CN101451162B - Primer, detecting probe and actual time fluorescent PCR kit for detecting Thielaviopsls basicola - Google Patents
Primer, detecting probe and actual time fluorescent PCR kit for detecting Thielaviopsls basicola Download PDFInfo
- Publication number
- CN101451162B CN101451162B CN2008102333121A CN200810233312A CN101451162B CN 101451162 B CN101451162 B CN 101451162B CN 2008102333121 A CN2008102333121 A CN 2008102333121A CN 200810233312 A CN200810233312 A CN 200810233312A CN 101451162 B CN101451162 B CN 101451162B
- Authority
- CN
- China
- Prior art keywords
- primer
- probe
- sequence
- pcr
- black root
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a primer, a probe and a real-time fluorescent PCR reagent kit for detecting Thielaviopsls basicola. The primer comprises a forward primer Tb1 and a reverse primer Tb2, and base sequences of the forward primer Tb1 and the reverse primer Tb2 are Tb1: 5'-ACC ATA TGT GAA CGT ACC TTT TCT-3' and Tb2: 5'-AGT TTA TAA ATG CTA CCG GCA GAA-3' respectively. A base sequence of the probe is any one nucleotide sequence in an area that shifting two basic groups toward an upstream or shifting three basic groups toward a downstream at both ends of 5'-CCC GAG AGG CAC CTG CCA AAG CA-3'; on the nucleotide sequence, a fluorescent reporter group is labeled at a 5' end, and a fluorescent quencher group is labeled at a 3' end to form the fluorescence-labeled probe; and the primer and the probe are used to be prepared into the corresponding reagent kit. The invention can quickly and accurately detect the Thielaviopsls basicola in field sick soil, thereby providing an accurate evidence for promptly adopting measures to cut off infection sources of plant diseases; and the invention can also quickly and accurately detect the plant diseases on plants.
Description
Technical field
The present invention relates to a kind of biotechnology that departments such as agriculture production, plant protection use that is suitable for, particularly a kind of soil-borne disease---primer, probe and real-time fluorescent PCR reagent case of black root of tobacco pathogenic bacteria Thielaviopsis basicola that is used for detecting tobacco production.
Background technology
Black root of tobacco is one of important disease on the tobacco, and it spreads all over the world and mainly produces the cigarette district, in states such as the U.S., Canada, Japan tobacco is produced and sustains losses severely, and also all there is generation in China main product cigarette district.Especially in recent years, this disease spreads in some cigarette districts, economizes in Yunnan Province of China, Guizhou, Hubei etc. to take place heavylier, and serious plot sickness rate can reach more than 30%.Also there is generation in provinces such as Shandong, Henan, Anhui, Jilin, Fujian, and the trend that increases the weight of to endanger is arranged in recent years.
Detection method for plant pathogenic fungi mainly contains immunological technique at present, molecular marking technique and conventional round pcr, but these technology can only be determined having or not of pathogenic micro-organism, but can not carry out quantitative accurately to the conidium and the thick spore number that extends of pathogenic fungi.And traditional plant pathogenic fungi detection quantivative approach is a selective medium cultivation counting process, but it is long that selective medium is cultivated the count cycle, labor intensive, and need the operator to have abundant morphologic knowledge, thereby limited the application of selective medium cultivation counting process.
After conventional PCR detection technique, emerging real-time fluorescence quantitative PCR technology relies on that it is accurately quantitative, highly sensitive to the original template amount, high specificity, advantage such as simple, convenient and rapid, become the mainstream technology in the domestic and international molecular biology research, equally also in the detection of phytopathogen and diagnosis, obtained being extensive use of.Begin successively both at home and abroad in recent years the real-time fluorescence PCR technology is applied to the Plant diseases diagnostic detection.
Real-time quantitative fluorescence PCR is to utilize fluorescent signal to be accompanied by the increase of PCR product and the enhanced principle, in the pcr amplification process, and the continuously variation of fluorescent signal in the detection reaction system.According to the mean value of fluorescent signal baseline and average standard deviation, when 99.7% degree of confidence, calculate fluorescent value threshold value, the PCR cycle index (Ct value) when collecting fluorescent signal then and being strengthened to predetermined threshold greater than mean value.Strict linear relationship is arranged between the logarithmic value of initiate dna template amount in this parameter and the PCR reaction system.Utilize the Ct value of positive gradient dilution standard substance to be depicted as typical curve, just can measure the starting template copy number of target pathogenic bacteria again according to the Ct value of test sample exactly.Real-time fluorescence PCR is divided into two class methods of using probe and not using probe according to its principle that produces fluorescence.Because probe and pathogenic bacteria dna sequence dna to be detected have very high specificity, and can effectively avoid the false positive problem of conventional PCR, so use fluorescence labeling probe method comparatively generally at present.
Real-time fluorescence PCR detection method is used for the detection of phytopathogen, at home and abroad all still is at the early-stage.At present, in the real-time fluorescence PCR of black root of tobacco bacterium detects, be primer, be that probe is prepared into corresponding real-time fluorescent PCR reagent case still do not have relevant report with 5 '-ACC ATA TGT GAA CGT ACC TTTTCT-3 ' and 5 '-AGT TTA TAA ATG CTA CCG GCA GAA-3 ' with 5 '-CCC GAG AGG CACCTG CCA AAG CA-3 ' sequence.
Summary of the invention
One of purpose of the present invention just provides a kind of primer that is used to detect the black root of tobacco bacterium, and it can be in the PCR testing process, qualitative detection black root of tobacco bacterium, and high specificity.
The primer that is used to detect the black root of tobacco bacterium provided by the present invention comprises that the primer that forward primer Tb1 and reverse primer Tb2 form is right, the nucleotide sequence of described forward primer Tb1 is seen shown in the sequence 1 in the sequence table, Tb1:5 '-ACC ATA TGTGAA CGT ACC TTT TCT-3 '; Described reverse primer Tb2 nucleotide sequence is seen shown in the sequence 2 in the sequence table, Tb2:5 '-AGT TTA TAA ATG CTA CCG GCA GAA-3 '.
The applicant is through long-term a large amount of experiment, filters out the above-mentioned primer of being made up of forward primer Tb1 and reverse primer Tb2 from numerous PCR primers that are used for detecting the black root of tobacco bacterium, and its amplified fragments size is 76bp.The characteristics of this primer are the black root of tobacco bacterium to be had the conservative property of height, and do not have significant homology between other nearly edge biological species, carry out pcr amplification with this primer, realize the accurate qualitative detection of black root of tobacco bacterium, and high specificity.
Two of purpose of the present invention just provides a kind of probe that is used to detect the black root of tobacco bacterium, and it can be in the real-time fluorescence PCR testing process, and detection by quantitative black root of tobacco bacterium is improved detection sensitivity significantly.The base sequence of this probe is any nucleotide sequence in 5 '-CCC GAG AGG CAC CTG CCA AAG CA-3 ' 2 bases of two ends upward displacement or 3 base zones of downstream displacement, described probe one end is connected with the fluorescent quenching group, and the other end is connected with the fluorescence report group.
Described fluorescence probe reporter group is marked at 5 ' end, and the fluorescent quenching group is marked at 3 ' end, constitutes fluorescence labeling probe.
Described probe is at upstream be shifted the nucleotide sequence 5 '-GCC CCG AGA GGC ACC TGC CAA AGC A-3 ' of 2 bases of 5 '-CCC GAG AGG CAC CTG CCA AAG CA-3 ', sees shown in the sequence 4 in the sequence table.
The nucleotide sequence of described probe is the sequence shown in the sequence 3 in the sequence table: 5 '-CCC GAG AGG CAC CTG CCAAAG CA-3 '.
Described probe is at be shifted downstream the nucleotide sequence 5 '-CCC GAG AGG CAC CTG CCA AAG CAG CT-3 ' of 3 bases of 5 '-CCC GAG AGG CAC CTG CCA AAG CA-3 ', sees shown in the sequence 5 in the sequence table.
Three of purpose of the present invention just provides a kind of test kit that utilizes the detection black root of tobacco bacterium that primer in the purpose one and the probe in the purpose two make, can be in the real-time fluorescence PCR testing process, in the detection by quantitative soil and the black root of tobacco bacterium on the plant, and reliable and stable, highly sensitive quickly and accurately.Described test kit comprises that also testing sample extracts reagent, quantitative criterion product, real-time fluorescence PCR reaction solution and detects articles for use, and described real-time fluorescence PCR reaction solution includes following component and corresponding final concentration thereof: PCR damping fluid: 10%; MgCl
2: 2.6mmol/L; DNTPs:0.2mmol/L; Taq archaeal dna polymerase: 1U/20 μ L; Forward primer Tb1:0.4 μ mol/L; Reverse primer Tb2:0.4 μ mol/L; Fluorescence labeling probe: 0.4 μ mol/L; Adopt aseptic ultrapure water to adjust final concentration.
According to practical situation, what each component adopted in the real-time fluorescence PCR reaction solution is: the PCR damping fluid is 10 * PCR damping fluid; MgCl
2Initial concentration be 25mmol/L; The initial concentration of dNTPs is 10mmol/L; The initial concentration of Taq archaeal dna polymerase is 2.5U/ μ L; The initial concentration of forward primer Tb1 is 10 μ mol/L; The initial concentration of reverse primer Tb2 is 10 μ mol/L; The initial concentration of fluorescence labeling probe is 5 μ mol/L; PCR damping fluid, MgCl
2, dNTPs, Taq archaeal dna polymerase, forward primer Tb1, reverse primer Tb2, fluorescence labeling probe volume ratio be: 20: 21: 4: 4: 8: 8: 16.This fluorescent quantitative PCR system has very high sensitivity, and the amount that detects object bacteria DNA reaches 100fg/ μ L.
In the real-time fluorescence PCR reaction solution, what each component adopted is: the PCR damping fluid is 10 * PCR damping fluid; MgCl
2Initial concentration be 25mmol/L; The initial concentration of dNTPs is 2mmol/L; The initial concentration of Taq archaeal dna polymerase is 1U/ μ L; The initial concentration of forward primer Tb1 is 5 μ mol/L; The initial concentration of reverse primer Tb2 is 5 μ mol/L; The initial concentration of fluorescence labeling probe is 5 μ mol/L; PCR damping fluid, MgCl
2, dNTPs, Taq archaeal dna polymerase, forward primer Tb1, reverse primer Tb2, fluorescence labeling probe volume ratio be: 20: 21: 20: 10: 16: 16: 16.This fluorescent quantitative PCR system has very high sensitivity, and the amount that detects object bacteria DNA reaches 100fg/ μ L.
Adopt the test kit among the present invention, on the one hand, in the testing sample preprocessing process, the black root of tobacco bacterium DNA that testing sample extraction reagent can directly extract in the pedotheque is used as the pcr amplification template, has saved the time; On the other hand, in the real-time fluorescence PCR amplification procedure of testing sample, directly the real-time fluorescence PCR reaction solution that will prepare by best component and volume ratio thereof adds in eight connecting legs, both saved a large amount of time, and reliable and stable, thus reached detect quickly and accurately in the soil and plant on the black root of tobacco bacterium.
Owing to adopted technique scheme, the present invention to have following advantage:
(1) high specificity: primer and probe all design according to black root of tobacco bacterium specific sequence among the present invention, and compare with other biological species, do not have remarkable homology, and detection specificity is strong;
(2) highly sensitive: utilize when real-time fluorescent PCR reagent case detects the black root of tobacco bacterium among the present invention, can be accurately quantitative, object bacteria DNA has good linear relationship in 10ng/ μ L-100fg/ μ L concentration range, highly sensitive;
(3) practicality is good: real-time fluorescent PCR reagent case has unlatching promptly with advantages such as, standard are unified, simple and convenient among the present invention, can detect quickly and accurately in the field soil with plant on the black root of tobacco bacterium, practicality is good;
(4) applied range: can detection by quantitative soil and plant on the black root of tobacco bacterium, be applicable to field dynamic monitoring of black root of tobacco bacterium and disease screening, can be used for the rate of propagation of black root of tobacco bacterium in tobacco plant and the detection by quantitative of seed-borne fungi simultaneously.
In sum, adopt the present invention, can be in the field in spite of illness in the soil and detect the black root of tobacco bacterium quickly and accurately on the plant, thus the source of infection that cuts off disease in time taking measures provides accurate foundation, also can detect the disease on the plant quickly and accurately, its meaning is very great.
Description of drawings
Fig. 1 fluorescent quantitation amplification system specific detection
Fig. 2 A quantitative fluorescent PCR positive criteria product amplification curve
Fig. 2 B quantitative fluorescent PCR typical curve
Fig. 3 actual sample detects
Embodiment
The invention will be further described below in conjunction with embodiment:
In the present invention, the primer that is used to detect the black root of tobacco bacterium comprises forward primer Tb1 and reverse primer Tb2, and its base sequence is respectively:
Tb1:5 '-ACC ATA TGT GAA CGT ACC TTT TCT-3 ' sees shown in the sequence 1 in the sequence table;
Tb2:5 '-AGT TTA TAA ATG CTA CCG GCA GAA-3 ' sees shown in the sequence 2 in the sequence table.
Special primer with design finds that to carrying out homology comparison back special primer is to only having the conservative property of height with the black root of tobacco bacterium, and do not have significant homology between other nearly edge biological species, and the possibility that the higher species of other homologys exist in soil is minimum, illustrates that the right specificity of this primer is very high.In order better to verify the specificity of primer, in fluorescent quantitative PCR experiment, adopt common soil-borne pathogen in the following soil, as Phytophthora nicotianae germ (Phytophthoranicotianae), sweet potato black rot (Ceratocystis fimbriata) Phytophthora cactorum (Phytophthora cactorum), frog eye leaf spot of tobacco bacterium (Cercospora nicotianae), cotton seedling blight germ (Fusaricum oxysporium), verticillium dahliae (Verticillium dahiliae), Irving's formula bacillus (Erwinia carotovora), subtilis (Bacillus subtilis), the soil dominant bacteria of tobacco ralstonia solanacearum (Ralstonia solanacearum) and separation and Culture is as negative control bacterium (table 1), extract its genomic dna respectively, after its concentration of UV spectrophotometer measuring and the purity, DNA concentration is adjusted to 50ng/ μ L.Fluorescent quantitation amplification curve (Fig. 1) is the result show, all negative control bacterium all do not have initial amplification (Ct=N/A), verified that once more designed primer is to having very high specific.
Table 1
The quantitative fluorescent PCR strains tested
a+ the positive
b-feminine gender
cN/A the unknown
In the present invention, be used to detect the probe of black root of tobacco bacterium, its base sequence is any nucleotide sequence in 5 '-CCC GAG AGG CACCTG CCA AAG CA-3 ' 2 bases of two ends upward displacement or 3 base zones of downstream displacement, list at this nucleotides sequence, the fluorescence report group is marked at 5 ' end, the fluorescent quenching group is marked at 3 ' end, to constitute fluorescence labeling probe.
In the amplification region of the primer of forming by forward primer Tb1 and reverse primer Tb2, principle of design and method of design according to probe, design the probe that is used for the detection of black root of tobacco bacterium real-time fluorescence PCR, its base sequence is any nucleotide sequence in 5 '-CCCGAG AGG CAC CTG CCA AAG CA-3 ' 2 bases of two ends upward displacement or 3 base zones of downstream displacement; Carry out the real-time fluorescence PCR amplification with designed probe, designed probe is screened; Result according to the real-time fluorescence PCR amplification filters out best probe at last.
At upstream the be shifted nucleotide sequence of 2 bases of 5 '-CCC GAG AGG CAC CTG CCA AAG CA-3 ' can be: 5 '-GCC CCG AGA GGC ACC TGC CAA AGC A-3 ', see shown in the sequence 4 in the sequence table.
The nucleotide sequence of fluorescence labeling probe also can be: 5 '-CCC GAG AGG CAC CTG CCA AAG CA-3 ', see shown in the sequence 3 in the sequence table that it is best embodiment.
At be shifted the downstream nucleotide sequence of 3 bases of 5 '-CCC GAG AGG CAC CTG CCA AAG CA-3 ' can also be: 5 '-CCC GAG AGG CAC CTG CCA AAG CAG CT-3 ', see shown in the sequence 5 in the sequence table.
5 ' end at fluorescence labeling probe indicates note reporter group FAM, and 3 ' end is marked with fluorescent quenching group TAMRA.When probe was complete, reporter group institute emitted fluorescence energy was absorbed by quenching group, and instrument detecting is less than signal.Along with PCR carries out, the Taq archaeal dna polymerase runs in the chain extension process and template bonded probe, its 5 ' → 3 ' exonuclease activity will cut off probe, reporter group is away from quenching group, and its energy can not be absorbed, and promptly produces fluorescent signal, the fluorescence that reporter group discharged can be detected by the photofluorometer in the detection by quantitative instrument, template is whenever duplicated once, just has a probe to be cut off, and follows the release of a fluorescent signal.Because d/d fluorophor number and PCR product quantity are man-to-man relations, so the accumulation volume of the increase of fluorescence volume and PCR product is proportionlity.The Ct value is the cycle number that the accumulation of fluorescence volume in the PCR process surpasses the substrate fluorescence volume.Utilize the Ct value of positive gradient standard form to make typical curve, again according to the Ct value of testing sample can quantitative exactly detected sample in the concentration of black root of tobacco bacterium, thereby the detection sensitivity of significantly improving.
The test kit of detection black root of tobacco bacterium of the present invention, it includes testing sample and extracts reagent, quantitative criterion product, real-time fluorescence PCR reaction solution and detect articles for use, and described real-time fluorescence PCR reaction solution includes following component and corresponding final concentration thereof: PCR damping fluid: 10%; MgCl
2: 2.6mmol/L; DNTPs:0.2mmol/L; Taq archaeal dna polymerase: 1U/25 μ L; Forward primer Tb1:0.4 μ mol/L; Reverse primer Tb2:0.4 μ mol/L; Fluorescence labeling probe: 0.4 μ mol/L; Adopt aseptic ultrapure water to adjust final concentration.
Testing sample extracts reagent to be made up of soil DNA extraction damping fluid and TE damping fluid, and wherein the TE damping fluid is conventional reagent, is formulated according to methods involving in " fine works molecular biology experiment guide (the 4th edition) ".
The quantitative criterion product comprise standard positive control template and the positive criteria product based on the peculiar nucleotide sequence design of black root of tobacco bacterium.The standard positive control template is prepared from by containing the pMD18-T carrier (Takara) that inserts target fragment.Preparation process is: adopt primer Tb1 and Tb2 amplification black root of tobacco bacterium genomic dna, cut glue behind the PCR product electrophoresis and reclaim target fragment, be connected under 16 ℃ with the pMD18-T carrier and spend the night, connect product transformed into escherichia coli JM109 competent cell, converted product is coated on the penbritin flat board that contains X-gal and IPTG and cultivates, the picking hickie shakes and adopts behind the bacterium plasmid extraction kit (Omega Biotek Inc.) to extract plasmid, by the enzyme evaluation positive colony of cutting and check order.The plasmid DNA of positive colony is through ultraviolet spectrophotometer A
260Quantitatively, be 10ng/ μ L-100fg/ μ L through 10 * gradient dilution, be stored in-20 ℃.
The real-time fluorescence PCR reaction solution includes following component and corresponding final concentration thereof: PCR damping fluid: 10%; MgCl
2: 2.6mmol/L; DNTPs:0.2mmol/L; Taq archaeal dna polymerase: 1U/20 μ L; Forward primer Tb1:0.4 μ mol/L; Reverse primer Tb2:0.4 μ mol/L; Fluorescence labeling probe: 0.4 μ mol/L; Adopt aseptic ultrapure water to adjust final concentration.Wherein, PCR damping fluid, MgCl
2, dNTPs, archaeal dna polymerase be the conventional reagent of PCR, all available from Beijing ancient cooking vessel state biotechnology responsibility company limited, primer is synthetic by Shanghai Ying Jun Bioisystech Co., Ltd, fluorescence labeling probe is synthetic in Takara company.
Detect articles for use and include self-sealing plastics bag and 0.2mL eight connecting legs.
On the basis of the best probe in primer in utilizing purpose one and the purpose two, optimizing reaction system and reaction conditions are determined best black root of tobacco bacterium real-time fluorescence PCR detection architecture.
The real-time fluorescence PCR reaction system cumulative volume of optimization of the present invention is 20 μ L, 10 * PCR damping fluid, 2.0 μ L wherein, 25mmol/L MgCl
22.6 μ L, 2mmol/L, dNTPs 2.0 μ L, 2.5U/ μ L Taq archaeal dna polymerase 0.4 μ L, each 1.6 μ L of 5 μ mol/L forward primer Tb1 and reverse primer Tb2,5 μ mol/L fluorescence labeling probes, 1.6 μ L, dna profiling 1.0 μ L, aseptic ultrapure water 8.8 μ L.Real-time fluorescence PCR adopts two-step approach, and the amplified reaction program is 94 ℃, 4min; 42 circulations then, each circulation is 94 ℃, 10s, 60 ℃, 20s collects fluorescence in each round-robin annealing/extension stage (60 ℃).
Utilize the real-time fluorescent PCR reagent case among the present invention that the black root of tobacco bacterium is detected, comprise the real-time fluorescence PCR reaction of testing sample pre-treatment and testing sample.
The preparation of testing sample pcr amplification template and the reaction of the real-time fluorescence PCR of testing sample.
The preparation that is prepared as pedotheque pcr amplification template of testing sample pcr amplification template.
The sample preparation product pcr amplification template of soil is equipped with:
(1) take by weighing 0.5 gram pedotheque in the 5mL centrifuge tube, add 0.2g polyvinylpolypyrrolidone (PVPP), vortex 30s adds 3mL DNA extraction buffer (100mmol/L Tris-HCl, 100mmol/L EDTA, 100mmol/L Na again
3PO
4, 1.5mol/L NaCl, 1% CTAB pH8.0) mixes;
(2) place-196 ℃ of freezing 2min of liquid nitrogen rapidly, take out the back and melt (about 3min, the attention time can not be oversize, melts the back and take out at once), 2-3 cracking cell so repeatedly in 65 ℃ of water-baths;
(3) add 20 μ L 10mg/mL Proteinase Ks, 0.5mL 50mg/mL N,O-Diacetylmuramidase, the mixing that turns upside down places the 30min (225r/min) that vibrates on 37 ℃ of shaking tables;
(4) add 600 μ L 10%SDS, 65 ℃ of water-bath 30min put upside down mixing gently every 5-10min therebetween;
(5) centrifugal (12,000r/min) 10min collects supernatant liquor to room temperature, transfers in another new 5mL centrifuge tube;
(6) soil precipitation adds 0.5mL extracting solution and 100 μ L 10%SDS again, vortex 30s, and 65 ℃ of water-bath 10min, room temperature is centrifugal, and (12,000r/min) 10min collects supernatant liquor and merges with supernatant liquor last time;
(7) add 0.05g PVPP in the supernatant liquor, room temperature in conjunction with 20min after, room temperature is centrifugal, and (12,000r/min) 10min collects supernatant liquor, transfers in another new 5mL centrifuge tube;
(8) supernatant liquor is with isopyknic chloroform/primary isoamyl alcohol (24: 1) extracting twice, centrifugal (12,000r/min, 10min) back is drawn water and is transferred in another 5mL centrifuge tube;
(9) with 0.6 times of volume Virahol precipitation at room temperature 30min of precooling, room temperature is centrifugal, and (12,000r/min) 10min collects the nucleic acid precipitation;
(10) with cold 70% absolute ethanol washing precipitation twice, dry up naturally, be dissolved in the ultrapure water of 100 μ L sterilization.
The real-time fluorescence PCR reactions steps of testing sample is:
(1) be provided with the start of real-time fluorescence PCR instrument standby;
(2) adding 18 μ L real-time fluorescence PCR reaction solutions and 1 μ L initial concentration are the bovine serum albumin (BSA) of 10mg/mL in 0.2mL eight connecting legs;
(3) add 1 μ L analyte sample fluid, the standard positive control template that test kit is provided adds in the positive control pipe, 10 * gradient dilution the liquid that adds the positive criteria product in the positive criteria pipe, 1 μ L sterilization deionized water replaces template as blank, and the consumption of control sample is every pipe 1 μ L;
(4) can carry out the PCR reaction after mixing;
(5) treat that pcr amplification finishes, the analysis software that adopts instrument to carry, analysing amplified result, production standard curve (Fig. 2), Y=-3.296X+4.327 calculates the DNA amount of black root of tobacco bacterium in the testing sample reaction system according to typical curve.
Adopt the test kit among the present invention, on the one hand, in the testing sample preprocessing process, the black root of tobacco bacterium DNA that testing sample extraction reagent can directly extract in the pedotheque is used as the pcr amplification template, has saved the time; On the other hand, in the real-time fluorescence PCR amplification procedure of testing sample, directly the real-time fluorescence PCR reaction solution that will prepare by best component and volume ratio thereof adds in eight connecting legs, both saved a large amount of time, and reliable and stable, thereby reached the black root of tobacco bacterium (Fig. 3) that detects quickly and accurately in the soil.
In above-mentioned real-time fluorescence PCR reaction solution, what each component adopted is: the PCR damping fluid is 10 * PCR damping fluid; MgCl
2Initial concentration be 25mmol/L; The initial concentration of dNTPs is 10mmol/L; The initial concentration of Taq archaeal dna polymerase is 2.5U/ μ L; The initial concentration of forward primer Tb1 is 10 μ mol/L; The initial concentration of reverse primer Tb2 is 10 μ mol/L; The initial concentration of fluorescence labeling probe is 5 μ mol/L; PCR damping fluid, MgCl
2, dNTPs, Taq archaeal dna polymerase, forward primer Tb1, reverse primer Tb2, fluorescence labeling probe volume ratio be: 20: 21: 4: 4: 8: 8: 16.
The PCR damping fluid is 10 * PCR damping fluid; MgCl
2Initial concentration be 25mmol/L; The initial concentration of dNTPs is 2mmol/L; The initial concentration of Taq archaeal dna polymerase is 1U/ μ L; The initial concentration of forward primer Tb1 is 5 μ mol/L; The initial concentration of reverse primer Tb2 is 5 μ mol/L; The initial concentration of fluorescence labeling probe is 5 μ mol/L; PCR damping fluid, MgCl
2, dNTPs, Taq archaeal dna polymerase, forward primer Tb1, reverse primer Tb2, fluorescence labeling probe volume ratio be: 20: 21: 20: 10: 16: 16: 16.
Sequence table
＜110〉University Of Chongqing
＜120〉be used to detect primer, probe and the real-time fluorescent PCR reagent case of black root of tobacco bacterium
＜130〉do not have
<160>5
<170>Patent?In?version?3.5
<210>1
<211>19
<212>DNA
＜213〉black root of tobacco pathogenic bacteria (Thielaviopsis basicola)
<400>1
accatatgtg?aacgtacctt?ttct 19
<210>2
<211>22
<212>DNA
＜213〉black root of tobacco pathogenic bacteria (Thielaviopsis basicola)
<400>2
agtttataaa?tgctaccggc?agaa 22
<210>3
<211>23
<212>DNA
＜213〉black root of tobacco pathogenic bacteria (Thielaviopsis basicola)
<400>3
cccgagaggc?acctgccaaa?gca 23
<210>4
<211>25
<212>DNA
＜213〉black root of tobacco pathogenic bacteria (Thielaviopsis basicola)
<400>4
gccccgagag?gcacctgcca?aagca 25
<210>5
<211>26
<212>DNA
＜213〉black root of tobacco pathogenic bacteria (Thielaviopsis basicola)
<400>5
cccgagaggc?acctgccaaa?gcagct 26
Claims (4)
1. primer that is used to detect the black root of tobacco bacterium, it comprises that the primer of forward primer Tb1 and reverse primer Tb2 composition is right, the nucleotide sequence of described forward primer Tb1 is seen shown in the sequence 1 in the sequence table, Tb1:5 '-ACC ATATGT GAA CGT ACC TTT TCT-3 '; Described reverse primer Tb2 nucleotide sequence is seen shown in the sequence 2 in the sequence table, Tb2:5 '-AGT TTA TAA ATG CTA CCG GCA GAA-3 '.
2. probe that is used to detect the black root of tobacco bacterium, the nucleotide sequence of described probe are the sequences shown in sequence 3, sequence 4 or the sequence 5 in the sequence table; Its fluorescence report group is marked at 5 ' end, and the fluorescent quenching group is marked at 3 ' end, constitutes fluorescence labeling probe.
3. a test kit that detects the black root of tobacco bacterium comprises described primer of claim 1 and the described probe of claim 2.
4. the test kit of detection black root of tobacco bacterium according to claim 3, it is characterized in that: described test kit comprises that also testing sample extracts reagent, quantitative criterion product, real-time fluorescence PCR reaction solution and detects articles for use, and described real-time fluorescence PCR reaction solution includes following component and corresponding final concentration thereof: PCR damping fluid: 10%; MgCl
2: 2.6mmol/L; DNTPs:0.2mmol/L; Taq archaeal dna polymerase: 1U/20 μ L; Forward primer Tbl:0.4 μ mol/L; Reverse primer Tb2:0.4 μ mol/L; Fluorescence labeling probe: 0.4 μ mol/L; Adopt aseptic ultrapure water to adjust final concentration.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008102333121A CN101451162B (en) | 2008-12-11 | 2008-12-11 | Primer, detecting probe and actual time fluorescent PCR kit for detecting Thielaviopsls basicola |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008102333121A CN101451162B (en) | 2008-12-11 | 2008-12-11 | Primer, detecting probe and actual time fluorescent PCR kit for detecting Thielaviopsls basicola |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101451162A CN101451162A (en) | 2009-06-10 |
| CN101451162B true CN101451162B (en) | 2011-06-29 |
Family
ID=40733650
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2008102333121A Expired - Fee Related CN101451162B (en) | 2008-12-11 | 2008-12-11 | Primer, detecting probe and actual time fluorescent PCR kit for detecting Thielaviopsls basicola |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101451162B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105112555A (en) * | 2015-10-08 | 2015-12-02 | 中国烟草总公司郑州烟草研究院 | Tobacco bacterial wilt real-time fluorescence quantitative PCR detection kit and detection method |
| CN105274241A (en) * | 2015-11-21 | 2016-01-27 | 云南省烟草公司大理州公司 | Molecular detection primer and quick detection method for thielaviopsis basicola |
| CN107937575A (en) * | 2017-11-24 | 2018-04-20 | 四川农业大学 | A kind of walnut alternaria real-time fluorescence PCR specific detection primer and method |
| CN109406778B (en) * | 2018-10-15 | 2021-06-29 | 国家烟草质量监督检验中心 | Time-resolved fluorescence quantitative test strip for detecting ralstonia solanacearum in tobacco leaves and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1904076A (en) * | 2006-08-03 | 2007-01-31 | 东北农业大学 | Detection method of soyabean phytophthora and special primer |
-
2008
- 2008-12-11 CN CN2008102333121A patent/CN101451162B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1904076A (en) * | 2006-08-03 | 2007-01-31 | 东北农业大学 | Detection method of soyabean phytophthora and special primer |
Non-Patent Citations (5)
| Title |
|---|
| D Bai et al.Identification of two RAPD markers tightly linked with the Nicotiana debneyi gene for resistance to black root rot of tobacco.《Theoretical and applied genetics》.1995,第91卷(第8期),1184-1189. * |
| K D Kenward et al.Isolation and characterization of Tnd-1, a retrotransposon marker linked to black root rot resistance in tobacco.《Theoretical and applied genetics》.1999,第98卷(第3-4期),387-395. * |
| KDKenwardetal.IsolationandcharacterizationofTnd-1 a retrotransposon marker linked to black root rot resistance in tobacco.《Theoretical and applied genetics》.1999 |
| 张驰宇等.荧光实时定量PCR的研究进展.《江苏大学学报（医学版）》.2006,第16卷(第3期),268-271. * |
| 赵永强等.烟草根黑腐病菌的ITS分子检测.《中国植物病理学会2008年学术年会》.2008, * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101451162A (en) | 2009-06-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101250580A (en) | Primer, probe and real-time fluorescent PCR reagent case for detecting tobacco ralstonia solanacearum | |
| CN105039586A (en) | Primer and kit for detecting duck type-II adenovirus | |
| CN111705158A (en) | LFD-RPA visual detection primer group for detecting sweet potato black spot pathogen and detection method | |
| CN101451162B (en) | Primer, detecting probe and actual time fluorescent PCR kit for detecting Thielaviopsls basicola | |
| CN101899521A (en) | Loop-mediated isothermal amplification (LAMP) detection method of Angiostrongylus cantonensis | |
| CN101775443B (en) | LAMP kit for detecting PRV and preparation method thereof | |
| CN101225435A (en) | Method for quickly detecting transgenic soybean | |
| CN104846124A (en) | CyHV-2 (cyprinid herpesvirus 2) specificity PCR (polymerase chain reaction) detection kit and detection method | |
| CN102344953B (en) | Primer for detecting peach-derived component in sample, method and kit | |
| CN106434989B (en) | The LAMP rapid detection method of tobacco brown spot pathogen | |
| CN103555842B (en) | Animal chlamydia TaqMan-MGB probe multiplex real-time fluorescent quantitative PCR (polymerase chain reaction) detection primers, kit and method | |
| CN101676405A (en) | Mycobacterium tuberculosis fluorescence quantitative PCR detection method and kit thereof | |
| CN105112558B (en) | The triple real time fluorescent quantitative RT PCR detection kits of the type of foot and mouth disease virus O, A and Asia I | |
| CN100404689C (en) | Real-time fluorescent PCR testing primer, probe and immobilized kit for citrus ulcer bacteria and testing process thereof | |
| CN104293932A (en) | Method for detecting HLA-B * 5801 allele based on real-time fluorescence PCR | |
| CN104232782A (en) | PCR (polymerase chain reaction) primer for detecting tobacco soil-borne fungal pathogens as well as application and method of PCR primer | |
| CN101492741A (en) | Method for quantitative detection of mycoplasma hyopneumoniae | |
| CN103074429B (en) | UU (ureaplasma urealyticum) detection kit | |
| CN105821159A (en) | Nested RT-PCR detection method for differentiating variant strains and classic strains of PEDV | |
| CN109234432A (en) | A kind of primer, probe and kit based on recombinase polymeric enzymatic amplification method detection soybean samping off | |
| CN102329885B (en) | Kit for detecting polymorphism of VKORC1 and CYP2C9 genes | |
| CN101974621B (en) | LAMP detection method for babesia bovis | |
| CN106884048A (en) | One kind detection fish Streptococcus iniae fluorescent PCR kit and its application | |
| CN102382890B (en) | Pythium splendens braun fluorescence quantitative PCR detection reagent and detection kit and application | |
| CN1912131B (en) | Telomere enzyme active quantitive detection method and kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20110629 Termination date: 20151211 |
|
| EXPY | Termination of patent right or utility model |