CN103205504A - Real-time fluorescence quantification PCR (Polymerase Chain Reaction) method for detecting ustilago scitaminea - Google Patents
Real-time fluorescence quantification PCR (Polymerase Chain Reaction) method for detecting ustilago scitaminea Download PDFInfo
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Abstract
The invention relates to a real-time fluorescence quantification PCR (Polymerase Chain Reaction) method for detecting ustilago scitaminea. The method comprises the steps of sugarcane genome DNA (Deoxyribonucleic Acid) extraction, real-time fluorescence quantification PCR detection system establishment, experiment effectiveness judgment and real-time fluorescence quantification PCR detection. According to the real-time fluorescence quantification PCR method, a specificity quantification PCR detection primer and a Taqman probe are designed according to a bE gene of the ustilago scitaminea; with the adoption of the real-time fluorescence quantification PCR method, the quantity of the ustilago scitaminea in a sample to be detected is calculated by a constructed standard curve equation; and the method for detecting the ustilago scitaminea is strong in specificity, high in sensitivity, easy and simple to operate, quick and efficient, and is applicable to resistance identification of the ustilago scitaminea, and to early detection and diagnosis of the ustilago scitaminea infection of farm sugarcane.
Description
Technical field
The present invention relates to the detection method of a kind of sugarcane pathogenic bacteria, be specifically related to a kind of real time fluorescence quantifying PCR method that detects ustilago scitaminea bacteria, belong to biological technical field.
Background technology
Smut of sugarcane has another name called sugarcane whip smut, smut disease, and the obvious characteristics of this disease is that the diseased plant tip of a branch grows a whiplike thing of black.Smut of sugarcane be by ustilago scitaminea bacteria (
Sporisorium scitamineum) a kind of gas borne fungus diseases of causing, its pathogenic bacteria is the sugarcane ustilago of the Basidiomycetes Ustilago in the fungi.Infect the kind sugarcane rudiment of black tassel bacteria early, leaf is elongated, pale green, and stem is tiny, tillers to increase, and also grows the dust-brand whip on after this tillering.The sick sugarcane of perennial root and the soil that carries disease germs are the primary source of infection of smut of sugarcane.Chlamydospore is by air-flow, rainwater, drop on after irrigation water and the entomochory and ensconce between scale on the sugarcane sugarcane bud, meet water sprouting formation and infect mycelia intrusion sugarcane bud, mycelia after temporary transient dormancy with the sugarcane bud sprout reactivate and with vegetative point to vertical spread, vegetative point is subjected to germ to stimulate the whiplike thing of initial stage adularescent to be formed up to and extracts the whiplike thing of black when chlamydospore forms out, the chlamydospore that produces is fallen on the lateral bud of sugarcane stem through media transmission again, stimulate the sugarcane bud to sprout, make sugarcane extract side shoot out or tiller and grow up to whiplike thing, hide the smut mycelia in the sugarcane bud and the chlamydospore that falls into soil become the primary source of infection of next sugarcane in season.When soil was moist, spore was almost all sprouted in 48 hours, then very fast death when the sprouting spore does not in time meet the sugarcane bud.Chlamydospore can survive in the soil of drying several months to 1 year.The Winter-Spring arid is conducive to the spore survival, and spring, high temperature and rainy was beneficial to smut generation and popular.The sugarcane field of perennial root is heavier than the morbidity of new plant cane field, and the perennial root time limit is more long, and disease takes place more serious, and the whole strain kind ground morbidity of tip of a branch seed ground is light.
So far, smut of sugarcane has become one of main disease of respectively planting the sugarcane district, and the trend that increases the weight of is year by year arranged, and the serious time is caused the loss of sugarcane yield 20 %～50 %.Because the sugarcane production cycle is long, plant is tall and big, cause of disease spore amount is big, cultivates and utilize the smut resistance kind to become the most economical effective measures of control smut of sugarcane.Country's " eight or five " key scientific and technological projects begin to pay close attention to the research of smut of sugarcane resistance, and from " 95 ", smut resistance are examined one of (mirror) fixed important indicator as sugar cane breed.
In the process of cultivating the smut resistance sugar cane breed, the sensitivity of ustilago scitaminea bacteria detection technique and efficient have directly influenced the process of smut resistance breeding.At present, whether sugarcane is subjected to cause of disease to infect decision method commonly used mainly is according to disease symptom, realizes by sickness rate and disease index investigation, external also determines that by catching the cause of disease spore variation of cause of disease quantity predicts the popular of disease.Though above-mentioned traditional method is comparatively directly perceived, but because there is latent infection in cause of disease, can not determine fully based on the decision method of phenotype symptom whether sugarcane infected by black tassel bacteria, also be difficult to judge spore amount wherein, field planting and data gathering simultaneously also has the cost height, long (need the investigation in disease generation and the popular phase in whole crop season of cycle, collect, together with plantation 8-10 consuming time month), be subjected to environment and human factor to influence bigger problem, and the method for seizure cause of disease spore, also be subjected to weather such as wind direction easily, the influence of aspects such as air quantity operation and sugarcane plant size and reguarity, bad weather waits the seizure that also can further influence the cause of disease spore as raining.In addition, by the traditional method that germ separation and purification cultivation and cause of disease morphology are identified, consuming time also the need is unfavorable in time detecting, judges and controls in a week usually.Real time fluorescence quantifying PCR method high specificity, highly sensitive, easy and simple to handle, rapidly and efficiently also can carry out multiple detection, efficiently solves the limitation that conventional P CR amplification can only end point determination.The present invention is intended to utilize real time fluorescence quantifying PCR method to detect black tassel bacteria fast, delicately before the smut of sugarcane symptom does not occur, for ustilago scitaminea bacteria rapid detection and early prevention provide scientific basis.
Summary of the invention
The purpose of this invention is to provide a kind of real time fluorescence quantifying PCR method that detects ustilago scitaminea bacteria, can be applicable to early detection and diagnosis that the evaluation of smut of sugarcane resistance and field sugarcane infect black tassel bacteria.
Purpose of the present invention is achieved through the following technical solutions.
A kind of real time fluorescence quantifying PCR method that detects ustilago scitaminea bacteria of the present invention comprises that sugarcane extracting genome DNA, the foundation of real-time fluorescence quantitative PCR detection architecture, experiment availability deciding and real-time fluorescence quantitative PCR detect; It is characterized in that:
(1) the real-time fluorescence quantitative PCR detection architecture is set up: the real-time fluorescence quantitative PCR reaction totally is 25 μ L, comprise 2 * TaqMan Universal Master Mix, 12.5 μ L, each 1 μ L of 10 μ mol/L quantitative PCR detection primer bEQ-F and bEQ-R, 10 μ mol/L Taqman probes, 0.2 μ L, sugarcane dna profiling 1 μ L, ddH
2O mends to 25 μ L; Amplification program is: 50 ℃, and 2 min, 95 ℃, 10 min; 95 ℃, 15 s, 60 ℃, 1 min, 40 circulations are carried out the single-point fluoroscopic examination at 60 ℃; The dna sequence dna of described primer and Taqman probe is as follows:
bEQ-F：5'-TGAAAGTTCTCATGCAAGCC-3'，
bEQ-R：5'-TGAGAGGTCGATTGAGGTTG-3'；
Taqman probe: 5'-FAM-TGCTCGACGCCAATTCGGAG-TAMRA-3';
(2) experiment availability deciding
The experiment that possesses a, b, three conditions of c simultaneously is effectively experiment, otherwise is invalid experiment; Described a, b, three conditions of c are as follows: a, blank, no Ct value and do not have amplification curve; B, negative control, no Ct value and do not have amplification curve; Ct value≤35.0 of c, positive control, and typical amplification curve appears: as shown in Figure 1, described typical amplification curve is S type kinetic curve; Threshold line refers to that the vertex with negative control sample amplification curve is benchmark, the straight line parallel with X-coordinate; The corresponding fluorescent quantitative PCR cycle number of intersection point of threshold line and typical amplification curve is the Ct value; Blank refers to use isopyknic ddH
2O replaces template DNA; Negative control is the known sample that does not contain ustilago scitaminea bacteria; Positive control is the known sample that contains ustilago scitaminea bacteria;
(3) real-time fluorescence quantitative PCR detects
During real-time fluorescence quantitative PCR detected, sample detection result was as follows:
A, no Ct value and do not have amplification curve do not contain ustilago scitaminea bacteria in the expression sample;
B, Ct value≤35.0, and typical amplification curve appears, contain ustilago scitaminea bacteria in the expression sample;
C, when Ct value＞35.0, then this sample is done repeated experiments; Repeated experiments result does not have the Ct value, does not then contain ustilago scitaminea bacteria in this sample; If the repeated experiments result has the Ct value, then contain ustilago scitaminea bacteria in this sample.
A kind of method of utilizing real-time fluorescence quantitative PCR to detect the ustilago scitaminea bacteria number is characterized in that utilizing above-mentioned Ct value and ustilago scitaminea bacteria
BEThe extension rate of plasmid makes up the typical curve equation, calculates contained ustilago scitaminea bacteria number in the sample to be checked.Described structure typical curve equation refers to that with the Ct value be ordinate zou, ustilago scitaminea bacteria
BEThe plasmid extension rate is the typical curve equation of X-coordinate, wherein,
BEThe plasmid extension rate refers to the ustilago scitaminea bacteria with prior art acquisition concentration known
BEPlasmid DNA is carried out the multiple proportions gradient dilution.
Advantage of the present invention is as follows:
1, first according to ustilago scitaminea bacteria
BEGene design specificity quantitative PCR detection primer and probe, and by ncbi database comparison retrieval, confirming does not have 100 % consistence with the other biological gene;
2, utilize real time fluorescence quantifying PCR method first, by the typical curve equation that makes up, the quantity of black tassel bacteria in the calculation sample;
3, the invention provides high specificity, highly sensitive, easy and simple to handle, ustilago scitaminea bacteria detection architecture rapidly and efficiently, can be applicable to early diagnosis and the smut resistance in sugarcane evaluation of field smut of sugarcane.
Description of drawings
Fig. 1 detects synoptic diagram for the ustilago scitaminea bacteria real-time fluorescence quantitative PCR.X-coordinate represents the quantitative pcr amplification cycle number, ordinate zou represents fluorescence signal intensity, 1 represents threshold line, the typical amplification curve of 2 representatives, 3 represent intersection point (the 3 corresponding quantitative PCR cycle numbers of threshold line 1 and amplification curve 2, be the Ct value), 4 represent negative control sample amplification curve, and 5 represent the vertex of negative control sample amplification curve.
Fig. 2 is the ustilago scitaminea bacteria of 8 10 times of gradient dilutions
BEThe amplification curve synoptic diagram of plasmid DNA standard substance real-time fluorescence quantitative PCR.X-coordinate represents the quantitative pcr amplification cycle number, and ordinate zou represents fluorescence signal intensity.
Fig. 3 is
BEThe typical curve synoptic diagram of plasmid DNA standard substance real-time fluorescence quantitative PCR amplification.The X-coordinate representative
BEThe multiple (10 of plasmid DNA gradient dilution
-3～10
-10), ordinate zou represents quantitative pcr amplification cycle number Ct value.
Fig. 4 is the electrophoresis result synoptic diagram of black tassel bacteria regular-PCR limit of detection concentration.M is 100 bp DNA Ladder marker, and 1～8 is copy number 1.987 * 10
8～1.987 * 10
18 10 times of gradient dilutions of copies/ μ L
BEThe regular-PCR amplified production of plasmid DNA standard substance.
Fig. 5 is the electrophoresis result synoptic diagram of black tassel bacteria regular-PCR limit of detection concentration in field smut of sugarcane disease plant ROC22+1 leaf.Wherein, 1～5 representative amplification of sugarcane+1 leaf genomic dna concentration behind 5 times of gradient dilutions of 500,100,20,4 and 0.8 ng/ μ L of catching an illness; 6 represent positive control, and 7 represent negative control, and 8 represent blank.
EmbodimentIn order further to illustrate the present invention rather than restriction the present invention, be illustrated below in conjunction with embodiment.
All available from giving birth to worker's biotechnology (Shanghai) limited-liability company, quantitative fluorescent PCR reagent is available from Roche company for various biochemical reagents, PDA substratum and the Streptomycin sulphate that the present invention uses; PMD18-T carrier, TAKALA
Ex Taq, fluorescence quantification PCR primer and probe be all available from TAKALA company; Glue reclaims test kit available from OMEGA company; Marker, bacillus coli DH 5 alpha are available from TIANGEN Biotech (Beijing) Co., Ltd.; Order-checking is finished in giving birth to worker's biotechnology (Shanghai) limited-liability company.
Embodiment one, a kind of real time fluorescence quantifying PCR method that detects ustilago scitaminea bacteria
1, the extraction of sugarcane genomic dna
Adopt the CTAB method to extract genomic dna, concrete operations are as follows:
(1) 7 sugarcane samples to be measured are 75 % alcohol wipe with volumetric concentration respectively after, be placed in the mortar, become fine powder with liquid nitrogen grinding, be sub-packed in 2 mL centrifuge tubes, each centrifuge tube sample-loading amount 0.2 g;
(2) add 900 μ L through 2 * CTAB of 65 ℃ of preheatings extracting solution vibration mixing, incubation 60 min put upside down mixing once every 5～10 min in 65 ℃ of water-baths;
(3) take out centrifuge tube 12,000 rpm/min, centrifugal 5 min after the water-bath.Draw supernatant liquor 750 μ L in 2 new mL centrifuge tubes, add equal-volume phenol/chloroform/primary isoamyl alcohol (volume ratio is 25:24:1), vibration, 12,000 rpm/min, centrifugal 10 min of room temperature;
(4) get supernatant liquor 600 μ L, change in the 1.5 new mL centrifuge tubes, add equal-volume chloroform/primary isoamyl alcohol (volume ratio is 24:1), vibration, 12,000 rpm/min, centrifugal 10 min of room temperature;
(5) draw supernatant liquor 500 μ L, change in the 1.5 new mL centrifuge tubes, add the Virahol of equal-volume-20 ℃ precooling, rock deposit D NA, 10,000 rpm/min, 4 ℃ of centrifugal 5 min;
(6) take out centrifuge tube, abandon liquid phase, the volumetric concentration that adds 4 ℃ of precoolings of 1,000 μ L is 75 % washing with alcohol precipitation twice, and absolute ethanol washing is once abandoned liquid phase, Bechtop drying 15～20 min;
(7) add 100 μ L ddH
2O(contains RNase A 100 μ g/mL) 37 ℃ of water-bath 30 min, electrophoresis detection is also measured the OD value, and-20 ℃ of preservations are standby.
2, the real-time fluorescence quantitative PCR detection architecture is set up
The real-time fluorescence quantitative PCR reaction totally is 25 μ L, comprise 2 * TaqMan Universal Master Mix, 12.5 μ L, each 1 μ L of 10 μ mol/L quantitative PCR detection primer bEQ-F and bEQ-R, 10 μ mol/L Taqman probes, 0.2 μ L, sugarcane dna profiling 1 μ L, ddH
2O mends to 25 μ L; Amplification program is: 50 ℃, and 2 min, 95 ℃, 10 min; 95 ℃, 15 s, 60 ℃, 1 min, 40 circulations are carried out the single-point fluoroscopic examination at 60 ℃; Described primer bEQ-F, bEQ-R and Taqman probe are with ustilago scitaminea bacteria
BEGene order is the basis, the ustilago scitaminea bacteria specificity detection by quantitative sequence of design, and the dna sequence dna of primer and probe is as follows:
bEQ-F：5'-TGAAAGTTCTCATGCAAGCC-3'，
bEQ-R：5'-TGAGAGGTCGATTGAGGTTG-3'；
Taqman probe: 5'-FAM-TGCTCGACGCCAATTCGGAG-TAMRA-3'.
3, experiment availability deciding
The experiment that possesses a, b, three conditions of c simultaneously is effectively experiment, otherwise is invalid experiment; Described a, b, three conditions of c are as follows: a, blank, no Ct value and do not have amplification curve; B, negative control, no Ct value and do not have amplification curve; Ct value≤35.0 of c, positive control, and typical amplification curve appears.
4, real-time fluorescence quantitative PCR detects
During real-time fluorescence quantitative PCR detected, sample detection result was as follows:
A, no Ct value and do not have amplification curve do not contain ustilago scitaminea bacteria in the expression sample;
B, Ct value≤35.0, and typical amplification curve appears, contain ustilago scitaminea bacteria in the expression sample;
C, when Ct value＞35.0, then this sample is done repeated experiments; Repeated experiments result does not have the Ct value, does not then contain ustilago scitaminea bacteria in this sample; If the repeated experiments result has the Ct value, then contain ustilago scitaminea bacteria in this sample.
5, real-time fluorescence quantitative PCR detects the ustilago scitaminea bacteria number
(1) ustilago scitaminea bacteria
BEThe clone of gene and typical curve make up
Be template with the ustilago scitaminea bacteria genomic dna, utilize the black tassel bacteria genomic dna to detect primer bE4:5'-CGCTCTGGTTCATCAACG-3' and bE8:5'-TGCTGTCGATGGAAGGTGT-3', pcr amplification, system is: template DNA (100 ng/ μ L) 1 μ L, 10 *
Ex TaqBuffer(Mg
2+Plus) 2.5 μ L, dNTP(2.5 mmol/L) 2.0 μ L,
Ex TaqEnzyme (5 U/ μ L) 0.125 μ L, each 1 μ L of the forward and reverse primer bE4 of 10 μ mol/L and bE8, ddH
2O mends to 25 μ L.Pcr amplification program: 94 ℃ of pre-sex change 4 min; 94 ℃ of sex change 30 s, 55 ℃ of annealing 30 s, 72 ℃ are extended 30 s, circulate 35 times; 72 ℃ are extended 10 min again.Amplified production carries out electrophoresis in containing 1.5 % sepharoses of ethidium bromide (EB), and glue reclaims purifying
BEGene fragment is connected on the PMD18-T carrier, and transformed into escherichia coli DH5 α selects positive colony then, extracts plasmid DNA, and order-checking and after the ncbi database comparison is errorless is preserved standby.The plasmid DNA of extracting is ustilago scitaminea bacteria
BEGene is measured plasmid DNA concentration, and-20 ℃ of preservations are standby.According to formula:
MW=base number * 660 dalton/bp
Copies/mL=6.02 * 10
23* (concentration g/mL)/(MW g/mol)
With concentration known
BEPlasmid DNA is converted into the copy number that comprises corresponding black tassel bacteria DNA.As being 459 bp for length in this experiment, concentration is the two strands of 100 ng/ μ L
BEPlasmid DNA adopts above calculation formula to get black tassel bacteria DNA copy number and is equivalent to 1.987 * 10
11Copies/ μ L.With being somebody's turn to do after quantitative
BEPlasmid DNA (1.987 * 10
11Copies/ μ L) is standard substance, carries out 10 times of gradient dilutions, be i.e. 1v standard substance stoste i+9v ddH
2O gets standard substance ii; 1v standard substance ii+9v ddH
2O gets standard substance iii, by that analogy, obtains copy number 10
-3～10
-10Standard substance.Getting final concentration then is 1.987 * 10
8～1.987 * 10
18 of copies/ μ L
BEThe plasmid DNA diluent is template, and 3 repetitions are all adopted in experiment, utilizes the real-time fluorescence quantitative PCR detection architecture production standard curve of setting up.
In the experiment 8 through 10 times of gradient dilutions
BEThe amplification curve that the plasmid DNA standard substance obtain by above-mentioned real-time fluorescence quantitative PCR detection architecture as shown in Figure 2, the Ct value is between 14～37, quantitative PCR can detect black tassel bacteria, its dilution point of accumulation is 10 ag/ μ L, corresponding Ct value is 36.283, utilizes Excel software that experimental data is generated typical curve as shown in Figure 3.Normal typical curve need satisfy: R
2(typical curve relation conefficient)〉0.99 ,-3.5＜slope(slope of standard curve)＜-3.0,0.9＜efficiency[reacts amplification efficiency (E)=10
(1/ slope)-1]＜1.2.This experiment exists
BEThe plasmid DNA copy number is 1.987 * 10
8～1.987 * 10
1The scope of copies/ μ L has good linear relationship, the typical curve R of structure
2Be that 0.9969, slope is that-3.2556, efficiency is 1.0284, draw
BEThe linear equation of plasmid DNA standard substance copy number and Ct value, namely the typical curve equation is: y=-3.2556x+4.6029(wherein, x representative
BEThe multiple of plasmid DNA gradient dilution, y represent the Ct value).
(2) the ustilago scitaminea bacteria number detects
Agricultural No. 40 (FN40) are example with the sugar cane breed good fortune.Experiment learnt from else's experience the FN40 robust growth behind tissue culture four months and the strengthening seedling and rooting, the sugarcane seedling of growing way unanimity after the clear water renaturation is cultivated 10 d, adopt the microsyringe of 0.5 μ L with 5 * 10
6Individual/mL smut spore suspension acupuncture injection inoculation 0.5 μ L places illumination box in the false basal part of stem of sugarcane seedling 0.5～2.0 cm shoot tip meristem place up, illumination 12 h, and dark 12 h, temperature is 28 ℃, humidity 65 % cultivate.From postvaccinal the 0th h(sample 1), 12 h(samples 2), 24 h(samples 3), 48 h(samples 4), 120 h(samples 5), 168 h(samples 6) and 336 h(samples 7), gather 3 strain sugarcane seedling inoculation positions and above stem stalk respectively, press the CTAB method and extract genomic dna.With sample 1 and the healthy negative contrast of sugarcane tissue-culture seedling FN39 blade genomic dna (1 μ g/ μ L), with the positive contrast of black tassel bacteria genomic dna (100 ng/ μ L), ddH
2O is blank, and the DNA detection final concentration of FN40 inoculation material is 1 μ g, and experiment is all adopted and repeated quantitative pcr amplification 3 times.Among the control experiment result, negative control does not have the Ct value and does not have amplification curve, does not contain ustilago scitaminea bacteria in the expression sample; Positive control Ct value is 15.774, less than 35.0, and typical amplification curve occurs, contains ustilago scitaminea bacteria in the expression sample; Blank does not have the Ct value and does not have amplification curve.Therefore, this experimental result meets three conditions of testing validity, and experiment is effective.
Use real-time fluorescence quantitative PCR detection architecture and the typical curve equation of above-mentioned structure, according to sugarcane testing sample Ct value (y), from typical curve, try to achieve corresponding black tassel bacteria
BEThe extension rate of plasmid DNA (x), final calculating obtains contained ustilago scitaminea bacteria DNA copy number (copies).Because the two strands of 100 ng/ μ L
BEPlasmid DNA adopts above-mentioned formula " MW=base number * 660 dalton/bp, copies/mL=6.02 * 10
23* (concentration g/mL)/(MW g/mol) " calculate black tassel bacteria DNA copy number and be equivalent to 1.987 * 10
11So copies/ μ L is testing sample ustilago scitaminea bacteria DNA copy number copies=10
x* 1.987 * 10
11
Real-time fluorescence quantitative PCR detects ustilago scitaminea bacteria and counts result's demonstration, and after the FN40 inoculation black tassel bacteria, ustilago scitaminea bacteria DNA copy number changes as shown in table 1 with acquisition time.Can detect black tassel bacteria DNA in postvaccinal sample 2, this moment, the Ct value was 35.214, and corresponding black tassel bacteria DNA copy number is 8.300 * 10
1Copies/ μ L.To between the sample 6, along with the prolongation of inoculation time, the black tassel bacteria amount is in rising trend at sample 2; Sample 3 and sample 4 black tassel bacteria content are respectively 4.951 * 10
2Copies/ μ L and 8.462 * 10
2Copies/ μ L; Sample 6 black tassel bacteria DNA copy numbers reach maximum value (1.293 * 10
4Copies/ μ L); Begin afterwards to descend, sample 7 black tassel bacteria DNA copy numbers are 3.407 * 10
3Copies/ μ L.Experimental result shows that black tassel bacteria is the critical period that the sugarcane resistance level plays a role in postvaccinal 48 h of sugarcane～168 h, has determined the propagation rate of black tassel bacteria, and therefore, this time may be the critical period that sugarcane and black tassel bacteria are done mutually.This result has detected the dynamic change of ustilago scitaminea bacteria quantity, for the fast quantification of ustilago scitaminea bacteria under the artificial inoculation conditions provides reliable method, simultaneously also for the early diagnosis of field ustilago scitaminea bacteria and disease is popular effective prediction is provided and prevents and treats foundation.
Table 1 ustilago scitaminea bacteria real-time fluorescence quantitative PCR detects the Ct value and measures with corresponding bacterium
The detection of embodiment two, real-time fluorescence quantitative PCR sensitivity
(1) positive plasmid detection limit simultaneous test
Reorganization with 100 ng/ μ L
BEPlasmid DNA is carried out 10 times of gradient dilutions, according to formula " MW=base number * 660 dalton/bp, copies/mL=6.02 * 10
23* (concentration g/mL)/(MW g/mol) ", be 100 * 10 with concentration
-3～100 * 10
-11It is 1.987 * 10 that the dilute sample of ng/ μ L is converted into the known copy number
8～1.987 * 10
18 10 times of gradients of copies/ μ L
BEThe plasmid DNA template is carried out real-time fluorescence quantitative PCR and regular-PCR then respectively and is detected.Wherein, the real-time fluorescence quantitative PCR reaction totally is 25 μ L, comprises 2 * TaqMan Universal Master Mix, 12.5 μ L, each 1 μ L of 10 μ mol/L quantitative PCR detection primer bEQ-F and bEQ-R, and 10 μ mol/L Taqman probes, 0.2 μ L,
BEPlasmid DNA template 1 μ L, ddH
2O mends to 25 μ L; Amplification program is: 50 ℃, and 2 min, 95 ℃, 10 min; 95 ℃, 15 s, 60 ℃, 1 min, 40 circulations are carried out the single-point fluoroscopic examination at 60 ℃.The regular-PCR amplification system is:
BEPlasmid DNA template 1 μ L, 10 *
Ex TaqBuffer(Mg
2+Plus) 2.5 μ L, dNTP(2.5 mmol/L) 2.0 μ L,
Ex TaqEnzyme (5 U/ μ L) 0.125 μ L, each 1 μ L of the forward and reverse primer bE4 of 10 μ mol/L and bE8, ddH
2O mends to 25 μ L; Amplification program is: 94 ℃ of pre-sex change 4 min; 94 ℃ of sex change 30 s, 55 ℃ of annealing 30 s, 72 ℃ are extended 30 s, circulate 35 times; 72 ℃ are extended 10 min again.In addition with the negative contrast of health cane tissue cultured seedling FN39 blade genomic dna (1 μ g/ μ L), with the positive contrast of black tassel bacteria genomic dna (100 ng/ μ L), ddH
2O is blank, and 3 repetitions are all adopted in experiment.The amplified production of regular-PCR compares quantitative fluorescent PCR and two kinds of methods of regular-PCR to gene fragment amplification result's minimum detectability with the detected through gel electrophoresis of 1.5 %.
The result as shown in Figure 2, the amplification of real-time fluorescence quantitative PCR has good linear relationship (y=-3.2556x+4.6029, R
2=0.9969, slope=-3.2556, efficiency=1.0284), lowest detection is limited to 10 ag/ μ L template concentrations (about 1.987 * 10
1Copies/ μ L); Agarose gel electrophoresis is observed, and can see significant concn gradient bands of a spectrum (459 bp), and observable lowest detection is limited to 10 fg/ μ L(1.987 * 10
4Copies/ μ L) (Fig. 4).Simultaneous test shows that the low energy of real-time fluorescence quantitative PCR detection method detects 1.987 * 10
1Individual DNA copy number is than regular-PCR 1.987 * 10
4The detection of L DNA copy number of copies/ μ is high 1000 times.
(2) sugarcane sample detection limit simultaneous test
3 strain field smut of sugarcane disease plants+1 leaf is got in experiment, adopts the CTAB method to extract sample gene group DNA, and electrophoresis detection is also measured the OD value, and-20 ℃ of preservations are standby.With 500 ng/ μ L+1 leaf genomic dna carries out 5 times of gradient dilutions, i.e. 1v standard substance stoste i+4v ddH
2O gets standard substance ii; 1v standard substance ii+4v ddH
2O gets standard substance iii, by that analogy, obtain concentration and be 500,100,20,4 and 0.8 ng/ μ L+1 leaf DNA dilute sample.Be template with these 5 dilute samples then, respectively get 2 μ L and carry out real-time fluorescence quantitative PCR (primer is bEQ-F, bEQ-R and Taqman probe) and regular-PCR (primer is bE4 and bE8) detection respectively, and with the detected through gel electrophoresis regular-PCR product of 1.5 %, the relatively detection sensitivity of quantitative fluorescent PCR and two kinds of method amplifications of regular-PCR and the further specificity of the detection technique that makes up of checking.Other is with the negative contrast of sugarcane tissue-culture seedling FN39 blade genomic dna (1 μ g/ μ L) of health, with the positive contrast of black tassel bacteria genomic dna (100 ng/ μ L), ddH
2O is blank, and 3 repetitions are all adopted in experiment.
Table 2 shows the real-time fluorescence quantitative PCR technology of utilizing, and the minimum black tassel bacteria of sugarcane testing sample (field smut morbidity strain+1 leaf genomic dna) is detected copy number, and to reach 41.125(DNA concentration be 0.8 ng/ μ L); As shown in Figure 5, the regular-PCR detected result shows that infected plant+1 leaf genomic dna concentration is 100 ng/ μ L, and it is unintelligible to detect bands of a spectrum.Illustrate that the ustilago scitaminea bacteria real-time fluorescence quantitative PCR detection technique that this experiment is set up can directly detect susceptible tissue, and do not need to carry out the separation and purification of pathogenic bacteria, easy and simple to handle, specificity is good, highly sensitive, the rapid detection that is suitable for sugarcane sample black tassel bacteria is identified.
Table 2 smut of sugarcane disease plant+1 leaf DNA real-time fluorescence quantitative PCR detects the Ct value and measures with corresponding bacterium
The present invention is directed to black tassel bacteria
BEThe specific real-time fluorescence quantitative PCR of gene design detects primer bEQ-F, bEQ-R and probe, to the positive
BEPlasmid and sugarcane sample are carried out the sensitivity simultaneous test, and the quantity dynamic change of the black tassel bacteria behind the real-time monitoring sugarcane inoculation black tassel bacteria is probed into sugarcane-black tassel bacteria and made situation mutually simultaneously.The result shows that detection method high specificity, highly sensitive, reliable results that the present invention sets up have application value widely in real work.
＜110〉University Of Agriculture and Forestry In Fujian
＜120〉a kind of real time fluorescence quantifying PCR method that detects ustilago scitaminea bacteria
<130>
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
＜213〉ustilago scitaminea bacteria (
Sporisorium scitamineum)
<400> 1
<210> 2
<211> 20
<212> DNA
＜213〉ustilago scitaminea bacteria (
Sporisorium scitamineum)
<400> 2
Claims (2)
1. a real time fluorescence quantifying PCR method that detects ustilago scitaminea bacteria comprises that sugarcane extracting genome DNA, the foundation of real-time fluorescence quantitative PCR detection architecture, experiment availability deciding and real-time fluorescence quantitative PCR detect; It is characterized in that:
(1) the real-time fluorescence quantitative PCR detection architecture is set up: the real-time fluorescence quantitative PCR reaction totally is 25 μ L, comprise 2 * TaqMan Universal Master Mix, 12.5 μ L, each 1 μ L of 10 μ mol/L quantitative PCR detection primer bEQ-F and bEQ-R, 10 μ mol/L Taqman probes, 0.2 μ L, sugarcane dna profiling 1 μ L, ddH
2O mends to 25 μ L; Amplification program is: 50 ℃, and 2 min, 95 ℃, 10 min; 95 ℃, 15 s, 60 ℃, 1 min, 40 circulations are carried out the single-point fluoroscopic examination at 60 ℃; The dna sequence dna of described primer and Taqman probe is as follows:
bEQ-F：5'-TGAAAGTTCTCATGCAAGCC-3'，
bEQ-R：5'-TGAGAGGTCGATTGAGGTTG-3'；
Taqman probe: 5'-FAM-TGCTCGACGCCAATTCGGAG-TAMRA-3';
(2) experiment availability deciding: the experiment that possesses a, b, three conditions of c simultaneously is effectively experiment, otherwise is invalid experiment; Described a, b, three conditions of c are as follows: a, blank, no Ct value and do not have amplification curve; B, negative control, no Ct value and do not have amplification curve; Ct value≤35.0 of c, positive control, and typical amplification curve appears; Described typical amplification curve is S type kinetic curve; Threshold line refers to that the vertex with negative control sample amplification curve is benchmark, the straight line parallel with X-coordinate; The corresponding fluorescent quantitative PCR cycle number of intersection point of threshold line and typical amplification curve is the Ct value; Blank refers to use isopyknic ddH
2O replaces template DNA; Negative control is the known sample that does not contain ustilago scitaminea bacteria; Positive control is the known sample that contains ustilago scitaminea bacteria;
(3) real-time fluorescence quantitative PCR detects: during real-time fluorescence quantitative PCR detected, sample detection result was as follows:
A, no Ct value and do not have amplification curve do not contain ustilago scitaminea bacteria in the expression sample;
B, Ct value≤35.0, and typical amplification curve appears, contain ustilago scitaminea bacteria in the expression sample;
C, when Ct value＞35.0, then this sample is done repeated experiments; Repeated experiments result does not have the Ct value and does not then contain ustilago scitaminea bacteria in this sample; If the repeated experiments result has the Ct value, then contain ustilago scitaminea bacteria in this sample.
2. method of utilizing real-time fluorescence quantitative PCR to detect the ustilago scitaminea bacteria number is characterized in that utilizing Ct value and the ustilago scitaminea bacteria of claim 1
BEThe extension rate of plasmid makes up the typical curve equation, calculates contained ustilago scitaminea bacteria number in the sample to be checked.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103555859A (en) * | 2013-11-07 | 2014-02-05 | 福建农林大学 | Fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) kit for detecting sugarcane streak mosaic virus |
| CN103937813A (en) * | 2014-04-14 | 2014-07-23 | 广西壮族自治区农业科学院植物保护研究所 | B-site mating-type gene of sugarcane smut as well as expression vector and construction method of b-site mating-type gene |
| CN105400890A (en) * | 2015-12-22 | 2016-03-16 | 贵州省亚热带作物研究所 | Method for detecting coix ustilago scitaminea |
| CN113981133A (en) * | 2021-12-09 | 2022-01-28 | 中国农业大学 | PCR quantitative detection method and kit for maize southern rust pathogen submerged period |
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| CN101880720A (en) * | 2010-07-20 | 2010-11-10 | 云南省农业科学院甘蔗研究所 | Method for rapidly detecting ustilago scitaminea bacteria |
| CN102329858A (en) * | 2011-08-02 | 2012-01-25 | 广州甘蔗糖业研究所 | Sugarcane smut bacteria nest type polymerase chain reaction (PCR) quick detection method |
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| CN101880720A (en) * | 2010-07-20 | 2010-11-10 | 云南省农业科学院甘蔗研究所 | Method for rapidly detecting ustilago scitaminea bacteria |
| CN102329858A (en) * | 2011-08-02 | 2012-01-25 | 广州甘蔗糖业研究所 | Sugarcane smut bacteria nest type polymerase chain reaction (PCR) quick detection method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103555859A (en) * | 2013-11-07 | 2014-02-05 | 福建农林大学 | Fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) kit for detecting sugarcane streak mosaic virus |
| CN103555859B (en) * | 2013-11-07 | 2015-01-21 | 福建农林大学 | Fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) kit for detecting sugarcane streak mosaic virus |
| CN103937813A (en) * | 2014-04-14 | 2014-07-23 | 广西壮族自治区农业科学院植物保护研究所 | B-site mating-type gene of sugarcane smut as well as expression vector and construction method of b-site mating-type gene |
| CN105400890A (en) * | 2015-12-22 | 2016-03-16 | 贵州省亚热带作物研究所 | Method for detecting coix ustilago scitaminea |
| CN113981133A (en) * | 2021-12-09 | 2022-01-28 | 中国农业大学 | PCR quantitative detection method and kit for maize southern rust pathogen submerged period |
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