTR4 genomic dna is for green that result shows only template, be indicated as the positive, all the other are orange, be indicated as feminine gender (see Fig. 3-C), the product of each PCR pipe is detected through 2% agarose gel electrophoresis, only template is that FocTR4 genomic dna shows gradient band, all the other are all without gradient band (see Fig. 3-A), and what show to only have positive reaction in LAMP detects just has gradient band, and negative reaction without gradient band, specific PCR with reference to No. 4 microspecies in the Pathogen Causing Banana Fusarium Wilt torrid zone of people's designs such as Dita detects primers F ocTR4-F/FocTR4-R, amplification is used for the confession examination pathogenic bacteria of specificity analyses, only template is the single band that FocTR4 genomic DNA amplification goes out that size is 463bp, and all the other are all without band (see Fig. 3-B).The tropical No. 4 microspecies specific detection results of banana rot pathogenic bacteria that the people such as LAMP colour developing and electrophoretic analysis and Dita design are consistent, show the specificity that LAMP detects.Amplification curve after ESE-QuantTubeScanner instrument carries out RealAmp amplified reaction only has 1, the PCR pipe that what corresponding numbering was corresponding is is template with FocTR4 genomic dna.
The structure of RealAmp detection sensitivity and typical curve:
To comprise pMD-18-T-TR4 plasmid DNA between RealAmp amplification region for template, according to 10 times of gradient series dilution methods by pMD-18-T-TR4 plasmid DNA from 4.3 × 10
1ng/ μ l is diluted to 4.3 × 10 successively
-5ng/ μ l, be negative control with sterilized water, result shows, pMD-18-T-TR4 plasmid DNA concentration is 4.3 × 10
1ng/ μ l ~ 4.3 × 10
-4all colour developings of ng/ μ l are green, and negative control colour developing, for orange, shows that the Monitoring lower-cut of RealAmp is about 1 × 10
-4ng/ μ l(is shown in Fig. 4-B); 2% agarose gel electrophoresis detects the product of each PCR pipe, and pMD-18-T-TR4 plasmid DNA concentration is 4.3 × 10
1ng/ μ l ~ 4.3 × 10
-4ng/ μ l shows gradient band, and concentration is 4.3 × 10
-4the gradient band of ng/ μ l is more weak, all without gradient band (see Fig. 4-A), all the other show that at RealAmp Monitoring lower-cut be 4.3 × 10
-4ng/ μ l, concentration is lower than 4.3 × 10
-4ng/ μ l then examines and does not measure.
Amplification curve display RealAmp after ESE-QuantTubeScanner amplified reaction can increase the template DNA (see Fig. 4-C) of 6 orders of magnitude.Linear relationship is there is between the starting template DNA of different concns and corresponding Tt value.Represent the logarithmic value of initial template concentration with X-axis, Y-axis represents that different concns template amplification reaches the threshold value time used (Tt), builds the typical curve y=-2.6429x+25.171 (R that RealAmp detects
2=0.9975) (see Fig. 4-D).During application, only need to know that amplification reaches the threshold value time used (Tt), just can obtain the content of No. 4 microspecies in the banana blight bacteria torrid zone in soil.
The Field information that RealAmp detects:
RealAmp detection is carried out at Hainan banana plantation random acquisition 6 pedotheques, arrange positive control and negative control (see figure 5), result shows, all detects No. 4 microspecies in the banana blight bacteria torrid zone in 6 samples, its content difference to some extent, between 468fg/g ~ 800fg/g.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (1)
1. from soil detection by quantitative banana blight bacteria the torrid zone No. 4 microspecies a method, it is characterized in that, should from soil detection by quantitative banana blight bacteria the torrid zone No. 4 microspecies method comprise the following steps:
Step one, pre-treatment is carried out to test sample, extract the DNA of test sample;
The preparation of step 2, Auele Specific Primer;
Step 3, fluorescence nucleic acid constant-temperature amplification detection technique RealAmp react;
The structure of step 4, RealAmp typical curve;
Step 5, result judge;
Carry out pre-treatment to test sample, the concrete grammar extracting the DNA of test sample is as follows:
The DNA nucleic acid extraction of test sample: add 300 μ lDNA extracting solutions in 0.1g detected sample, after grinding to form pasty state, after 95 DEG C of water bath with thermostatic control 10min, 25 DEG C, under 10000rpm centrifugal 2 minutes, gets 50 μ l supernatant liquors as detection template DNA;
DNA extraction liquid comprises: 100mMTris-HClpH7.4,1MKC1,10mMEDTA and 2%w/vPVPP;
The preparation of Auele Specific Primer designs the LAMP detection primer of No. 4 microspecies in the banana blight bacteria torrid zone with reference to the 463bp fragment that banana blight bacteria No. 4, the torrid zone microspecies specific detection primers that Dita designs obtain, obtain RealAmp Auele Specific Primer 2 right, inner primer and each a pair of outer primer;
The primer mixed solution be made up of two pairs of primers, two pairs of primers are respectively: outer primer FocTR4-F3 i.e. 5 '-CACGTTTAAGGTGCCATGAGAG-3 ', outer primer FocTR4-B3 i.e. 5 '-CGCACGCCAGGACTGCCTCGTGA-3 ', inner primer FocTR4-FIP i.e. 5 '-ATTCAAGCCGGATTGACGGATTGGATATGTAGAGAATGTGGTGG-3 ', inner primer FocTR4-BIP i.e. 5 '-GGGAGCCAAGAAGAAGCAGGACCTTCGATTCTTGTATC-3 ', the concentration of described outer primer FocTR4-F3 and described outer primer FocTR4-B3 is respectively 5pmol/ μ l; The concentration of described inner primer FocTR4-FIP and described inner primer FocTR4-BIP is 40pmol/ μ l;
The reaction of step 3 fluorescence nucleic acid constant-temperature amplification detection technique is specific as follows:
Mix through the sample of pre-treatment, primer, reaction buffer with BstDNA polysaccharase, carry out endless chain replacement(metathesis)reaction at 63 DEG C of insulation 90min;
RealAmp25 μ L reaction system: the nucleic acid DNA of 1 μ L extracting is as template, 2.5 μ L10 × BstDNApolymersebuffer, 1.4mMdNTPs, 0.8mM trimethyl-glycine, 8mMMgSO4, the outer primer of 0.2 μM, inner primer, the 1 μ LBstDNApolymerase of 1.6 μMs, 0.2 μM of SYTO-9 fluorescence dye, moisturizing to 25 μ L, and then add isopyknic paraffin oil and cover whole reaction solution;
RealAmp reaction is carried out on ESE-QuantTubeScanner, 80 DEG C of reaction 10min termination reactions after 63 DEG C of reaction 90min;
RealAmp reaction before in reaction tubes, cover the SYBRGreenI fluorescence dye added as 1 μ L1:10 doubly dilutes, question response terminate rear brief centrifugation SYBRGreenI is added in LAMP reaction solution carry out colour developing observation; When positive control reaction is set, replace by the oranges and tangerines genome DNA infecting HLB; When negative control reaction is set, replace with 100mMTris-HClpH8.0 and 50mMEDTA, the reaction tubes containing the reaction soln prepared is placed in 63 DEG C of reaction 90min; Reaction terminates rear display screen display amplification curve, and X-axis represents proliferation time, Y-axis display fluorescent value;
The construction process of step 4 RealAmp typical curve is as follows:
Adopt No. 4 microspecies Fusariumoxysporiumf.sp.cubenseTripicalrace4 in the banana blight bacteria torrid zone of Dita design, FocTR4 specific detection primer FocTR4-F5 '-CACGTTTAAGGTGCCATGAGAG-3 '/FocTR4-R5 '-CGCACGCCAGGACTGCCTCGTGA-3 ', amplified fragments size is 463bp; Plasmid after cloned sequence is correct to the order-checking of pMD18-T carrier, called after pMD18-T-TR4, for building typical curve;
The PCR detection reaction system method of No. 4 microspecies in the banana blight bacteria torrid zone is as follows:
2.5 μ L10 × PCRbuffer, 2 μ LdNTPs, 0.25 μ LTaq polysaccharase, each 1 μ L of primers F ocTR4-F/FocTR4-R, 1 μ LDNA, moisturizing to 25 μ L;
The condition of PCR reaction: 94 DEG C of denaturation 3min, 94 DEG C of sex change 30s, 57 DEG C of annealing 30s, 72 DEG C extend 60s, carry out 35 circulations; 72 DEG C extend 7min;
After reaction terminates, get 5 μ L reaction product, EB dyeing observations after 1% agarose gel electrophoresis;
Pcr amplification band is cut after glue reclaims connection pMD-18-T carrier and is checked order correctly, by plasmid called after pMD18-T-TR4, after 10 times of gradient dilutions, build the typical curve of RealAmp of No. 4 microspecies in the banana blight bacteria torrid zone as template thus for assessment of the detection sensitivity of RealAmp;
Result judges employing two kinds of method judged results, and a kind of is the judgement of detection by quantitative result, and directly after ESE-QuantTubeScanner reaction terminates, instrument is automatically according to typical curve display quantitative result; In addition, reaction terminates centrifugally to be mixed in reaction solution by the SYBRGreenI covered in reaction tubes afterwards, and adopt covered colour developing to carry out judged result, if reaction solution color is orange, represent that result be negative, if reaction solution color be green, expression result is the positive.
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| CN104611451B (en) * | 2015-02-16 | 2017-01-18 | 中国热带农业科学院环境与植物保护研究所 | Complete set primers for identification or assisted identification of fusarium oxysporum f.sp.cubense and application of complete set primers |
| CN107164462A (en) * | 2017-04-24 | 2017-09-15 | 江苏省农业科学院 | The quantitative detecting method of withered germ of water-melon in a kind of soil |
| CN108060211B (en) * | 2018-01-23 | 2018-11-13 | 中国热带农业科学院环境与植物保护研究所 | A method of quantitatively detecting mango anthrax-bacilus |
| CN111944919B (en) * | 2020-07-31 | 2023-05-26 | 广东省农业科学院果树研究所 | Banana fusarium wilt tropical No.4 small species visual detection technology system capable of being operated in field and at normal temperature |
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