CN101041853A - Primer, probe and real-time fluorescent PCR reagent case for detecting tobacco black shank bacterium - Google Patents
Primer, probe and real-time fluorescent PCR reagent case for detecting tobacco black shank bacterium Download PDFInfo
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Abstract
The invention discloses a primer, probe and real-time fluorescence PCR agent box to check black shank bacterium of tobacco, which is characterized by the following: making the primer with forward primer Pn1 and backward primer Pn2; setting the base sequence as Pn1:5'- GAA CAA TGC AAC TTA TTG GAC GTT-3', Pn2:5'-AAC CGA AGC TGC CAC CCT AC -3'; setting the base sequence of probe as a random nucleic acid sequence in the zone of upper river shift two bases or lower river shift three bases on two ends of 5'-TTC ACC AGT CCA TCA CGC CAC AGC-3'; signing fluorescence report group on 5' end; signing fluorescence quenched group on 3' end; forming fluorescence labeling probe; preparing agent box with the primer and probe. This invention can quickly test black shank bacterium of tobacco in soil, which possesses important meaning.
Description
(1), affiliated 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 great destructive disease---primer, probe and real-time fluorescent PCR reagent case of black shank pathogenic fungi Phytophthora nicotianae (Phytophthora nicotianae) that is used for detecting tobacco production.
(2), technical background
Black shank is the great destructive disease of the global tobacco industry development of influence, takes place comparatively general in China various places.This disease can endanger all cultivation tobaccos such as comprising flue-cured tobacco, air-curing of tobacco leaves, suncured tabacco, burley tobaccos and Turkish tobaccos, can infect harm at any growing stage of tobacco, but main harm field period cigarette strain, the morbidity in seedling stage often causes the cigarette seedling dead in flakes, shape becomes to damping off, field period morbidity then can cause whole cigarette strain wilting suddenly and with root or basal part of stem blackening symptom, often causes irredeemable loss.This disease is based on the soil biography, and wind and rain, flowing water, farming operation etc. all are its routes of transmission.
Detection method for tobacco black shank bacterium has conventional sense methods such as classical symptom appearance method, tissue pathological slice method, pathogenic assay method.The advantage of ordinary method is directly perceived, simple and easy to do, but its shortcoming is that sense cycle is long, the person's of lacking experience False Rate height, and can not detect at the cause of disease initial stage of infecting.In addition, the detection of tobacco black shank bacterium is also had enzyme-linked immunosorbent assay, spot immune absorption method etc., but exist process complexity, sensitivity low, detect problems such as cost height, cycle be long.
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, stopped pipe operation, advantage such as simple, convenient and rapid, become the mainstream technology in the domestic and international molecular biology research, it has equally also obtained being extensive use of in the detection of phytopathogen and diagnosis gradually.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 tobacco black shank bacterium detects, be primer, classify probe as with any nucleotides sequence in 3 base zones of 2 bases or downstream displacement that upstream are shifted at 5 '-TTC ACC AGT CCA TCA CGC CAC AGC-3 ' two ends and be prepared into corresponding real-time fluorescent PCR reagent case still do not have relevant report with 5 '-GAA CAA TGCAAC TTA TTG GAC GTT-3 ' and 5 '-AAC CGA AGC TGC CAC CCT AC-3 '.
(3), summary of the invention
One of purpose of the present invention just provides a kind of primer that is used to detect tobacco black shank bacterium, and it can be in the PCR testing process, qualitative detection tobacco black shank bacterium, and high specificity.
Two of purpose of the present invention just provides a kind of probe that is used to detect tobacco black shank bacterium, and it can be in the real-time fluorescence PCR testing process, and the detection by quantitative tobacco black shank bacterium improves detection sensitivity significantly.
Three of purpose of the present invention just provides a kind of test kit that utilizes the detection tobacco black shank 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 tobacco black shank bacterium on the plant, and reliable and stable, highly sensitive quickly and accurately.
First purpose of the present invention is to realize that by such technical scheme it comprises forward primer Pn1 and reverse primer Pn2, it is characterized in that its base sequence is respectively:
Pn1：5’-GAA?CAA?TGC?AAC?TTA?TTG?GAC?GTT-3’
Pn2：5’-AAC?CGA?AGC?TGC?CAC?CCT?AC-3’。
Through long-term a large amount of experiment, filter out the above-mentioned primer of forming by forward primer Pn1 and reverse primer Pn2 from numerous PCR primers that are used for detecting tobacco black shank bacterium, its amplified fragments size is 120 bp.The characteristics of this primer are tobacco black shank 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 tobacco black shank bacterium, and high specificity.
Second purpose of the present invention is to realize by such technical scheme, its base sequence is any nucleotide sequence in 5 '-TTC ACC AGT CCA TCA CGC CAC AGC-3 ' two ends upstream are shifted 2 bases 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 Pn1 and reverse primer Pn2, principle of design and method of design according to probe, design the probe that is used for the detection of tobacco black shank bacterium real-time fluorescence PCR, its base sequence is any nucleotide sequence in 5 '-TTC ACC AGT CCA TCA CGC CAC AGC-3 ' two ends upstream are shifted 2 bases or 3 base zones of downstream displacement; Carry out the real-time fluorescence PCR amplification with all designed probes, designed probe is screened; Result according to the real-time fluorescence PCR amplification filters out best probe at last, and its nucleotide sequence is: 5 '-TTC ACC AGT CCA TCA CGCCAC AGC-3 '.
5 ' end at fluorescence labeling probe is marked with 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 tobacco black shank bacterium, thereby the detection sensitivity of significantly improving.
The 3rd purpose of the present invention is to realize by such technical scheme, it includes testing sample and extracts reagent, quantitative criterion product, real-time fluorescence PCR reaction solution and detect articles for use, it is characterized in that the real-time fluorescence PCR reaction solution includes following component and corresponding final concentration thereof: PCR damping fluid: 10%; MgCl
2: 2.5mmol/L; DNTPs:0.3mmol/L; Taq archaeal dna polymerase: 1U/25 μ L; Forward primer Pn1:0.4 μ mol/L; Reverse primer Pn2:0.4 μ mol/L; Fluorescence labeling probe: 0.2 μ mol/L; Adopt aseptic double-distilled water to adjust concentration.
Testing sample extracts reagent to be made up of TES damping fluid and TE damping fluid, and TES damping fluid and TE damping fluid all are 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, no tobacco black shank bacterium soil sample and the healthy plant negative control template based on the peculiar nucleotide sequence design of tobacco black shank bacterium.The standard positive control template forms by containing pUCm-T (available from the Sangon company) preparing carriers of inserting target fragment.Preparation process is: the ITS district that adopts primer I TS4 and ITS5 amplification tobacco black shank bacterium, downcut the gel that contains target fragment behind the PCR product electrophoresis, target fragment reclaims test kit with gel and reclaims behind the purifying to spend the night for 16 ℃ with the pUCm-T carrier and be connected transformed into escherichia coli DH5 α competent cell.Converted product is coated with dull and stereotyped the cultivation, and the picking hickie adopts primer Pn1-Pn2 to carry out PCR and identifies.Positive colony is by the reliability of the further qualification result of sequential analysis.Select PCR and sequential analysis to be the male clone, be inoculated into the LB substratum incubated overnight that contains acillin, with SDS alkaline hydrolysis legal system prepare plasmid DNA.DNA is through ultraviolet spectrophotometer A
260Quantitatively, be 250ng-250fg/ 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: 2.5mmol/L; DNTPs:0.3mmol/L; Taq archaeal dna polymerase: 1U/25 μ L; Forward primer Pn1:0.4 μ mol/L; Reverse primer Pn2:0.4 μ mol/L; Fluorescence labeling probe: 0.2 μ mol/L; Adopt aseptic double-distilled water to adjust concentration.Wherein, PCR damping fluid, MgCl
2, dNTPs, Taq 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 match Parkson, Beijing gene engineering company limited, fluorescence labeling probe is synthetic in Takara company.
Detect articles for use and include self-sealing plastics bag, compound membrane filter and 0.2mL eight connecting legs.
On the basis of the probe in primer in utilizing purpose one and the purpose two, optimizing reaction system and reaction conditions are determined best tobacco black shank bacterium real-time fluorescence PCR detection architecture.
The real-time fluorescence PCR reaction system cumulative volume of optimization of the present invention is 25 μ L, 10 * PCR damping fluid, 2.5 μ L wherein, 25 mmol/L MgCl
22.5 μ L, 5mmol/L dNTPs1.5 μ L, 2U/ μ L Taq archaeal dna polymerase 0.5 μ L, each 2 μ L of 5 μ mol/L forward primer Pn1 and reverse primer Pn2,5 μ mol/L fluorescence labeling probes, 1 μ L, aseptic double-distilled water 11 μ L, dna profiling 2 μ L.Real-time fluorescence PCR adopts two-step approach, and the amplified reaction program is 95 ℃, 10min; 40 circulations then, each circulation is 95 ℃, 10s, 60 ℃, 30s collects fluorescence in each round-robin annealing/extension stage (60 ℃).
Utilize the real-time fluorescent PCR reagent case among the present invention that tobacco black shank bacterium is detected, comprise the real-time fluorescence PCR reaction of testing sample pre-treatment and testing sample.
The testing sample pre-treatment step is:
(1) with 70% alcohol wipe worktable, to guarantee under sterile state, operating whole process;
(2) choose pedotheque or tobacco leaf randomly, add and to be equipped with in the plastics bag of 10mL TES damping fluid, soaks 15 minutes (during vibration 2-3 time), the 50mL centrifuge tube of packing into then, the centrifugal 1min of 5000r/min is then with compound membrane filter filtration;
(3) wash thalline from filter membrane, filter membrane is immersed 100 μ L TE damping fluids, thermal agitation, the bacterium liquid of wash-out is follow-up PCR template to be measured.
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) in 0.2mL eight connecting legs, add 23 μ L real-time fluorescence PCR reaction solutions;
(3) add 2 μ L analyte sample fluids, no tobacco black shank bacterium soil sample or health tobacco plant negative control that test kit is provided add in the negative control pipe, the positive control pipe adds 10 * gradient dilution liquid of positive control standard substance, and the consumption of control sample is every pipe 2 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, the production standard curve calculates the DNA amount of tobacco black shank 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, testing sample extraction reagent can directly extract the tobacco black shank bacterium in pedotheque or the tobacco leaf, do not come to have saved the time and do not need to extract tobacco black shank bacterium DNA as the pcr amplification template; 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 tobacco black shank bacterium.
Owing to adopted technique scheme, the present invention to have following advantage:
(1), high specificity: primer and probe all design according to the tobacco black shank 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 tobacco black shank bacterium among the present invention, can be accurately quantitative, object bacteria DNA has good linear relationship in 100ng-10fg/ 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 tobacco black shank bacterium, practicality is good;
(4), applied range: can detection by quantitative soil and plant on tobacco black shank bacterium, be applicable to dynamic monitoring of tobacco black shank bacterium field and disease screening, can be used for the rate of propagation of tobacco black shank bacterium in tobacco plant and the detection by quantitative of seed-borne fungi simultaneously.
In sum, adopt the present invention, can detect tobacco black shank bacterium quickly and accurately in the soil in spite of illness in the field, thereby 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.
(4), embodiment
The invention will be further described below in conjunction with embodiment:
In the present invention, the primer that is used to detect tobacco black shank bacterium comprises forward primer Pn1 and reverse primer Pn2, it is characterized in that its base sequence is respectively:
Pn1：5’-GAA?CAA?TGC?AAC?TTA?TTG?GAC?GTT-3’
Pn2：5’-AAC?CGA?AGC?TGC?CAC?CCT?AC-3’。
Through long-term a large amount of experiment, filter out the above-mentioned primer of forming by forward primer Pn1 and reverse primer Pn2 from numerous PCR primers that are used for detecting tobacco black shank bacterium, its amplified fragments size is 120bp.The characteristics of this primer are tobacco black shank 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 tobacco black shank bacterium, and high specificity.
In the present invention, be used to detect the probe of tobacco black shank bacterium, its base sequence is any nucleotide sequence in 5 '-TTC ACCAGT CCA TCA CGC CAC AGC-3 ' two ends upstream are shifted 2 bases 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 Pn1 and reverse primer Pn2, principle of design and method of design according to probe, design the probe that is used for the detection of tobacco black shank bacterium real-time fluorescence PCR, its base sequence is any nucleotide sequence in 5 '-TTC ACC AGT CCA TCA CGC CAC AGC-3 ' two ends upstream are shifted 2 bases or 3 base zones of downstream displacement; Carry out the real-time fluorescence PCR amplification with all designed probes, 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 '-TTC ACC AGT CCA TCA CGC CAC AGC-3 ' can be: 5 '-GGT TCA CCA GTC CAT CAC GCC ACA GC-3 '.
The nucleotide sequence of fluorescence labeling probe also can be: 5 '-TTC ACC AGT CCA TCA CGC CAC AGC-3 ', it is a preferred forms.
At be shifted the downstream nucleotide sequence of 3 bases of 5 '-TTC ACC AGT CCA TCA CGC CAC AGC-3 ' can also be: 5 '-TTC ACC AGT CCA TCA CGC CAC AGC AGG-3 '.
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 tobacco black shank bacterium, thereby the detection sensitivity of significantly improving.
The test kit of detection tobacco black shank 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, it is characterized in that the real-time fluorescence PCR reaction solution includes following component and corresponding final concentration thereof: PCR damping fluid: 10%; MgCl
2: 2.5mmol/L; DNTPs:0.3mmol/L; Taq archaeal dna polymerase: 1U/25 μ L; Forward primer Pn1:0.4 μ mol/L; Reverse primer Pn2:0.4 μ mol/L; Fluorescence labeling probe: 0.2 μ mol/L; Adopt aseptic double-distilled water to adjust concentration.
Testing sample extracts reagent to be made up of TES damping fluid and TE damping fluid, and TES damping fluid and TE damping fluid all are 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, no tobacco black shank bacterium soil sample and the healthy plant negative control template based on the peculiar nucleotide sequence design of tobacco black shank bacterium.The standard positive control template forms by containing pUCm-T (available from the Sangon company) preparing carriers of inserting target fragment.Preparation process is: the ITS district that adopts primer I TS4 and ITS5 amplification tobacco black shank bacterium, downcut the gel that contains target fragment behind the PCR product electrophoresis, target fragment reclaims test kit with gel and reclaims behind the purifying to spend the night for 16 ℃ with the pUCm-T carrier and be connected transformed into escherichia coli DH5 α competent cell.Converted product is coated with dull and stereotyped the cultivation, and the picking hickie adopts primer Pn1-Pn2 to carry out PCR and identifies.Positive colony is by the reliability of the further qualification result of sequential analysis.Select PCR and sequential analysis to be the male clone, be inoculated into the LB substratum incubated overnight that contains acillin, with SDS alkaline hydrolysis legal system prepare plasmid DNA.DNA is through ultraviolet spectrophotometer A
260Quantitatively, be 250ng-250fg/ 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: 2.5mmol/L; DNTPs:0.3mmol/L; Taq archaeal dna polymerase: 1U/25 μ L; Forward primer Pn1:0.4 μ mol/L; Reverse primer Pn2:0.4 μ mol/L; Fluorescence labeling probe: 0.2 μ mol/L; Adopt aseptic double-distilled water to adjust concentration.Wherein, PCR damping fluid, MgCl
2, dNTPs, Taq 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 match Parkson, Beijing gene engineering company limited, fluorescence labeling probe is synthetic in Takara company.
Detect articles for use and include self-sealing plastics bag, compound membrane filter 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 tobacco black shank bacterium real-time fluorescence PCR detection architecture.
The real-time fluorescence PCR reaction system cumulative volume of optimization of the present invention is 25 μ L, 10 * PCR damping fluid, 2.5 μ L wherein, 25mmol/L MgCl
22.5 μ L, 5mmol/L dNTPs 1.5 μ L, 2U/ μ L Taq archaeal dna polymerase 0.5 μ L, each 2 μ L of 5 μ mol/L forward primer Pn1 and reverse primer Pn2,5 μ mol/L fluorescence labeling probes, 1 μ L, aseptic double-distilled water 11 μ L, dna profiling 2 μ L.Real-time fluorescence PCR adopts two-step approach, and the amplified reaction program is 95 ℃, 10min; 40 circulations then, each circulation is 95 ℃, 10s, 60 ℃, 30s collects fluorescence in each round-robin annealing/extension stage (60 ℃).
Utilize the real-time fluorescent PCR reagent case among the present invention that tobacco black shank bacterium is detected, comprise the real-time fluorescence PCR reaction of testing sample pre-treatment and testing sample.
The testing sample pre-treatment step is:
(1) with 70% alcohol wipe worktable, to guarantee under sterile state, operating whole process;
(2) picked at random pedotheque or tobacco leaf add and to be equipped with in the plastics bag of 10mL TES damping fluid, soak 15 minutes (during vibration 2-3 time), the 50mL centrifuge tube of packing into then, and the centrifugal 1min of 5000r/min is then with compound membrane filter filtration;
(3) wash thalline from filter membrane, filter membrane is immersed 100 μ L TE damping fluids, thermal agitation, the bacterium liquid of wash-out is follow-up PCR template to be measured.
The real-time fluorescence PCR reactions steps of testing sample is:
(1) with real-time fluorescence PCR instrument (model: ICycler
TMIQ, Bole's analysis of biochemical Instr Ltd. (Shanghai) produces) start is provided with standby;
(2) in 0.2mL eight connecting legs, add 23 μ L real-time fluorescence PCR reaction solutions;
(3) add 2 μ L analyte sample fluids, no tobacco black shank bacterium soil sample or health tobacco plant negative control that test kit is provided add in the negative control pipe, the positive control pipe adds 10 * gradient dilution liquid of positive control standard substance, and the consumption of control sample is every pipe 2 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, the production standard curve calculates the DNA amount of tobacco black shank bacterium in the testing sample reaction system according to typical curve.
In above-mentioned real-time fluorescence PCR reaction solution, what each component can adopt is: the PCR damping fluid is 10 * PCR damping fluid; MgCl
2Initial concentration be 25mmol/L; The initial concentration of dNTPs is 5mmol/L; The initial concentration of Taq archaeal dna polymerase is 2U/ μ L; The initial concentration of forward primer Pn1 and reverse primer Pn2 respectively 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 Pn1, reverse primer Pn2, fluorescence labeling probe volume ratio be: 5: 5: 3: 1: 4: 4: 2.
In above-mentioned real-time fluorescence PCR reaction solution, what each component also can adopt 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 TaqDNA polysaccharase is 5U/ μ L; The initial concentration of forward primer Pn1 is 10 μ mol/L; The initial concentration of reverse primer Pn2 is 10 μ mol/L; The initial concentration of fluorescence labeling probe is 10 μ mol/L; PCR damping fluid, MgCl
2, dNTPs, Taq archaeal dna polymerase, forward primer Pn1, reverse primer Pn2, fluorescence labeling probe volume ratio be: 50: 50: 15: 4: 20: 20: 10.
SEQUENCE?LISTING
＜110〉University Of Chongqing
＜120〉a kind of primer, probe and real-time fluorescent PCR reagent case that is used to detect tobacco black shank bacterium
＜130〉do not have
<160>5
<170>PatentIn?version?3.4
<210>1
<211>24
<212>DNA
<213>Phytophthora?nicotianae
<400>1
gaacaatgca?acttattgga?cgtt 24
<210>2
<211>20
<212>DNA
<213>Phytophthora?nicotianae
<400>2
aaccgaagct?gccaccctac 20
<210>3
<211>24
<212>DNA
<213>Phytophthora?nicotianae
<400>3
ttcaccagtc?catcacgcca?cagc 24
<210>4
<211>26
<212>DNA
<213>Phytophthora?nicotianae
<400>4
ggttcaccag?tccatcacgc?cacagc 26
<210>5
<211>27
<212>DNA
<213>Phytophthora?nicotianae
<400>5
ttcaccagtc?catcacgcca?cagcagg 27
Claims (8)
1. primer that is used to detect tobacco black shank bacterium, it comprises forward primer Pn1 and reverse primer Pn2, it is characterized in that its base sequence is respectively:
Pn1：5’-GAA?CAA?TGC?AAC?TTA?TTG?GAC?GTT-3’
Pn2：5’-AAC?CGA?AGC?TGC?CAC?CCT?AC-3’。
2. probe that is used to detect tobacco black shank bacterium, the base sequence that it is characterized in that it is any nucleotide sequence in 5 '-TTC ACC AGT CCA TCA CGC CAC AGC-3 ' two ends upstream are shifted 2 bases 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.
3. the probe that is used to detect tobacco black shank bacterium as claimed in claim 2 is characterized in that at upstream the be shifted nucleotide sequence of 2 bases of 5 '-TTC ACC AGT CCA TCA CGC CAC AGC-3 ' being: 5 '-GGT TCA CCA GTC CAT CAC GCC ACA GC-3 '.
4. the probe that is used to detect tobacco black shank bacterium as claimed in claim 2 is characterized in that its nucleotide sequence is: 5 '-TTC ACC AGT CCA TCA CGC CAC AGC-3 '.
5. the probe that is used to detect tobacco black shank bacterium as claimed in claim 2 is characterized in that at be shifted the downstream nucleotide sequence of 3 bases of 5 '-TTC ACC AGT CCA TCA CGC CAC AGC-3 ' being: 5 '-TTC ACC AGT CCA TCA CGC CAC AGC AGG-3 '.
6. test kit that utilizes the detection tobacco black shank bacterium that described primer of claim 1 and the described probe of claim 2 make, it includes testing sample and extracts reagent, quantitative criterion product, real-time fluorescence PCR reaction solution and detect articles for use, it is characterized in that the real-time fluorescence PCR reaction solution includes following component and corresponding final concentration thereof: PCR damping fluid: 10%; MgCl
2: 2.5mmol/L; DNTPs:0.3mmol/L; Taq archaeal dna polymerase: 1U/25 μ L; Forward primer Pn1:0.4 μ mol/L; Reverse primer Pn2:0.4 μ mol/L; Fluorescence labeling probe: 0.2 μ mol/L; Adopt aseptic double-distilled water to adjust final concentration.
7. the test kit of detection tobacco black shank bacterium as claimed in claim 6 is characterized in that in the real-time fluorescence PCR reaction solution, and 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 5mmol/L; The initial concentration of Taq archaeal dna polymerase is 2U/ μ L; The initial concentration of forward primer Pn1 is 5 μ mol/L; The initial concentration of reverse primer Pn2 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 Pn1, reverse primer Pn2, fluorescence labeling probe volume ratio be: 5: 5: 3: 1: 4: 4: 2.
8. the test kit of detection tobacco black shank bacterium as claimed in claim 6 is characterized in that: 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 10mmol/L; The initial concentration of Taq archaeal dna polymerase is 5U/ μ L; The initial concentration of forward primer Pn1 is 10 μ mol/L; The initial concentration of reverse primer Pn2 is 10 μ mol/L; The initial concentration of fluorescence labeling probe is 10 μ mol/L; PCR damping fluid, MgCl
2, dNTPs, Taq archaeal dna polymerase, forward primer Pn1, reverse primer Pn2, fluorescence labeling probe volume ratio be: 50: 50: 15: 4: 20: 20: 10.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2006100953506A CN101041853A (en) | 2006-12-26 | 2006-12-26 | Primer, probe and real-time fluorescent PCR reagent case for detecting tobacco black shank bacterium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2006100953506A CN101041853A (en) | 2006-12-26 | 2006-12-26 | Primer, probe and real-time fluorescent PCR reagent case for detecting tobacco black shank bacterium |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399896A (en) * | 2011-12-07 | 2012-04-04 | 中国农业科学院烟草研究所 | Method for detecting phytophthora parasitica in soil |
| CN108949916A (en) * | 2018-08-28 | 2018-12-07 | 华中农业大学 | Rape black shank bacterium specific sequence and LAMP detection primer and application |
| CN113430290A (en) * | 2021-06-15 | 2021-09-24 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Probe gene, primer pair, kit and application for detecting tobacco black shank |
| CN114942331A (en) * | 2022-07-06 | 2022-08-26 | 安徽农业大学 | Enzyme linked immunosorbent assay kit for detecting phytophthora parasitica and application thereof |
| CN117802267A (en) * | 2024-03-01 | 2024-04-02 | 中国计量科学研究院 | Quantitative detection method and proliferation prediction system for phytophthora nicotianae |
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2006
- 2006-12-26 CN CNA2006100953506A patent/CN101041853A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399896A (en) * | 2011-12-07 | 2012-04-04 | 中国农业科学院烟草研究所 | Method for detecting phytophthora parasitica in soil |
| CN108949916A (en) * | 2018-08-28 | 2018-12-07 | 华中农业大学 | Rape black shank bacterium specific sequence and LAMP detection primer and application |
| CN113430290A (en) * | 2021-06-15 | 2021-09-24 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Probe gene, primer pair, kit and application for detecting tobacco black shank |
| CN113430290B (en) * | 2021-06-15 | 2022-08-26 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Probe gene, primer pair, kit and application for detecting tobacco black shank |
| CN114942331A (en) * | 2022-07-06 | 2022-08-26 | 安徽农业大学 | Enzyme linked immunosorbent assay kit for detecting phytophthora parasitica and application thereof |
| CN117802267A (en) * | 2024-03-01 | 2024-04-02 | 中国计量科学研究院 | Quantitative detection method and proliferation prediction system for phytophthora nicotianae |
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