CN103589794A - Method for real-time fluorescence isothermal quantitative detection of citrus huanglongbing - Google Patents
Method for real-time fluorescence isothermal quantitative detection of citrus huanglongbing Download PDFInfo
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Abstract
The invention discloses a method for real-time fluorescence isothermal quantitative detection of citrus huanglongbing. The method includes the steps: pretreating a detected sample, and rapidly extracting DNA; according to an HLB16SrDNA conserved sequence, designing two pairs of specific primers including an inner side primer HLB-F3, an inner side primer HLB-B3, an outer primer HLB-FIP and an outer primer HLB-BIP, and preparing the specific primers; carrying out a fluorescence nucleic acid isothermal amplification detection technology reaction; constructing a fluorescence nucleic acid isothermal amplification detection standard curve according to a relationship between plasmid concentrations of a template pMD18-T-HLB with different dilution and corresponding Tt values, wherein the X axis represents log values of starting template concentrations, and the Y axis represents the time taken for amplifying the different-concentration templates to reach thresholds; adopting quantitative detection result judgment, after ending an ESE-Quant TubeScanner reaction, displaying quantitative results by an instrument automatically according to the standard curve, after ending the reaction, instantaneously centrifuging to mix SYBRGreenI on an inner cover of a reaction tube into a reaction liquid, and judging results by adopting color development without opening the cover. The method has the advantages of high sensitivity, high specificity, low pollution, and stable reaction.
Description
Technical field
The invention belongs to Citrus Huanglongbing pathogen detection technique field, relate in particular to a kind of method that real-time fluorescence constant-temperature quantitative detects Citrus Huanglongbing pathogen.
Background technology
Citrus Huanglongbing pathogen (CitrusHuanglongbing, HLB) is one of destructive disease of tool on world's Orange Producing, the important object of Plant Quarantine both at home and abroad.HLB is mainly distributed in nearly 50 countries and regions of Asia, Africa, Oceania, South America and North America at present.Citrus huanglongbing pathogen bacterium can be infected various citrus, and symptom has mottled type yellow, evenly type yellow, the yellow of nutritional deficiency type and " rubber " fruit etc., shortens the plant economical life, reduces yield and quality, causes huge financial loss.19 oranges and tangerines of China produce and economize Weihai that in (city, autonomous region), great majority are received this disease, seriously restrict the sound development of Citrus Industry.
Citrus huanglongbing pathogen bacterium still can not artificial culture, generally believe that at present prokaryotic organism Gracilicutes distortion Gammaproteobacteria (Proteobacteria) α-modification Gammaproteobacteria (Alphaproteobacteriacea) root nodule Zoopagales (Rhizobiales) Rhizobiaceae (Rhizobiaceae) phloem Bacillaceae (' CandidatusLiberibacter ') is the main pathogenic fungi that causes yellow twig, its pathogenic bacteria is divided into Asia kind (' Ca.L.asiaticus '), (' Ca.L.africanus ') and America kind (' Ca.L.americanus ') are planted in Africa, China's citrus huanglongbing pathogen is mainly Asia kind (' Ca.L.asiaticus ').Because Citrus Huanglongbing pathogen there is no specific treatment medicament, also not anti-(resistance to) sick kind can be for application.Therefore, strict quarantine measures, plant anosis nursery stock, excavate disease tree in time, and system and to prevent and treat wood louse be to prevent and treat at present comparatively effective means of yellow twig comprehensively, and fast, stable, sensitive detection method is the important guarantee that nontoxic seedling is produced.
In recent years, there is method that report confirms PCR-based successfully for the detection of HLB, PCR method improves a lot compared with ordinary method on the operating time and aspect the specificity detecting and sensitivity, but, PCR detection technique needs special plant and instrument and testing cost higher, and its detection time still long (2～3 hours), be not suitable for the large-scale application fast in field.Therefore,, in strengthening oranges and tangerines nursery stock quarantine monitoring, in the urgent need to developing a kind of highly sensitive, easy and simple to handle, with low cost and detection method that result is swift with judgement, can replace to a certain extent the detection method of PCR.
Japanese scholars Notomi equals to have developed first for 2000 ring mediated isothermal nucleic acid amplification reaction (loop-mediatedisothermalamplification, 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-timefluorescenceloop-mediatedisothermalamplificatio nassay, 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, 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 a kind of auxiliary judgment measure using SYBRGreenI 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, analysis judgement reaction product result.
At present, the tufB-secE-nusG-rplKAJL-rpoB gene of external Okudaetal. (2005) yellow twig Asia kind has been set up the LAMP detection technique of HLB Asia kind, its detection sensitivity can detect about 300 gene copies, and Japan and Indonesian HLB have been carried out detecting analysis.At home, Huang Li etc. (2012) adopt ribosomal outer membrane protein gene (Genebank accession number: AY842429) set up the LAMP method for quick of HLB, plasmid DNA copy number is 3.9 * 10
1template still can effectively increase, higher than the LAMP detection sensitivity of Okudaetal. (2005), author infers it may is the result of primer amplification efficiency and reaction system optimization.Kong Deying etc. (2013) adopt ribosomal outer membrane protein gene (Genebank accession number: the LAMP detection method of HQ2627229.1) having set up Asia kind for target equally, its sensitivity can reach 25pg/ μ L, identical with the sensitivity of contrast real time fluorescent PCR method.
Summary of the invention
A kind of method that the object of the embodiment of the present invention is to provide real-time fluorescence constant-temperature quantitative to detect Citrus Huanglongbing pathogen, is intended to solve that the sensitivity that existing Citrus Huanglongbing pathogen detection method exists is low, specificity is low, high pollution, the unsettled problem of reaction.
The embodiment of the present invention is achieved in that the real-time fluorescence constant-temperature quantitative of the embodiment of the present invention detects the method for Citrus Huanglongbing pathogen, and the method steps of this real-time fluorescence constant-temperature quantitative detection Citrus Huanglongbing pathogen is as follows:
The structure of step 4, fluorescence nucleic acid constant-temperature amplification examination criteria curve; Adopt HLB Asia Species specific PCR primer OI1 and OI2c, this primer amplification rDNA sequence comprise whole RealAmp amplification region between, clone the plasmid after this sequence checks order correctly to pMD18-T carrier, called after pMD18-T-HLB, for building typical curve;
The method of the PCR detection reaction system of HLB: 2.5 μ L10 * PCRbufferMg2+, 2 μ L dNTPs(2.5mMeach), 0.25 μ LTaq polysaccharase (5U/ μ L), primer OI1(5 '-GCGCGTATTTTATACGAGCGGCA-3 ') and OI2c(5 '-GCCTCGCGACTTCGCAACCCAT-3 ') each 1 μ L(10pM), 1 μ LDNA, moisturizing to 25 μ L; The condition of PCR reaction: 94 ℃ of denaturation 3min, 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ extend 90s, 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-HLB10ng/ μ L, after 10 times of gradient dilutions as template for assessment of the detection sensitivity of fluorescence nucleic acid constant-temperature amplification and build thus the typical curve of HLBRealAmp;
The judgement of step 5, employing detection by quantitative result, after ESE-QuantTubeScanner reaction finishes, instrument shows quantitative result according to typical curve automatically, reaction finishes the rear instantaneous centrifugal SYBRGreenI covering in reaction tubes is blended in reaction solution, adopts covered colour developing to carry out judged result.
Further, in step 1, DNA extraction liquid comprises: 100mMTris-HClpH8.0,20mM EDTApH8.0 and 2%w/vSDS.
Further, in step 2, the primer mixed solution being formed by two pairs of primers, wherein HLB-F3 is that 5 '-GGCCTTAGGGTTGTAAAGC-3 ' and HLB-B3 are that 5 '-CACCTCTACACTCGGAATTC-3 ' is outside primer, HLB-FIP i.e. 5 '-GGCTGCTGGCACGAAGTTACGCCGGAGAAGATAATGAC-3 ' is that 5 '-GCGAGCGTTGTTCGGAATAACGTTGAGCCCTGGGATTTC-3 ' is inner side primer with HLB-BIP, and the concentration of outer primer HLB-F3 and described outer primer HLB-B3 is respectively 5pmol/ μ l; The concentration of inner primer HLB-FIP and inner primer HLB-BIP is 40pmol/ μ l.
Further, in step 3, fluorescence nucleic acid constant-temperature amplification 25 μ L reaction systems are: the nucleic acid DNA of 1 μ L extracting is as template, the inner primer FIP of the outer primer B3 of 2.5 μ L10 * BstDNApolymersebuffer, 1.4mMdNTPs, 0.8mM trimethyl-glycine, 8mMMgSO4,0.2 μ M and F3,1.6 μ M and BIP, 1 μ LBstDNApolymerase8U/ 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;
Fluorescence nucleic acid isothermal amplification reactions: 80 ℃ of reaction 10min termination reactions after 63 ℃ of reaction 90min, before RealAmp reaction, in reaction tubes, cover and add the SYBRGreenI fluorescence dye doubly diluting as 1 μ L1:10, question response finishes instantaneously centrifugal SYBRGreenI 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 TE, be that 100mMTris-HClpH8.0,50mMEDTA 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.
Further, in step 4, typical curve is to build according to the relation between the plasmid concentration of the template pMD18-T-HLB 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.
Real-time fluorescence constant-temperature quantitative provided by the invention detects the method for Citrus Huanglongbing pathogen, adopting real-time fluorescence nucleic acid constant-temperature amplification detection technique is 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, there is highly sensitive, high specific, low pollution, the advantage of stable reaction, adopt impact and the energy Site Detection pedotheque of covered not vulnerable to pollution thing, the method of analyzing judgement reaction product is very simple, be suitable for applying widely, by adopting rrna 16SrDNA, be the LAMP detection system that target sequence is set up HLB Asia kind, detection sensitivity is 1pg/ μ L plasmid DNA, improves SYBRGreenI dyeing process simultaneously and prevents from polluting, and is applicable to high-throughout Fields detection analysis.
Accompanying drawing explanation
Fig. 1 is the method flow diagram that the real-time fluorescence constant-temperature quantitative that provides of the embodiment of the present invention detects Citrus Huanglongbing pathogen;
Fig. 2 is the specificity analyses schematic diagram that the oranges and tangerines HLBRealAmp that provides of the embodiment of the present invention detects;
Fig. 3 is that the susceptibility that the oranges and tangerines HLBRealAmp that provides of the embodiment of the present invention detects is analyzed schematic diagram;
Fig. 4 is the structure schematic diagram of the oranges and tangerines HLBRealAmp typical curve that provides of the embodiment of the present invention;
Fig. 5 is the field sample segment quantitative detecting analysis schematic diagram that the embodiment of the present invention provides.
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.
Below in conjunction with drawings and the specific embodiments, application principle of the present invention is further described.
As shown in Figure 1, the method for the real-time fluorescence constant-temperature quantitative of embodiment of the present invention detection Citrus Huanglongbing pathogen comprises the following steps:
S101: test sample is carried out to pre-treatment, DNA rapid extraction;
S102: design 2 pairs of Auele Specific Primers according to HLB16SrDNA conserved sequence, each is a pair of to comprise inner side primer HLB-F3, HLB-B3 and outside primer HLB-FIP, HLB-BIP, prepares Auele Specific Primer;
S103: fluorescence nucleic acid constant-temperature amplification detection technique reaction;
S104: the structure of fluorescence nucleic acid constant-temperature amplification examination criteria curve, according to the relation between the plasmid concentration of the template pMD18-T-HLB of difference dilution and corresponding Tt value, build, 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;
S105: adopt the judgement of detection by quantitative result, after ESE-QuantTubeScanner reaction finishes, instrument shows quantitative result according to typical curve automatically, reaction finishes the rear instantaneous centrifugal SYBRGreenI covering in reaction tubes is blended in reaction solution, adopts covered colour developing to carry out judged result.
Concrete steps of the present invention are:
Fluorescence nucleic acid isothermal amplification reactions: 80 ℃ of reaction 10min termination reactions after 63 ℃ of reaction 90min, before RealAmp reaction, in reaction tubes, cover and add the SYBRGreenI fluorescence dye doubly diluting as 1 μ L1:10, question response finishes instantaneously centrifugal SYBRGreenI 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 TE, be that 100mMTris-HClpH8.0,50mMEDTA 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.
The structure of step 4, fluorescence nucleic acid constant-temperature amplification examination criteria curve; Adopt HLB Asia Species specific PCR primer OI1 and OI2c, this primer amplification rDNA sequence comprise whole RealAmp amplification region between, clone the plasmid after this sequence checks order correctly to pMD18-T carrier, called after pMD18-T-HLB, for building typical curve;
The method of the PCR detection reaction system of HLB: 2.5 μ L10 * PCRbufferMg2+, 2 μ L dNTPs(2.5mMeach), 0.25 μ LTaq polysaccharase (5U/ μ L), primer OI1(5 '-GCGCGTATTTTATACGAGCGGCA-3 ') and OI2c(5 '-GCCTCGCGACTTCGCAACCCAT-3 ') each 1 μ L(10pM), 1 μ LDNA, moisturizing to 25 μ L; The condition of PCR reaction: 94 ℃ of denaturation 3min, 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ extend 90s, 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-HLB10ng/ μ L, after 10 times of gradient dilutions as template for assessment of the detection sensitivity of fluorescence nucleic acid constant-temperature amplification and build thus the typical curve of HLBRealAmp; Typical curve is to build according to the relation between the plasmid concentration of the template pMD18-T-HLB 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.
The judgement of step 5, employing detection by quantitative result, after ESE-QuantTubeScanner reaction finishes, instrument shows quantitative result according to typical curve automatically, reaction finishes the rear instantaneous centrifugal SYBRGreenI covering in reaction tubes is blended in reaction solution, adopts covered colour developing to carry out judged result.
The present invention is described further in conjunction with specific embodiments:
Method for extracting nucleic acid: get the nearly petiole of the arteries and veins 5-10mg of place in the aging blade of this season and be placed in 1.5mL centrifuge tube, in liquid nitrogen, be ground to after Powdered and add 60 μ LTES damping fluids, after 70 ℃ of water bath with thermostatic control 7min, add again 60 μ L phenol: chloroform: primary isoamyl alcohol mixed solution (25:24:1), vortex mixes the centrifugal 5min of rear 12000rpm, getting 40 μ L supernatant liquors adds in the microtrabeculae consisting of SephadexG-50-80,4 ℃, the centrifugal 4min of 5000rpm, with the centrifuge tube of a new aseptic 1.5mL, collect filtrate ,-20 ℃ save backup;
DNA extraction liquid comprises: 100mMTris-HCl(pH8.0), 20mMEDTA(pH8.0) and 2%(w/v) SDS;
The preparation of step 2, Auele Specific Primer
According to HLB16SrDNA conserved sequence, design 2 pairs of Auele Specific Primers, each is a pair of to comprise inner side primer (HLB-F3 and HLB-B3) and outside primer (HLB-FIP and HLB-BIP), the primer mixed solution being formed by two pairs of primers, HLB-F3(5 '-GGCCTTAGGGTTGTAAAGC-3 ' wherein) and HLB-B3(5 '-CACCTCTACACTCGGAATTC-3 ') be outside primer, HLB-FIP(5 '-GGCTGCTGGCACGAAGTTACGCCGGAGAAGATAATGAC-3 ') and HLB-BIP(5 '-GCGAGCGTTGTTCGGAATAACGTTGAGCCCTGGGATTTC-3 ') be inner side primer, the concentration of outer primer HLB-F3 and described outer primer HLB-B3 is respectively 5pmol/ μ l, the concentration of inner primer HLB-FIP and inner primer HLB-BIP is 40pmol/ μ l,
Table 1HLBRealAmp specific detection primer sequence
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-QuantTubeScanner, ESE-QuantTubeScanner 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 amplification finishes, at the amplification curve that shows screen display, have several numerical value:
Tt value (thresholdtime, 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, 2.5 μ L10 * Bst DNApolymersebuffer, 1.4mMdNTPs, 0.8mM trimethyl-glycine, 8mMMgSO4, the outer primer of 0.2 μ M (B3 and F3), the inner primer of 1.6 μ M (FIP and BIP), 1 μ LBstDNApolymerase(8U/ μ L), 0.2 μ MSYTO-9 fluorescence dye, moisturizing to 25 μ L, and then the paraffin oil that the system such as adds covers whole reaction solution and prevents volatilization, RealAmp reaction is at ESE-QuantTubeScanner (ESE Gmbh, Stockach, Germany) on, carry out, 80 ℃ of reaction 10min termination reactions after 63 ℃ of reaction 90min, before RealAmp reaction, in reaction tubes, cover and add the SYBRGreenI fluorescence dye doubly diluting as 1 μ L1:10, question response finishes instantaneously centrifugal SYBRGreenI to be added to the observation that develops the color in LAMP reaction solution afterwards, the detection Aerosol Pollution of avoiding uncapping, when positive control reaction is set, by the oranges and tangerines genome DNA that infects HLB, replace, when negative control reaction is set, with TE(100mMTris-HCl(pH8.0), 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,
The structure of step 4, RealAmp typical curve
For the detection sensitivity of accurate assessment RealAmp and build thus typical curve, adopt HLB Asia Species specific PCR primer OI1 and OI2c, this primer amplification rDNA sequence comprise whole RealAmp amplification region between, therefore, clone the plasmid after this sequence checks order correctly to pMD18-T carrier, called after pMD18-T-HLB, for building typical curve;
The PCR detection reaction system of HLB is with reference to the method for Jagoueix etc.: 2.5 μ L10 * PCRbuffer(Mg2+), 2 μ LdNTPs(2.5mMeach), 0.25 μ LTaq polysaccharase (5U/ μ L), primer OI1(5 '-GCGCGTATTTTATACGAGCGGCA-3 ') and OI2c(5 '-GCCTCGCGACTTCGCAACCCAT-3 ') each 1 μ L(10pM), 1 μ LDNA, moisturizing to 25 μ L, the condition of PCR reaction: 94 ℃ of denaturation 3min, 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ extend 90s, 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-HLB(10ng/ μ L), after 10 times of gradient dilutions as template for assessment of the detection sensitivity of RealAmp and build thus the typical curve of HLB RealAmp, typical curve is to build according to the relation between the plasmid concentration of the template pMD18-T-HLB of difference dilution and corresponding Tt value, X-axis represents the logarithmic value of starting template concentration, Y-axis represents that different concns template amplification reaches the threshold value time used (Tt),
The present invention adopts two kinds of method judged results, a kind of is the judgement of detection by quantitative result, directly after ESE-QuantTubeScanner 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 the instantaneous centrifugal SYBRGreenI 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 specificity, susceptibility and the field applied analysis that LAMP detects;
The specificity analyses that LAMP detects:
With other pathogen of oranges and tangerines, there is Citrus exocortis viroid (Citrusexocortisviroid, CEVd), broken mosaic virus (the Citrustatterleafvirus of oranges and tangerines, CTLV) and citrus anthracnose bacterium (Colletotrichum gloeosporioides), cucumber mosaic virus (Cucumbermosaicvirus, CMV), ring spot virus (the Papayaringspotvirus of papaya, PRSV), abaca bunchy top virus (Bananabunchytopvirus, BBTV) and No. 4 microspecies of banana blight bacteria (Fusariumoxysporumf.sp.cubenserace4), RNA template extra 0.2 μ LAMV ThermoScript II of adding in LAMP reaction system is carried out reverse transcription RealAmp reaction,
Result show template be only HLB genomic dna colour developing for green, be indicated as the positive, all the other be orange, be indicated as feminine gender (seeing Fig. 2-C); Through 2% agarose gel electrophoresis, detect the product of each PCR pipe, only template is that HLB genomic dna shows gradient band, all the other are all without gradient band (seeing Fig. 2-A), and what show to only have positive reaction in RealAmp detects just has a gradient band, and negative reaction without gradient band; The amplification curve that ESE-QuantTubeScanner instrument carries out after RealAmp amplified reaction only has 1, and corresponding numbering is corresponding is to take the PCR pipe that HLB genome is template,
HLB Asia species-specific primer OI1/OI2c with reference to designs such as Jagoueix, amplification is for the confession examination pathogenic bacteria of specificity analyses, only template is that to amplify size be the single band of 1167bp to HLB genomic dna, all the other are all without band (seeing Fig. 2-B), two kinds of detection methods that RealAmp adopts, the HLB specific PCR detected result that the people such as SYBRGreenI colour developing and agarose gel electrophoresis analytical results and Jagoueixa design is consistent, shows that the designed primer of RealAmp is the Auele Specific Primer of HLB;
(A) the corresponding Citrus exocortis viroid (Citrusexocortisviroid of 1-8 difference, CEVd), citrus anthracnose bacterium (Colletotrichumgloeosporioides), the ring spot virus of papaya (Papayaringspot virus, PRSV), abaca bunchy top virus (Bananabunchytopvirus, BBTV) and the DNA of No. 4 microspecies of banana blight bacteria (Fusariumoxysporumf.sp.cubenserace4) and the broken mosaic virus (Citrustatterleafvirus of oranges and tangerines, CTLV) and cucumber mosaic virus (Cucumbermosaicvirus, CMV) RNA, RNA template extra 0.2 μ LAMV ThermoScript II of adding in LAMP reaction system is carried out RT-LAMP reaction, 2% agarose gel electrophoresis detected result shows the only amplified production electrophoresis showed gradient band of HLBDNA, all the other contrasts all do not amplify product,
(B) the PCR product of each template in HLB Asia strain specific PCR primer OI1/OI2c amplification (A), only has HLBDNA to amplify single 1167bp band,
(C) SYBRGreenI staining detects the RealAmp amplified production of each template in (A), and green is positive, and orange be negative;
(D) each template adopts ESE-QuantTubeScanner instrument to carry out the amplification curve after RealAmp amplified reaction, and X-axis represents proliferation time (min), and Y-axis represents the fluorescence signal intensity (mV) of response.
The sensitivity analysis that LAMP detects:
The pMD-18-T-HLB plasmid DNA comprising between RealAmp amplification region of take is template, according to 10 times of gradient series dilution methods, pMD-18-T-HLB plasmid DNA is diluted to 1 * 10 successively from 10ng/ μ l
-6ng/ μ l, with the negative contrast of sterilized water, result shows, pMD-18-T-HLB plasmid DNA concentration is 10ng/ μ l～1 * 10
-3all 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
-3ng/ μ l(is shown in Fig. 3-D); 2% agarose gel electrophoresis detects the product of each PCR pipe, and pMD-18-T-HLB plasmid DNA concentration is 10ng/ μ l～1 * 10
-3ng/ μ l shows gradient band, and all the other show to be limited to 1 * 10 under RealAmp detects all without gradient band (seeing Fig. 3-A)
-3ng/ μ l, concentration is lower than 1 * 10
-3ng/ μ l examines and does not measure;
(A) take pMD18-T-HLB plasmid DNA carries out RealAmp sensitivity analysis as template (10ng/ μ L), and agarose gel electrophoresis detected result shows that the detection lower limit of RealAmp is about 1pg/ μ L;
(B) take pMD18-T-HLB plasmid DNA carries out PCR detection sensitivity as template (10ng/ μ L), and the detection lower limit of PCR is about 100pg/ μ L, and the detection sensitivity of RealAmp is about 100 times of PCR;
(C) SYBRGreenI staining detects the RealAmp amplified production of each template in (A), auxiliary judgment result, green is positive, and orange be negative, coloration result is consistent with the electrophoresis detection result in (A);
(D) each concentration pMD18-T-HLB plasmid DNA ESE-QuantTubeScanner instrument carries out the amplification curve after RealAmp amplified reaction, and X-axis represents proliferation time (min), and Y-axis represents the fluorescence signal intensity (mV) of response;
The Specification Curve of Increasing that LAMP detects
To comprise the pMD-18-T-HLB plasmid DNA between RealAmp amplification region, according to being used as the detection sensitivity of template assessment RealAmp and the amplification curve after ESE-QuantTubeScanner amplified reaction after 10 times of gradient series dilutions, can the increase template DNA of five 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=-7.4x+82 (R that LAMP detects
2=0.9884) (Fig. 4), during application, only need to know that amplification reaches the threshold value time used (Tt), just can obtain the content of HLB pathogenic bacteria in oranges and tangerines plant tissue;
(A) after 10 times of gradient dilution pMD18-T-HLB plasmid DNA, be used as the detection sensitivity of template assessment RealAmp, amplification curve after ESE-QuantTubeScanner amplified reaction, can the increase template DNA of five orders of magnitude of RealAmp, X-axis represents proliferation time (min), and Y-axis represents the fluorescence signal intensity (mV) of response;
(B) structure of HLBRealAmp amplification curve, relation between the starting template DNA concentration of different concns and corresponding Tt value builds, 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);
The field application that LAMP detects:
Doubtful HLB blade in symptoms such as 224 performance chrysanthemums of oranges and tangerines plantation random acquisition carries out RealAmp detection, positive control and negative control are set, result shows, in 224 samples, the positive that PCR and LAMP detect is respectively 201(89.7%) and 211(94.2%), at PCR, detect in 23 negative samples, LAMP detects 11 positive more, show that LAMP detected result is reliable and stable, recall rate is higher than PCR method, and the content of HLB pathogenic bacteria in can quantitative analysis oranges and tangerines plant tissue, the results are shown in Figure 5 and table 2
(A) after part field sample DNA RealAmp reaction, 2% agarose gel electrophoresis detected result;
(B) SYBRGreenI staining detects the RealAmp amplified production of each template in (A), and green is positive, and orange be negative;
(C) each sample DNA adopts ESE-QuantTubeScanner instrument to carry out the amplification curve after RealAmp amplified reaction, and X-axis represents proliferation time (min), and Y-axis represents the fluorescence signal intensity (mV) of response;
(D) each sample detection by quantitative result.
The comparison of table 2PCR and two kinds of detection methods of LAMP
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 (5)
1. real-time fluorescence constant-temperature quantitative detects a method for Citrus Huanglongbing pathogen, it is characterized in that, the method steps of this real-time fluorescence constant-temperature quantitative detection Citrus Huanglongbing pathogen is as follows:
Step 1, test sample is carried out to pre-treatment, extract DNA, get the nearly petiole of the arteries and veins 5mg～10mg of place in aging blade and be placed in 1.5mL centrifuge tube, in liquid nitrogen, be ground to after Powdered and add 60 μ LTES damping fluids, after 70 ℃ of water bath with thermostatic control 7min, add again 60 μ L phenol: chloroform: primary isoamyl alcohol mixed solution, phenol in mixed solution: chloroform: the weight ratio of primary isoamyl alcohol is 25:24:1, vortex mixes the centrifugal 5min of rear 12000rpm, getting 40 μ L supernatant liquors adds in the microtrabeculae consisting of SephadexG-50-80, 4 ℃, the centrifugal 4min of 5000rpm, with the centrifuge tube of a new aseptic 1.5mL, collect filtrate,-20 ℃ save backup,
Step 2, according to HLB16SrDNA conserved sequence, design 2 pairs of Auele Specific Primers, each is a pair of to comprise inner side primer HLB-F3, HLB-B3 and outside primer HLB-FIP, HLB-BIP, prepares Auele Specific Primer;
Step 3, fluorescence nucleic acid constant-temperature amplification detection reaction; Sample through pre-treatment, primer, reaction buffer are mixed with BstDNA polysaccharase, at 63 ℃ of insulation 90min, carry out endless chain replacement(metathesis)reaction;
The structure of step 4, fluorescence nucleic acid constant-temperature amplification examination criteria curve; Adopt HLB Asia Species specific PCR primer OI1 and OI2c, primer amplification rDNA sequence comprise whole RealAmp amplification region between, cloned sequence is to the plasmid after the order-checking correctly of pMD18-T carrier, called after pMD18-T-HLB, for building typical curve;
The method of the PCR detection reaction system of HLB: 2.5 μ L10 * PCRbufferMg2+, 2 μ LdNTPs(2.5mMeach), 0.25 μ LTaq polysaccharase (5U/ μ L), primer OI1(5 '-GCGCGTATTTTATACGAGCGGCA-3 ') and OI2c(5 '-GCCTCGCGACTTCGCAACCCAT-3 ') each 1 μ L(10pM), 1 μ LDNA, moisturizing to 25 μ L; The condition of PCR reaction: 94 ℃ of denaturation 3min, 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ extend 90s, 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-HLB10ng/ μ L, after 10 times of gradient dilutions as template for assessment of the detection sensitivity of fluorescence nucleic acid constant-temperature amplification and build the typical curve of HLB RealAmp;
The judgement of step 5, employing detection by quantitative result, after ESE-QuantTubeScanner reaction finishes, instrument shows quantitative result according to typical curve automatically, reaction finishes the rear instantaneous centrifugal SYBRGreenI covering in reaction tubes is blended in reaction solution, adopts covered colour developing to carry out judged result.
2. real-time fluorescence constant-temperature quantitative as claimed in claim 1 detects the method for Citrus Huanglongbing pathogen, it is characterized in that, in step 1, DNA extraction liquid comprises: 100mMTris-HClpH8.0,20mMEDTA pH8.0 and 2%w/vSDS.
3. real-time fluorescence constant-temperature quantitative as claimed in claim 1 detects the method for Citrus Huanglongbing pathogen, it is characterized in that, in step 2, the primer mixed solution being formed by two pairs of primers, wherein HLB-F3 is that 5 '-GGCCTTAGGGTTGTAAAGC-3 ' and HLB-B3 are that 5 '-CACCTCTACACTCGGAATTC-3 ' is outside primer, HLB-FIP i.e. 5 '-GGCTGCTGGCACGAAGTTACGCCGGAGAAGATAATGAC-3 ' is that 5 '-GCGAGCGTTGTTCGGAATAACGTTGAGCCCTGGGATTTC-3 ' is inner side primer with HLB-BIP, the concentration of outer primer HLB-F3 and described outer primer HLB-B3 is respectively 5pmol/ μ l, the concentration of inner primer HLB-FIP and inner primer HLB-BIP is 40pmol/ μ l.
4. real-time fluorescence constant-temperature quantitative as claimed in claim 1 detects the method for Citrus Huanglongbing pathogen, it is characterized in that, in step 3, fluorescence nucleic acid constant-temperature amplification 25 μ L reaction systems are: the nucleic acid DNA of 1 μ L extracting is as template, 2.5 μ L10 * BstDNApolymersebuffer, 1.4mMdNTPs, 0.8mM trimethyl-glycine, 8mMMgSO4, outer primer B3 and the F3 of 0.2 μ M, inner primer FIP and the BIP of 1.6 μ M, 1 μ LBstDNApolymerase8U/ 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,
Fluorescence nucleic acid isothermal amplification reactions: 80 ℃ of reaction 10min termination reactions after 63 ℃ of reaction 90min, before RealAmp reaction, in reaction tubes, cover and add the SYBRGreenI fluorescence dye doubly diluting as 1 μ L1:10, question response finishes instantaneously centrifugal SYBRGreenI 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 TE, be that 100mMTris-HClpH8.0,50mMEDTA 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.
5. real-time fluorescence constant-temperature quantitative as claimed in claim 1 detects the method for Citrus Huanglongbing pathogen, it is characterized in that, in step 4, typical curve is to build according to the relation between the plasmid concentration of the template pMD18-T-HLB 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.
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