CN103740806A - Method for quantitatively detecting Fusarium oxysporium f.sp.cubense Tripical race 4 from soil - Google Patents
Method for quantitatively detecting Fusarium oxysporium f.sp.cubense Tripical race 4 from soil Download PDFInfo
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Abstract
The present invention discloses a method for quantitatively detecting Fusarium oxysporium f.sp.cubense Tripical race 4 from soil. The method comprises: carrying out a pre-treatment on a sample requiring detection, and extracting DNA of the sample requiring detection; preparing specific primers; carrying out a fluorescent nucleic acid isothermal amplification detection technology RealAmp reaction; constructing a RealAmp standard curve; and judging results. The method has the following characteristics that: defects of the gene amplification method in the prior art are overcome, characteristics of high specificity, high sensitivity, quantitative detection lower limitation of 4.3*10<-4> ng/mul, easy operation, short time consumption (only requires 2-3 h), pollutant influence resistance, on site soil sample detection, no requirement of expensive and precise temperature cycling equipment, and only requirement of thermostatic equipment capable of achieving a temperature of 63 DEG C are provided, the reaction product analysis judgment method is simple and only requires observation by naked eye, the Fusarium oxysporium f.sp.cubense Tripical race 4 content in the soil can be quantitatively determined, and the method is suitable for wide promotion application.
Description
Technical field
The invention belongs to No. 4 microspecies detection fields in the banana blight bacteria torrid zone, relate in particular to a kind of from soil the method for No. 4 microspecies in the detection by quantitative banana blight bacteria torrid zone.
Background technology
Banana is Musaceae banana, is important cash crop and the food crop in tropical and subtropical zone area.Banana blight is the destructive disease of banana industry, is seriously restricting China's banana industry and is developing in a healthy way.
Pathogen Causing Banana Fusarium Wilt is Fusarium oxysporum Schlevht.f.sp.cubense (E.F.Smith) Snyd. & Hans., former name Fusarium cubense E.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 little speciogenesis is with a long history, can solve by replanting Cavendish class banana the harm of No. 1 microspecies.No. 4 microspecies were imported China, the safety of serious harm China banana industry in 1996.No. 4 microspecies are subdivided into again No. 4 microspecies of No. 4, subtropics microspecies and the torrid zone, in China, are mainly No. 4 microspecies in the torrid zone.
The propagation harm of banana blight minute long-distance communications and closely propagating, take at a distance pathogenic bacteria with the allocation and transportation of tissue culture seedlings of bananas secondary seedling as main, closely propagation is with soil its spread in china in pathogenic bacteria Jiao garden.In production, in the urgent need to pathogenic soil bacterium Fast Detection Technique, be used for detecting tissue cultured seedling secondary seedling soil, newly open up whether Banana soil carries disease germs and the quick diagnosis of doubtful diseased plant.Therefore, setting up No. 4, the torrid zone of a set of Pathogen Causing Banana Fusarium Wilt quick, the stable detection method of microspecies is the important guarantee of China's banana safety in production.
In recent years, there is report to confirm that the method for PCR-based is successful for detection of No. 4 microspecies in the Pathogen Causing Banana Fusarium Wilt torrid zone, PCR method improves a lot compared with ordinary method on the operating time and aspect the specificity detecting and sensitivity, 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 make this method can not meet the needs of actual detection.Therefore, need to develop a kind of highly sensitive and willing method, can replace the detection method of PCR to a certain extent, and, common PCR method can only qualitative detection pathogenic bacteria have or nothing, number that can not detection by quantitative pathogenic bacteria, more can not have how many pathogenic bacterias in clear and definite soil? therefore, the newest fruits of biotech development is applied to No. 4, the torrid zone of Pathogen Causing Banana Fusarium Wilt microspecies and detects, significant.
Japanese scholars Notomi equals to have developed first for 2000 ring mediated isothermal nucleic acid amplification reaction (loop-mediated isothermal amplification, LAMP), this technology relies on six special primers and a kind of archaeal dna polymerase with strand displacement characteristic, quick under isothermal condition, efficiently, high special, amplified target sequence with sensitivity, this technology is first utilized two inner primers and two outer primers, by strand displacement effect, form stem cyclic DNA structure, then a large amount of amplified target sequences of automated cycle strand replacement reaction that mediate by ring under the effect of inner primer, 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-time fluorescence loop-mediated isothermal amplification assay, be called for short RealAmp) be a kind of novel nucleic acids detection technique that nucleic acid constant-temperature amplification technology of 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 pollution, stable reaction.In addition, SYBR Green I is one of detection method that current LAMP product is the sensitiveest, but SYBR Green I'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 a kind of auxiliary judgment measure using SYBR Green I dyed color judged result as detection by quantitative.
The method is normally carried out as follows: step 1, tested sample is carried out to pre-treatment, the DNA of the tested sample of rapid extraction; The preparation of step 2, Auele Specific Primer: determine after the target gene of sample to be measured, obtain 2 pairs of Auele Specific Primers, each is a pair of for inner primer and outer primer; Step 3, real-time fluorescence nucleic acid constant-temperature amplification (RealAmp) reaction: the sample through pre-treatment, primer, reaction buffer are mixed with BstDNA polysaccharase, 63 ℃ of insulations, within 1.5 hours, carry out endless chain replacement(metathesis)reaction; Step 4, drawing standard curve: typical curve is to build according to the relation between the template concentrations of difference dilution and corresponding Tt value, X-axis represents the logarithmic value of starting template concentration, and Y-axis represents that different concns template amplification reaches the threshold value time used (Tt).Step 5, analysis judgement reaction product result.
The people such as Dita are by the difference between the transcribed spacer of 20 Different Nutrition affinity types of analysis Pathogen Causing Banana Fusarium Wilt, designed the Auele Specific Primer of 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, only the method by PCR detects No. 4 microspecies in the Pathogen Causing Banana Fusarium Wilt torrid zone qualitatively, is not from soil, to detect quantitatively.The method of the people such as Li by SCAR used loop-mediated isothermal amplification (LAMP) to detect No. 4 microspecies of banana blight bacteria (Fusarium oxysporium f.sp.cubense race4) in China, but No. 4 microspecies in the detection by quantitative banana blight bacteria torrid zone from soil not.
The PCR detection technique of using the people such as Dita to set up, sample gene group DNA, the used time that need to extract higher degree are longer, can not Site Detection pedotheque, more can not from soil, detect quantitatively pathogenic bacteria, and need expensive and accurate temperature cycling device, must could be to differentiating the positive or negative of amplified production by electrophoresis apparatus, cost is high, not easy to operate, the professional need to certain state of the art could carry out testing.The people such as Li have detected No. 4 microspecies of Chinese banana blight bacteria 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 quantitatively the pathogenic bacteria content in soil yet, can not clear and definite soil in the content of pathogenic bacteria.
Summary of the invention
The object of the present invention is to provide a kind of from soil the method for No. 4 microspecies in the detection by quantitative banana blight bacteria torrid zone, being intended to solve normal PCR detection technique, need to extract sample gene group DNA, used time of higher degree longer, can not Site Detection pedotheque, can not from soil, detect quantitatively pathogenic bacteria, and need expensive and accurate temperature cycling device, cost is high, not easy-operating problem.
The present invention be achieved in that a kind of from soil the method for No. 4 microspecies in the detection by quantitative banana blight bacteria torrid zone, should from soil, the method for No. 4 microspecies in the detection by quantitative banana blight bacteria torrid zone comprise the following steps:
The preparation of step 2, Auele Specific Primer;
The structure of step 4, RealAmp typical curve;
Further, test sample is carried out to pre-treatment, the concrete grammar of the DNA of extraction test sample is as follows:
The DNA nucleic acid extraction of test sample: add 300 μ l DNA extraction liquid in 0.1g detected sample, grind to form after pasty state, after 95 ℃ of water bath with thermostatic control 10min, under 25 ℃, 10000rpm centrifugal 2 minutes, get 50 μ l supernatant liquors as detecting template DNA;
DNA extraction liquid comprises: 100mM Tris-HClpH7.4,1M KC1,10mM EDTA and 2%w/vPVPP.
Further, the LAMP of No. 4 microspecies in the 463bp fragment design banana blight bacteria torrid zone that No. 4, the torrid zone of the banana blight bacteria microspecies specific detection primers that the preparation of Auele Specific Primer designs with reference to Dita obtain detects primer, obtain 2 pairs of RealAmp Auele Specific Primers, each is a pair of for inner primer and outer primer;
The primer mixed solution being formed by two pairs of primers, two pairs of primers are respectively: outer primer FocTR4-F3 i.e. 5 '-CACGTTTAAGGTGCCATGAGAG-3 ', outer primer FocTR4-B3 is 5 '-CGCACGCCAGGACTGCCTCGTGA-3 ', inner primer FocTR4-FIP i.e. 5 '-ATTCAAGCCGGATTGACGGATTGGATATGTAGAGAATGTGGTGG-3 ', inner primer FocTR4-BIP is 5 '-GGGAGCCAAGAAGAAGCAGGACCTTCGATTCTTGTATC-3 ', and 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:
Sample through pre-treatment, primer, reaction buffer are mixed with BstDNA polysaccharase, at 63 ℃ of insulation 90min, carry out endless chain replacement(metathesis)reaction;
RealAmp25 μ L reaction system: the nucleic acid DNA of 1 μ L extracting is as template, 2.5 μ L10 * Bst DNA polymerse buffer, 1.4mM dNTPs, 0.8mM trimethyl-glycine, the outer primer of 8mM MgSO4,0.2 μ M, the inner primer of 1.6 μ M, 1 μ L Bst DNA polymerase, 0.2 μ M SYTO-9 fluorescence dye, moisturizing to 25 μ L, and then the paraffin oil of system such as add to cover whole reaction solution;
RealAmp reaction is carried out on ESE-Quant Tube Scanner, 80 ℃ of reaction 10min termination reactions after 63 ℃ of reaction 90min;
Before RealAmp reaction, cover and add the SYBR Green I fluorescence dye doubly diluting as 1 μ L1:10 in reaction tubes, question response finishes instantaneously centrifugal SYBR Green I to be added to the observation that develops the color in LAMP reaction solution afterwards; When positive control reaction is set, by the oranges and tangerines genome DNA that infects HLB, replace; When negative control reaction is set, with 100mM Tris-HCl pH8.0 and 50mM EDTA, replace, the reaction tubes that contains the reaction soln preparing is placed in to 63 ℃ of reaction 90min; Reaction finishes rear demonstration screen display amplification curve, and X-axis represents proliferation time, and Y-axis shows fluorescent value.
Further, it is characterized in that, the construction process of step 4 RealAmp typical curve is as follows:
Adopt No. 4 microspecies Fusarium oxysporium f.sp.cubense Tripical race4 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 checks order correctly to pMD18-T carrier, 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 * PCR buffer, 2 μ L dNTPs, 0.25 μ L Taq polysaccharase, each 1 μ L of primers F ocTR4-F/FocTR4-R, 1 μ L DNA, moisturizing to 25 μ L;
The condition of PCR reaction: 94 ℃ of denaturation 3min, 94 ℃ of sex change 30s, 57 ℃ of annealing 30s, 72 ℃ extend 60s, carry out 35 circulations; 72 ℃ extend 7min;
After reaction finishes, get 5 μ L reaction product, EB dyeing observations after 1% agarose gel electrophoresis;
Pcr amplification band cut glue reclaim to connect after pMD-18-T carrier order-checking correct after, by this plasmid called after pMD18-T-TR4, after 10 times of gradient dilutions, as template, for assessment of the detection sensitivity of RealAmp, also build thus the typical curve of the RealAmp of No. 4 microspecies in the banana blight bacteria torrid zone.
Further, result judgement adopts two kinds of method judged results, and a kind of is the judgement of detection by quantitative result, and directly, after ESE-Quant Tube Scanner reaction finishes, instrument shows quantitative result according to typical curve automatically; In addition, reaction finishes rear centrifugal the SYBR Green I covering in reaction tubes is blended in reaction solution, adopts covered colour developing to carry out judged result, if reaction solution color is orange, represents that result is negative, and if reaction solution color be green, expression result is positive.
Real-time fluorescence nucleic acid constant-temperature amplification detection technique of the present invention (Real-time fluorescence loop-meidated isothermal amplification assay, be called for short RealAmp) be a kind of novel nucleic acids detection technique that nucleic acid constant-temperature amplification technology of 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 pollution, the advantages such as stable reaction, adopt covered (closed-tube detection technique) not impact and energy Site Detection pedotheque of vulnerable to pollution thing, the method of analyzing judgement reaction product is very simple, be suitable for applying widely.
Of the present invention a kind of from soil the method for No. 4 microspecies in the detection by quantitative banana blight bacteria torrid zone overcome the deficiency of gene amplification method in the past, there is high specific, under highly sensitive, detection by quantitative, be limited to 4.3 * 10
-4ng/ μ l, easy to operate, used time is short, only need 2-3h, not the impact of vulnerable to pollution thing and can Site Detection pedotheque, also do not need costliness and accurate temperature cycling device, only need to reach the thermostat of 63 ℃, the method for analyzing judgement reaction product is very simple, only needs visual inspection, the content that can differentiate quantitatively again No. 4 microspecies in the banana blight bacteria torrid zone in soil, is suitable for applying widely.
Accompanying drawing explanation
Fig. 1 be provided by the invention from soil the method flow diagram of No. 4 microspecies in the detection by quantitative banana blight bacteria torrid zone.
Fig. 2 is that No. 4, the torrid zone of the Pathogen Causing Banana Fusarium Wilt microspecies LAMP that the embodiment of the present invention provides detects design of primers schematic diagram.
Fig. 3 is the specificity analyses that No. 4, Pathogen Causing Banana Fusarium Wilt torrid zone microspecies RealAmp that the embodiment of the present invention provides detect;
(A) with No. 1 microspecies (race1) of Pathogen Causing Banana Fusarium Wilt (Fusarium oxysporum f.sp.cubense (Foc)), No. 4, subtropics microspecies (Subtropical Race4, ST4), No. 4 microspecies (the Tropical Race4 in the torrid zone, TR4), melon didymella bryoniae (Mycosphaerella melonis), cucumber fusarium axysporum (Fusarium oxysporum f.sp.cucumerium), DNA and the soil of withered germ of water-melon (Fusarium oxysporum f.sp.niveum), the specificity (1-8 of reference numeral respectively) that water is used for testing LAMP for contrast, 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 " Marker DL5000,
(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, all the other are all without band; " M " MarkerDL2000;
(C) color reaction of RealAmp detection shows, only template is that the colour developing of FocTR4 genomic dna is green (positive), and all the other are orange (feminine gender);
(D) amplification curve that ESE-Quant Tube Scanner instrument carries out after RealAmp amplified reaction only has 1, and corresponding numbering is corresponding is to take the PCR pipe that FocTR4 genomic dna is template.
Fig. 4 is sensitivity and the typical curve that No. 4, Pathogen Causing Banana Fusarium Wilt torrid zone microspecies RealAmp that the embodiment of the present invention provides detect;
(A) take the pMD-18-T-TR4 plasmid DNA comprising between RealAmp amplification region is 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, with the negative contrast of sterilized water (1-8 of reference numeral respectively), 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 a little less than, all the other are all without gradient band " M " Marker DL2000;
(B) color reaction that RealAmp detects shows, take pMD-18-T-TR4 plasmid DNA concentration as 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 that ESE-Quant Tube Scanner instrument carries out after RealAmp amplified reaction has 6, and what corresponding numbering was corresponding is to take pMD-18-T-TR4 plasmid DNA concentration as 4.3 * 10
1ng/ μ l～4.3 * 10
-4the PCR pipe of ng/ μ l;
(D) typical curve building there is linear relationship between the starting template DNA of different concns and corresponding Tt value.
Fig. 5 is the field application that No. 4, Pathogen Causing Banana Fusarium Wilt torrid zone microspecies RealAmp that the embodiment of the present invention provides detect;
(A) ESE-Quant Tube Scanner instrument carries out the amplification curve after RealAmp amplified reaction, take concentration as 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 that establishing criteria Curves calculates.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearer, 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, is not intended to limit the present invention.
Fig. 1 show provided by the invention from soil the method flow of No. 4 microspecies in the detection by quantitative banana blight bacteria torrid zone.For convenience of explanation, only show part related to the present invention.
Below in conjunction with drawings and the specific embodiments, application principle of the present invention is further described.
The DNA nucleic acid extraction of test sample: add 300 μ l DNA extraction liquid in 0.1g detected sample, grind to form after pasty state, after 95 ℃ of water bath with thermostatic control 10min, under 25 ℃, 10000rpm centrifugal 2 minutes, get 50 μ l supernatant liquors as detecting template DNA.
DNA extraction liquid comprises: 100mM Tris-HCl(pH7.4), 1MKC1,10mM EDTA and 2%(w/v) PVPP.
The preparation of step 2, Auele Specific Primer: with reference to No. 4 microspecies (the Fusarium oxysporium f.sp.cubense Tripical race4 in the banana blight bacteria torrid zone of people's designs such as Dita, the LAMP of No. 4 microspecies in the 463bp fragment design banana blight bacteria torrid zone that FocTR4) specific detection primer obtains detects primer, obtain 2 pairs of RealAmp Auele Specific Primers, each is a pair of for inner primer and outer primer.The sequence that the fragment of specific 463bp has comprised whole RealAmp.
The primer mixed solution being formed by 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
Sample through pre-treatment, primer, reaction buffer are mixed with BstDNA polysaccharase, at 63 ℃ of insulation 90min, carry out endless chain replacement(metathesis)reaction.RealAmp is at the enterprising line operate of ESE-Quant Tube Scanner, ESE-Quant Tube Scanner is the RealAmp amplification instrument of a thermal cycling and fluoroscopic examination and one, there are 8 bottoming holes, instrument self can directly show detected result with display screen, can also be connected on computer and be controlled by computer.After finishing, amplification has several numerical value at the amplification curve that shows screen display:
Tt value (threshold time, Tt) refers to the time that fluorescent signal in each reaction tubes experiences while arriving the thresholding of setting, with 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 is measured, and with milli-volts (mV), represents.
RealAmp25 μ L reaction system: the nucleic acid DNA of 1 μ L extracting is as template, the inner primer (FIP and BIP) of the outer primer of 2.5 μ L10 * BstDNApolymersebuffer, 1.4mMdNTPs, 0.8mM trimethyl-glycine, 8mM MgSO4,0.2 μ M (B3 and F3), 1.6 μ M, 1 μ L Bst DNA polymerase(8U/ μ L), 0.2 μ MSYTO-9 fluorescence dye, moisturizing to 25 μ L, and then the paraffin oil of system such as add to cover whole reaction solution to prevent volatilization.RealAmp reaction is carried out on ESE-Quant Tube Scanner (ESE Gmbh, Stockach, Germany), 80 ℃ of reaction 10min termination reactions after 63 ℃ of reaction 90min.Before RealAmp reaction, cover and add the SYBR Green I fluorescence dye doubly diluting as 1 μ L1:10 in reaction tubes, question response finishes instantaneously centrifugal SYBR Green I to be added to the observation that develops the color in LAMP reaction solution, the detection Aerosol Pollution of avoiding uncapping afterwards.When positive control reaction is set, by the oranges and tangerines genome DNA that infects HLB, replace; When negative control reaction is set, use TE(100mM Tris-HCl(pH8.0), 50mMEDTA) replace, the reaction tubes that contains the reaction soln preparing is placed in to 63 ℃ of reaction 90min.Reaction finishes rear demonstration screen display amplification curve, and X-axis represents proliferation time, and Y-axis shows fluorescent value.
The structure of step 4, RealAmp typical curve
For the detection sensitivity of accurate assessment RealAmp and build thus typical curve, adopt No. 4 microspecies (the Fusarium oxysporium f.sp.cubense Tripical race4 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 sequence that the fragment of specific 463bp has comprised whole RealAmp, therefore, clones the plasmid after this sequence checks order correctly to pMD18-T carrier, and called after pMD18-T-TR4, for building typical curve.
The PCR detection reaction system of No. 4 microspecies in the banana blight bacteria torrid zone is with reference to the method for Dita etc.: 2.5 μ L10 * PCR buffer(Mg
2+), 2 μ L dNTPs(2.5mM each), 0.25 μ L Taq polysaccharase (5U/ μ L), each 1 μ L(10pM of primers F ocTR4-F/FocTR4-R), 1 μ L DNA, moisturizing to 25 μ L; The condition of PCR reaction: 94 ℃ of denaturation 3min, 94 ℃ of sex change 30s, 57 ℃ of annealing 30s, 72 ℃ extend 60s, carry out 35 circulations; 72 ℃ extend 7min.After reaction finishes, get 5 μ L reaction product, EB dyeing observations after 1% agarose gel electrophoresis.Pcr amplification band cut glue reclaim to connect after pMD-18-T carrier order-checking correct after, by this plasmid called after pMD18-T-TR4(10ng/ μ L), after 10 times of gradient dilutions, as template, for assessment of the detection sensitivity of RealAmp, also build thus the typical curve of the RealAmp of No. 4 microspecies in the banana blight bacteria torrid zone.Typical curve is to build according to the relation between the template pMD18-T-TR4 plasmid concentration of difference dilution and corresponding Tt value, and X-axis represents the logarithmic value of starting template concentration, and Y-axis represents that different concns template amplification reaches the threshold value time used (Tt).
This experiment adopts two kinds of method judged results, and a kind of is the judgement of detection by quantitative result, and directly, after ESE-Quant Tube Scanner reaction finishes, instrument shows quantitative result according to typical curve automatically.In addition, in order to adapt to the fast results judgement in field, reaction finishes with hand, to get rid of or instantaneous centrifugal the SYBR Green I covering in reaction tubes is blended in reaction solution afterwards, adopt covered colour developing 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 be avoided the crossed contamination between sample, and can be used as the auxiliary judgment mode outside detection by quantitative result.
According to above-mentioned steps, carry out structure and the field applied analysis of specificity, susceptibility and the typical curve of RealAmp detection.
The specificity analyses that RealAmp detects:
With No. 1 microspecies (race1) of Pathogen Causing Banana Fusarium Wilt (Fusarium oxysporum f.sp.cubense (Foc)), No. 4, subtropics microspecies (SubtropicalRace4, ST4), No. 4 microspecies (the Tropical Race4 in the torrid zone, TR4), melon didymella bryoniae (Mycosphaerella melonis), cucumber fusarium axysporum (Fusarium oxysporum f.sp.cucumerium), DNA and the healthy Banana soil of withered germ of water-melon (Fusarium oxysporum f.sp.niveum), negative control is tested the specificity of LAMP, result shows that template is only that the colour developing of FocTR4 genomic dna is for green, be indicated as the positive, all the other are orange, be indicated as feminine gender (seeing Fig. 3-C), through 2% agarose gel electrophoresis, detect the product of each PCR pipe, only template is that FocTR4 genomic dna shows gradient band, all the other are all without gradient band (seeing Fig. 3-A), and what show to only have positive reaction in LAMP detects just has a gradient band, and negative reaction without gradient band, the specific PCR of No. 4 microspecies in the Pathogen Causing Banana Fusarium Wilt torrid zone that design with reference to people such as Dita detects primers F ocTR4-F/FocTR4-R, amplification is for the confession examination pathogenic bacteria of specificity analyses, only template be FocTR4 genomic dna to amplify size be the single band of 463bp, all the other are all without band (seeing Fig. 3-B).No. 4 microspecies specific detection results in the banana rot pathogenic bacteria torrid zone of people's designs such as LAMP colour developing and electrophoretic analysis and Dita are consistent, show the specificity that LAMP detects.The amplification curve that ESE-Quant Tube Scanner instrument carries out after RealAmp amplified reaction only has 1, and corresponding numbering is corresponding is to take the PCR pipe that FocTR4 genomic dna is template.
The structure of RealAmp detection sensitivity and typical curve:
The pMD-18-T-TR4 plasmid DNA comprising between RealAmp amplification region of take is 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, with the negative contrast of 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 detection lower limit 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 a little less than, all the other are all without gradient band (seeing Fig. 4-A), show to be limited to 4.3 * 10 under RealAmp detects
-4ng/ μ l, concentration is lower than 4.3 * 10
-4ng/ μ l examines and does not measure.
Amplification curve after ESE-Quant Tube Scanner amplified reaction shows can the increase template DNA (seeing Fig. 4-C) of 6 orders of magnitude of RealAmp.Between the starting template DNA of different concns and corresponding Tt value, there is linear relationship.The logarithmic value that represents starting 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 application that RealAmp detects:
At 6 pedotheques of Hainan banana plantation random acquisition, carry out RealAmp detection, positive control and negative control (see figure 5) are set, and result shows, all detects No. 4 microspecies in the banana blight bacteria torrid zone in 6 samples, its content is 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 modifications of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.
Claims (7)
1. a method for No. 4 microspecies in the detection by quantitative banana blight bacteria torrid zone from soil, is characterized in that, should from soil, the method for No. 4 microspecies in the detection by quantitative banana blight bacteria torrid zone comprise the following steps:
Step 1, test sample is carried out to pre-treatment, 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 reaction;
The structure of step 4, RealAmp typical curve;
Step 5, result judgement.
As claimed in claim 1 from soil the method for No. 4 microspecies in the detection by quantitative banana blight bacteria torrid zone, it is characterized in that, test sample is carried out to pre-treatment, and the concrete grammar of DNA that extracts test sample is as follows:
The DNA nucleic acid extraction of test sample: add 300 μ l DNA extraction liquid in 0.1g detected sample, grind to form after pasty state, after 95 ℃ of water bath with thermostatic control 10min, under 25 ℃, 10000rpm centrifugal 2 minutes, get 50 μ l supernatant liquors as detecting template DNA;
DNA extraction liquid comprises: 100mM Tris-HClpH7.4,1MKC1,10mM EDTA and 2%w/vPVPP.
As claimed in claim 1 from soil the method for No. 4 microspecies in the detection by quantitative banana blight bacteria torrid zone, it is characterized in that, the LAMP of No. 4 microspecies in the 463bp fragment design banana blight bacteria torrid zone that No. 4, the torrid zone of the banana blight bacteria microspecies specific detection primers that the preparation of Auele Specific Primer designs with reference to Dita obtain detects primer, obtain 2 pairs of RealAmp Auele Specific Primers, each is a pair of for inner primer and outer primer;
The primer mixed solution being formed by two pairs of primers, two pairs of primers are respectively: outer primer FocTR4-F3 i.e. 5 '-CACGTTTAAGGTGCCATGAGAG-3 ', outer primer FocTR4-B3 is 5 '-CGCACGCCAGGACTGCCTCGTGA-3 ', inner primer FocTR4-FIP i.e. 5 '-ATTCAAGCCGGATTGACGGATTGGATATGTAGAGAATGTGGTGG-3 ', inner primer FocTR4-BIP is 5 '-GGGAGCCAAGAAGAAGCAGGACCTTCGATTCTTGTATC-3 ', and 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.
As claimed in claim 1 from soil the method for No. 4 microspecies in the detection by quantitative banana blight bacteria torrid zone, it is characterized in that, the reaction of step 3 fluorescence nucleic acid constant-temperature amplification detection technique is specific as follows:
Sample through pre-treatment, primer, reaction buffer are mixed with BstDNA polysaccharase, at 63 ℃ of insulation 90min, carry out endless chain replacement(metathesis)reaction;
RealAmp25 μ L reaction system: the nucleic acid DNA of 1 μ L extracting is as template, 2.5 μ L10 * Bst DNA polymerse buffer, 1.4mM dNTPs, 0.8mM trimethyl-glycine, the outer primer of 8mM MgSO4,0.2 μ M, the inner primer of 1.6 μ M, 1 μ L BstDNA polymerase, 0.2 μ M SYTO-9 fluorescence dye, moisturizing to 25 μ L, and then the paraffin oil of system such as add to cover whole reaction solution;
RealAmp reaction is carried out on ESE-Quant Tube Scanner, 80 ℃ of reaction 10min termination reactions after 63 ℃ of reaction 90min;
Before RealAmp reaction, cover and add the SYBR Green I fluorescence dye doubly diluting as 1 μ L1:10 in reaction tubes, question response finishes instantaneously centrifugal SYBR Green I to be added to the observation that develops the color in LAMP reaction solution afterwards; When positive control reaction is set, by the oranges and tangerines genome DNA that infects HLB, replace; When negative control reaction is set, with 100mM Tris-HCl pH8.0 and 50mM EDTA, replace, the reaction tubes that contains the reaction soln preparing is placed in to 63 ℃ of reaction 90min; Reaction finishes rear demonstration screen display amplification curve, and X-axis represents proliferation time, and Y-axis shows fluorescent value.
As claimed in claim 1 from soil the method for No. 4 microspecies in the detection by quantitative banana blight bacteria torrid zone, it is characterized in that, the construction process of step 4 RealAmp typical curve is as follows:
Adopt No. 4 microspecies Fusarium oxysporium f.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.
As claimed in claim 1 from soil the method for No. 4 microspecies in the detection by quantitative banana blight bacteria torrid zone, it is characterized in that, the PCR detection reaction system method of No. 4 microspecies in the banana blight bacteria torrid zone is as follows:
2.5 μ L10 * PCR buffer, 2 μ L dNTPs, 0.25 μ L Taq polysaccharase, each 1 μ L of primers F ocTR4-F/FocTR4-R, 1 μ L DNA, moisturizing to 25 μ L;
The condition of PCR reaction: 94 ℃ of denaturation 3min, 94 ℃ of sex change 30s, 57 ℃ of annealing 30s, 72 ℃ extend 60s, carry out 35 circulations; 72 ℃ extend 7min;
After reaction finishes, get 5 μ L reaction product, EB dyeing observations after 1% agarose gel electrophoresis;
Pcr amplification band cut glue reclaim to connect after pMD-18-T carrier order-checking correct after, by plasmid called after pMD18-T-TR4, after 10 times of gradient dilutions, as template, for assessment of the detection sensitivity of RealAmp, also build thus the typical curve of the RealAmp of No. 4 microspecies in the banana blight bacteria torrid zone.
As claimed in claim 1 from soil the method for No. 4 microspecies in the detection by quantitative banana blight bacteria torrid zone, it is characterized in that, result judgement adopts two kinds of method judged results, a kind of is the judgement of detection by quantitative result, directly, after ESE-Quant Tube Scanner reaction finishes, instrument shows quantitative result according to typical curve automatically; In addition, reaction finishes rear centrifugal the SYBR Green I covering in reaction tubes is blended in reaction solution, adopts covered colour developing to carry out judged result, if reaction solution color is orange, represents that result is negative, and if reaction solution color be green, expression result is positive.
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| 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 |
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| WO2022102726A1 (en) * | 2020-11-11 | 2022-05-19 | 国立研究開発法人理化学研究所 | Method of diagnosing fusarium oxysporum soil by pathogen genomics |
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| CN104611452A (en) * | 2015-02-16 | 2015-05-13 | 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 |
| CN104611451A (en) * | 2015-02-16 | 2015-05-13 | 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 |
| CN108060211A (en) * | 2018-01-23 | 2018-05-22 | 中国热带农业科学院环境与植物保护研究所 | A kind of method for quantitatively detecting mango anthrax-bacilus |
| 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 |
| CN111944919A (en) * | 2020-07-31 | 2020-11-17 | 广东省农业科学院果树研究所 | Visual detection technology system for banana vascular wilt germ tropical No.4 microspecies capable of being operated in field at normal temperature |
| WO2022102726A1 (en) * | 2020-11-11 | 2022-05-19 | 国立研究開発法人理化学研究所 | Method of diagnosing fusarium oxysporum soil by pathogen genomics |
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