CN1824801A - Real-time fluorescent PCR testing primer, probe and immobilized kit for citrus ulcer bacteria and testing process thereof - Google Patents
Real-time fluorescent PCR testing primer, probe and immobilized kit for citrus ulcer bacteria and testing process thereof Download PDFInfo
- Publication number
- CN1824801A CN1824801A CN 200510057477 CN200510057477A CN1824801A CN 1824801 A CN1824801 A CN 1824801A CN 200510057477 CN200510057477 CN 200510057477 CN 200510057477 A CN200510057477 A CN 200510057477A CN 1824801 A CN1824801 A CN 1824801A
- Authority
- CN
- China
- Prior art keywords
- probe
- ulcer bacteria
- citrus ulcer
- pcr
- real
- 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.)
- Granted
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
This invention relates to oligonucleotide prime, mandarin orange ulcer germ real time fluorescence PCR detecting probe, solid phase reagent and its detecting method of mandarin orange ulcer germ detecting. It is a special quick detecting method direct towards mandarin orange ulcer disease, there is only 3 hours from sampling to detection, and detecting sensitivity is high, specificity is strong, stable and reliable. Misjudging in existing normal diagnosis method, missed diagnosis in electrical glass by asymmetry of germ and hard antibody preparation in single clone antibody and lack of specific reaction are conquered by this method, and false negative and false positive of normal PCR are avoided. Operation of solid phase kit is convenient, and its using is simple, and detecting sensitivity is high, specificity is strong, stable and reliable. So it is propitious to port detection of mandarin orange of import and export. Also it is propitious to allocation and transportation of mandarin orange inoculation, scion and other asymptomatic germ material, and early stage warning detection of non epidemic-stricken area orange ulcer disease.
Description
Technical field
The present invention relates to real-time fluorescence PCR immobilization test kit and the quarantine and examination technology thereof of the great epidemic disease biological citrus ulcer pathogenetic bacteria of evil (Xanthomonas axonopodis pv.citri); Being suitable for departments such as port quarantine, agriculture production, plant protection uses.Belong to technical field of bioengineering.
Technical background
Citrus ulcer is the great bacterial disease of the global citrus industry development of influence, also is domestic and international emphasis quarantine venereal disease evil.Citrus ulcer bacteria (Xanthomonas axonopodis pv.citri) is domestic and international important quarantine harmful organisms; Its tens of kinds of rutaceae of causing harm, comprise the most commercialization Cultivars in the both citrus, the over-ground part (blade, branch, prickle, trunk and fruit) of main infringement citrus, though seldom can directly kill trees, can cause fallen leaves, shedding, tree vigo(u)r is weak and fruit ulcer venereal disease spot.
Disease is mainly carried out long-distance communications by the allocation and transportation of citrus materials such as the seedling that carries disease germs, scion, fruit; The field disease is mainly pressed from both sides the rain diffusion with the wind by germ.Carry disease germs clothing, tr and the also certain diffusibility of tool of instrument of gathering.According to reports, U.S. scientist confirms, can propagate ulcer bacteria by agricultural transportation means such as vehicles, and uncommon Storms such as tropical storm etc. also can cause the long-distance communications of disease.
Method of inspection for the c itrus canker germ has conventional sense methods such as classical symptom appearance method, tissue pathological slice method, separate tissue cultivation, pathogenic assay method.Its advantage is directly perceived, simple and easy to do.Wherein pathogenic assay method can also be measured germ alive, thereby can reflect the danger of quarantine harmful organisms propagation diffusion truly.For determining of neopathy point, finally also need by pathogenic mensuration checking.But the shortcoming of conventional method is that sense cycle is long, and the person's of lacking experience False Rate height can have the disease material to citrus and detects, and is difficult to satisfy the basic demand of Plant Quarantine " fast, accurately, economy ".Detection to citrus bacterial canker disease also has phage method of inspection, enzyme-linked immunosorbent assay, spot immune absorption method etc. in addition, 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 using widely in the detection of phytopathogen and diagnosis gradually.Begin successively both at home and abroad in recent years real-time fluorescence PCR 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.As the fluorescence labeling probe method of high specificity, can effectively avoid the false positive problem of conventional PCR; Highly sensitive SYBRGreen I fluorescence dye method can be passed through melting curve analysis verification specificity, and promptly the distribution situation of the melting curve peak value by observation sample and each reference substance judges that whether the growth of fluorescent signal in the amplification curve is that amplification by target sequence causes.
Real-time fluorescence PCR detection method is used for the detection of plant pathogenetic bacteria, and all still at the early-stage at home and abroad, the real-time fluorescence PCR assay kit that does not still have citrus ulcer bacteria at present comes out, and existing test kit all must cryopreservation with send; Reaction reagent needs preparation voluntarily; This patent meticulously optimize and the basis of test repeatedly on, develop can the normal temperature accumulating premix freeze-drying immobilization test kit, have the i.e. usefulness of unlatching, advantage such as standard is unified, simple and convenient.Can satisfy the scientific research of port allocation and transportation, agriculture production, plant protection plant quarantine department and quick, accurate, the safe basic demand of inspection and quarantine.
Summary of the invention
The present invention is intended to overcome above-mentioned the deficiencies in the prior art, proposes primer, probe, immobilization test kit and the real-time fluorescence PCR detection method of the citrus ulcer bacteria of quick, reliable, highly sensitive, high specificity.
Realize the technical scheme of above-mentioned purpose:
Be used for the Oligonucleolide primers that citrus ulcer bacteria detects, comprise:
Oligonucleolide primers is right: 5 '-CGG CAG ATT GGA AGT CA-3 '
5 '-TCC AGC ACA TAC GGG TC-3 ' (sequence number: NO.1);
Oligonucleolide primers is right: 5 '-GTT CGG CGT CAA CAA C-3 '
5 '-CGG TAG GCG ACT CAT TT-3 ' (sequence number: NO.2);
Oligonucleolide primers is right: 5 '-GAG TCG CCT ACC GAG AAA TCC-3 '
5 '-ACC ACG GCA GGG TGA AGA C-3 ' (sequence number: NO.3);
Oligonucleolide primers is right: 5 '-CGT CAA CAA CCT GGA GAG CA-3 '
5 '-GGT AGG CGA CTC ATT TCC CA-3 ' (sequence number: NO.4).
A kind of oligonucleotide probe that the citrus ulcer bacteria real-time fluorescence PCR detects that is used for provided by the invention, its specific probe sequence be at 5 '-CCA GAC AGT GTC TCG GAA ATT CGA CCT CTC CGA AC-3 ' two ends upstream or the nucleotide sequence of formation in the zone of 3-6 base of downstream displacement (probe is numbered: NO.1).The probe sequence of its optimization is 5 '-AGT GTC TCG GAA ATT CGA CCT CTC CGA AC-3 '.
Another kind provided by the invention is used for the oligonucleotide probe that the citrus ulcer bacteria real-time fluorescence PCR detects, its specific probe sequence be at 5 '-AGC ATG CGA CGT TTC TAC CTC TCA TAC TCC CAG CC-3 ' two ends upstream or the nucleotide sequence of formation in the zone of 3-6 base of downstream displacement (probe is numbered: NO.4); The probe sequence of its optimization is 5 '-CGA CGT TTC TAC CTC TCA TAC TCC CAG CC-3 '.
Be used for the fluorescent mark oligonucleotide probe that the citrus ulcer bacteria real-time fluorescence PCR detects, fluorescence report group FAM is marked at 5 ' end, and fluorescent quenching group TAMRA is marked at 3 ' end.Can replace with self non-luminous Eclipse at 3 ' end fluorescent quenching group.
Be used for the test kit that the citrus ulcer bacteria real-time fluorescence PCR detects, comprise sample extraction reagent, nucleic acid amplification immobilization reagent, the employed primer of kit that innoxious quantitative criterion product, healthy plant negative control. wherein use SYBR Green I real-time fluorescence PCR detection method is NO.1 or NO.2 Oligonucleolide primers pair; And the employed primer of the kit that uses the fluorescence labeling probe real-time fluorescence PCR detection method be NO.3 and NO.4 Oligonucleolide primers pair, employed probe sequence be at 5 '-CCA GAC AGT GTC TCG GAA ATT CGA CCT CTC CGA AC-3 ' two ends upstream or the nucleotide sequence that forms in the zone of 3-6 base of downstream displacement or probe sequence be the fluorescence labeling probe of the nucleotide sequence of formation upstream or in 3-6 base of downstream displacement regional at 5 '-AGC ATG CGA CGT TTC TAC CTC TCA TAC TCC CAG CC-3 ' two ends.
Citrus ulcer bacteria real-time fluorescence PCR detection architecture makes up, may further comprise the steps: (1) earlier carries out sequence comparing analysis to citrus ulcer bacteria and allied species thereof or relevant kind, finds out the distinctive base sequence that citrus ulcer bacteria is different from other allied species or relevant kind; (2) according to the principle of design and the method for design of primer and probe in SYBR Green fluorescence dye method and the fluorescence labeling probe method,, design primer and probe that the citrus ulcer bacteria real-time fluorescence PCR detects according to the specific nucleic acid fragment of germ; (3) to designed primer with probe screens and the optimization of reaction system and reaction conditions, filter out Auele Specific Primer and probe, and suitable reaction system and the reaction conditions of optimization.(4) primer and the probe described in claim 2 or 4 (SYBR Green only needs to add primer and fluorescence dye gets final product) in the adding claim 1.(5) add innoxious positive criteria product.Carry out the reaction of citrus ulcer bacteria real-time fluorescence PCR.(6) carry out before the real-time fluorescence PCR reaction, use the sample extraction reagent that provides in the test kit to soak earlier and filter rich long-pending sample, again it is entered step (4) as template afterwards from the citrus tissue surface.
The citrus ulcer bacteria real-time fluorescence PCR detection method may further comprise the steps:
(1) earlier citrus ulcer bacteria and allied species thereof or relevant kind are carried out sequence comparing analysis, find out citrus ulcer bacteria and be different from other distinctive base sequence of relevant kind;
(2) according to the principle of design and the method for design of primer and probe in SYBR Green fluorescence dye method and the fluorescence labeling probe method, design primer and probe that the citrus ulcer bacteria real-time fluorescence PCR detects;
(3) designed probe is screened and the optimization of reaction system and reaction conditions, filter out the specific probe of optimization, and suitable reaction system and the reaction conditions of optimization.
(4) primer and the probe described in claim 2 or 4 (SYBR Green only needs to add primer and fluorescence dye gets final product) that adds in the claim 1 carries out the reaction of citrus ulcer bacteria real-time fluorescence PCR.
Enter step (4) and carry out before the real-time fluorescence PCR reaction, use the sample preparation reagents that provides in the test kit from plant sample, to soak earlier and filter the c itrus canker germ that enriched sample is carried, be re-used as template afterwards and enter step (4).
Adopt technique scheme, the technical progress that the present invention gives prominence to is:
1, the present invention utilize primer that conservative gene sequences Design in the known full genome of citrus ulcer bacteria is suitable for pathogenic bacteria real-time fluorescence PCR rapid detection to and fluorescent probe, high specificity.
2, the present invention can directly carry out real-time fluorescence PCR to citrus ulcer bacteria and detects, and is highly sensitive, can detect the no disease sample that carries disease germs.
3, the present invention adopts the filter membrane enrichment to filter sample making technology, has got rid of the influence of sample P CR supressor, has shortened the sample preparation time, and detecting whole process only needs 3 hours, the false negative problem of having avoided conventional PCR to detect effectively.
4,, develop innoxious recon positive control, epidemic situation diffusion and the pollution of having avoided the live body positive control to cause according to pathogenic bacteria specific DNA sequences segment.
5, premix freeze-drying immobilization test kit that can the normal temperature accumulating has the i.e. usefulness of unlatching, advantage such as standard is unified, simple and convenient.
In sum, adopt the present invention, can satisfy reproductive material entry and exit quarantine and examinations such as large carry disease germs fresh orange and seedling scion, the demands such as molecule discriminating of Pest-or disease-free area orchard HLB early warning monitoring and different citrus ulcer bacterias, its meaning is very great.
Embodiment
Embodiment one:
A kind of primer sequence and test kit that is used for the detection of citrus ulcer bacteria (Xanthomonas axonopodis pv.citri) SYBR Green I real-time fluorescence PCR, be used for the specific detection of citrus ulcer bacteria, it consists of: sample extraction reagent: TES damping fluid 100mL; 70% ethanol 100mL; Nucleic acid elutriant 10mL.Nucleic acid amplification immobilization reagent: be utilize the macromole stablizer through vacuum lyophilization be prepared from can normal temperature the pcr amplification immobilization mixture freeze-drying glue pearl of storing, contain following reagent in its composition:
The component final concentration
PCR damping fluid 1X;
MgCl
2 3mmol/L；
dNTPs 0.2mmol/L；
Primer is to each 0.5umol/Lol/L;
Taq polysaccharase 1Unit/25uL;
The biomacromolecule stablizer adds to the 23uL/ pipe;
SYBR Green I fluorescence dye 10umol/Lol/L;
Employed primer is right: 5 '-GAAGCTGGTGGAGGTG-3 '
5 '-GACTGCCCAACGAAAA-3 ' (sequence number: NO.1).
Innoxious quantitative criterion product: based on the innoxious recon positive control of the peculiar nucleotide sequence design of citrus ulcer bacteria; Citrus ulcer bacteria recon positive control dried frozen aquatic products wherein, healthy citrus plant negative control dried frozen aquatic products.
Detect articles for use: the self-sealing plastics bag; Filter apparatus (strainer that comprises compound filter membrane); 1.5ml plastic.
Embodiment two:
A kind of primer sequence and immobilization test kit that is used for the detection of citrus ulcer (Xanthomonas axonopodis pv.citri) SYBR Green real-time fluorescence PCR, the specific detection that is used for citrus ulcer bacteria Asia fungus strain, its composition comprises: sample extraction reagent; The immobilization reagent of nucleic acid amplification; Innoxious quantitative criterion product: citrus ulcer bacteria recon positive control dried frozen aquatic products, citrus healthy plant DNA dried frozen aquatic products.
Wherein primer sequence is: 5 '-TTTCGTTGGGCAGTCT-3 '
5 '-ATCGGGTAAAGAAGCA-3 ' (sequence number: NO.2).
The immobilization reagent of described sample extraction reagent, nucleic acid amplification, innoxious quantitative criterion product and detection articles for use are identical with embodiment one.
Embodiment three:
A kind of primer, probe sequence and immobilization test kit that is used for citrus ulcer bacteria (Xanthomonas axonopodis pv.citri) fluorescence labeling probe PCR detection, the rapid detection that is used for citrus ulcer bacteria, its composition comprises: sample extraction reagent: TES damping fluid 100mL; 70% ethanol 100mL; Nucleic acid elutriant 10mL.Nucleic acid amplification immobilization reagent: be utilize the macromole stablizer through vacuum lyophilization be prepared from can normal temperature the pcr amplification immobilization mixture freeze-drying glue pearl of storing, contain following reagent in its composition:
The component final concentration
PCR damping fluid 1X;
MgCl
2 3mmol/L；
dNTPs 0.2mmol/L；
Primer is to each 0.5umol/Lol/L;
Probe 0.2umol/Lol/L;
Taq polysaccharase 1Unit/25uL;
The biomacromolecule stablizer adds to 23uL;
Employed primer is to being: 5 '-TGGAGGTGTAAAAGTTGCCAAA-3 '
5 '-ACGAAAAGATCAGATATTCCTCTA-3 ' (sequence number: NO.3);
Employed fluorescence labeling probe sequence is: 5 '-AGT GTC TCG GAA ATT CGA CCT CTC CGAAC-3 ' (probe numbering: NO.3).
Innoxious quantitative criterion product: based on the innoxious recon positive control of the peculiar nucleotide sequence design of citrus ulcer bacteria; Citrus ulcer bacteria recon positive control dried frozen aquatic products wherein, healthy citrus plant negative control dried frozen aquatic products.
Detect articles for use: the self-sealing plastics bag; Filter apparatus (strainer that comprises compound filter membrane); 1.5ml plastic.
Embodiment four:
A kind ofly be used for primer, probe sequence and the immobilization test kit that citrus ulcer bacteria (Xanthomonas axonopodis pv.citri) fluorescence labeling probe real-time fluorescence PCR detects, be used for the specific detection of citrus ulcer bacteria, its composition comprises:
Sample extraction reagent, nucleic acid amplification immobilization reagent, innoxious quantitative criterion product: citrus ulcer bacteria recon positive control dried frozen aquatic products, healthy citrus plant negative control dried frozen aquatic products.
Wherein primer sequence is: 5 '-GAGGTGTAAAAGTTGCCAAA-3 '
5 '-CGAAAAGATCAGATATTCCTCTA-3 ' (sequence number: NO.4).
The fluorescence labeling probe sequence is: 5 '-CGA CGT TTC TAC CTC TCA TAC TCC CAG CC-3 ' (probe numbering: NO.6).
The immobilization reagent of described sample extraction reagent, nucleic acid amplification, innoxious quantitative criterion product and detection articles for use are identical with embodiment three.
The sample pretreatment step is:
1. cleaning work platform: wipe worktable and hand to reach the effect of sterilization with medical alcohol;
2. at random choose citrus leaves, add in the sterilized water 10ml plastics bag, soaked the 50mL centrifuge tube of packing into then 15 minutes; The centrifugal back of 8000xg membrane filtration;
3. wash thalline from filter membrane, filter membrane is immersed the 100uL elutriant, thermal agitation, the bacterium liquid of wash-out is follow-up PCR template to be measured.
The detection of nucleic acids step is:
1. be provided with the start of real-time fluorescence PCR instrument standby.
2. in eight pipes that immobilization freeze-drying PCR reaction mix reagent is housed, add 23uL and recover liquid.
3. add 2uL analyte sample fluid or template to be measured, the healthy citrus plant negative control that test kit is provided adds in the negative control pipe, the positive control pipe adds the 10x gradient dilution liquid (detection by quantitative) of innoxious positive control standard substance (the general detection) or standard substance according to circumstances, and the consumption of control sample is every pipe 2uL.
4. can directly carry out the PCR reaction after mixing; SYBR Greeno I dye method must be done the melting curve analysis after the PCR reaction, with the specificity of checking detection.
5. treat pcr amplification finish, according to amplification data, analyzing and testing result.
Each component is respectively in the system of detection reaction:
(1) SYBR Green I fluorescence dye method immobilization real-time fluorescence PCR system is that real-time fluorescence mixed solution 15uL (contains 1xPCR damping fluid, 3mmol/L MgCl
2, 0.2mmol/L dNTPs, 1Unit Taq enzyme, 0.5umol/Lol/L primer to each one and fluorescence dye 10umol/Lol/L), strengthen stabilizer molecule to 23uL, mixing-40 ℃ of refrigerator quick-frozens 30 minutes, places the interior freeze-drying of vacuum freezing drying device to transparent jelly.Pack under the normal temperature and preserve.Add 23ul before using and recover liquid, after mixing with the 2uL testing sample, carry out pcr amplification.
(2) fluorescence labeling probe method immobilization real-time fluorescence PCR system is that nucleic acid amplification mixed solution 15uL (contains 1x PCR damping fluid, 3mmol/L MgCl
2, 0.2mmol/L dNTPs, 1Unit Taq enzyme, 0.5umol/Lol/L primer to each one and the 0.2mmol/L probe), add the macromole stablizer to 23uL, mixing pre-freeze places in the vacuum freezing drying device freeze-drying to transparent jelly.Pack under the normal temperature and preserve.Add 23ul before using and recover liquid, after mixing with the 2uL testing sample, the control sample pipe is set as requested, carry out pcr amplification.
The detection reaction condition is:
(1) SYBR Green I fluorescence dye method: 95 ℃, 1min; 40 circulations then, each circulation is 95 ℃, 15s, 59 ℃, 15s, 72 ℃, 45s extends the stage (72 ℃) at each round-robin and collects fluorescence; Last 72 ℃, 7min.The reaction conditions of doing melting curve is: 95 ℃, and 1min; 55 ℃, 1min; Keep 10s for 0.5 ℃ since 55 ℃ of every risings, raise continuously 80 times (till 95 ℃).
(2) fluorescence labeling probe method: 95 ℃, 1min; 40 circulations then, each circulation is 95 ℃, 5s, 59 ℃, 30s collects fluorescence in each round-robin annealing/extension stage (59 ℃).
Criterion as a result is:
(1) reaction of use fluorescence labeling probe method
If it is that the positive control report fluorescence of template has clear signal to increase that the quantitative PCR instrument detects with citrus ulcer bacteria specific fragment recon; The report fluorescence of negative control and blank does not have fluorescent signal to increase, and test result of samples is to report that fluorescent signal rises appreciably, and then contains citrus ulcer bacteria in the sample that expression is detected; If it is that the positive control report fluorescence of template has clear signal to increase that the quantitative PCR instrument detects with citrus ulcer bacteria specific fragment recon, the report fluorescence of negative control and blank does not have fluorescent signal to increase, and test result of samples is to report that fluorescent signal does not rise appreciably, and does not then contain citrus ulcer bacteria in the sample that expression is detected.
(2) reaction of use SYBR Green I fluorescence dye method
If contain citrus ulcer bacteria in the sample that expression is detected first of the following two kinds of situations of appearance:
1. to detect with citrus ulcer bacteria specific fragment recon be that the positive control report fluorescence of template has clear signal to increase to the quantitative PCR instrument, the report fluorescence of negative control and blank does not have fluorescent signal to increase, and test result of samples is to report that fluorescent signal rises appreciably, and in the melting curve that carries out was subsequently analyzed, sample had the main peak the same with positive control;
2. positive control report fluorescence has clear signal to increase, the report fluorescence of negative control and blank has slight fluorescent signal to increase, and test result of samples is to report that fluorescent signal rises appreciably, and in the melting curve that carries out was subsequently analyzed, sample had a main peak identical with positive control and a side peak identical with negative control;
If do not contain citrus ulcer bacteria in the sample that expression is detected first of the following two kinds of situations of appearance:
1. to detect with citrus ulcer bacteria specific fragment recon be that the positive control report fluorescence of template has clear signal to increase to the quantitative PCR instrument, the report fluorescence of negative control and blank does not have fluorescent signal to increase, and test result of samples is the not growth of report fluorescent signal;
2. positive control report fluorescence has clear signal to increase, the report fluorescence of negative control and blank has slight fluorescent signal to increase, and test result of samples is to report that fluorescent signal also has marginal increase, and in the melting curve that carries out was subsequently analyzed, sample had a side peak identical with negative control.
The Oligonucleolide primers and the probe that are used for the citrus ulcer bacteria detection comprise:
Oligonucleolide primers is to sequence number: NO.1:
5’-CGG?CAG?ATT?GGA?AGT?CA-3’
5’-TCC?AGC?ACA?TAC?GGG?TC-3’
Oligonucleolide primers is to sequence number: NO.2:
5’-GTT?CGG?CGT?CAA?CAA?C-3’
5’-CGG?TAG?GCG?ACT?CAT?TT-3
Oligonucleolide primers is to sequence number: NO.3:
5’-GAG?TCG?CCT?ACC?GAG?AAA?TCC-3’
5’-ACC?ACG?GCA?GGG?TGA?AGA?C-3’
Oligonucleolide primers is to sequence number: NO.4:
5’-CGT?CAA?CAA?CCT?GGA?GAG?CA-3’
5’-GGT?AGG?CGA?CTC?ATT?TCC?CA-3’
The longest probe sequence numbering NO.1:
5’-CCA?GAC?AGT?GTC?TCG?GAA?ATT?CGA?CCT?CTC?CG-3
The shortest probe sequence numbering NO.2:
5’-CCA?GAC?AGT?GTC?TCG?GAA?ATT?CGA?CCT-3’
The probe sequence numbering NO.3 that optimizes:
5’-AGT?GTC?TCG?GAA?ATT?CGA?CCT?CTC?CGA?AC-3’
The longest probe sequence numbering NO.4:
5’-AGC?ATG?CGA?CGT?TTC?TAC?CTC?TCA?TAC?TCC?CA-3’
The shortest probe sequence numbering NO.5:
5’-GTT?TCT?ACC?TCT?CAT?ACT?CCC?AGC?CC-3’，
The probe sequence numbering NO.6 that optimizes
5’-CGA?CGT?TTC?TAC?CTC?TCA?TAC?TCC?CAG?CC-3’
Sequence table
＜110〉University Of Chongqing
Biotech development company limited of University Of Chongqing
Chongqing, Sichuan high-tech limited liability company
＜120〉citrus ulcer bacteria real-time fluorescent PCR testing primer, probe, immobilization test kit and detection method thereof
<130>
<160>12
<170>PatentIn?version?3.3
<210>1
<211>17
<212>DNA
<213>Xanthomonas?citri
<400>1
cggcagattg?gaagtca 17
<210>2
<211>17
<212>DNA
<213>Xanthomonas?citri
<400>2
tccagcacat?acgggtc 17
<210>3
<211>16
<212>DNA
<213>Xanthomonas?citri
<400>3
gttcggcgtc?aacaac 16
<210>4
<211>17
<212>DNA
<213>Xanthomonas?citri
<400>4
cggtaggcga?ctcattt 17
<210>5
<211>21
<212>DNA
<213>Xanthomonas?citri
<400>5
gagtcgccta?ccgagaaatc?c 21
<210>6
<211>19
<212>DNA
<213>Xanthomonas?citri
<400>6
accacggcag?ggtgaagac 19
<210>7
<211>20
<212>DNA
<213>Xanthomonas?citri
<400>7
cgtcaacaac?ctggagagca 20
<210>8
<211>20
<212>DNA
<213>Xanthomonas?citri
<400>8
ggtaggcgac?tcatttccca 20
<210>9
<211>35
<212>DNA
<213>Xanthomonas?citri
<400>9
ccagacagtg?tctcggaaat?tcgacctctc?cgaac 35
<210>10
<211>29
<212>DNA
<213>Xanthomonas?citri
<400>10
agtgtctcgg?aaattcgacc?tctccgaac 29
<210>11
<211>35
<212>DNA
<213>Xanthomonas?citri
<400>11
agcatgcgac?gtttctacct?ctcatactcc?cagcc 35
<210>12
<211>29
<212>DNA
<213>Xanthomonas?citri
<400>12
cgacgtttct?acctctcata?ctcccagcc 29
Claims (9)
1, a kind of Oligonucleolide primers that is used for the citrus ulcer bacteria detection comprises:
Oligonucleolide primers is right: 5 '-CGG CAG ATT GGA AGT CA-3 '
5 '-TCC AGC ACA TAC GGG TC-3 ' (sequence number: NO.1);
Oligonucleolide primers is right: 5 '-GTT CGG CGT CAA CAA C-3 '
5 '-CGG TAG GCG ACT CAT TT-3 ' (sequence number: NO.2);
Oligonucleolide primers is right: 5 '-GAG TCG CCT ACC GAG AAA TCC-3 '
5 '-ACC ACG GCA GGG TGA AGA C-3 ' (sequence number: NO.3);
Oligonucleolide primers is right: 5 '-CGT CAA CAA CCT GGA GAG CA-3 '
5 '-GGT AGG CGA CTC ATT TCC CA-3 ' (sequence number: NO.4).
2 ,-kind be used for the oligonucleotide probe that the citrus ulcer bacteria real-time fluorescence PCR detects, it is characterized in that, the fluorescence labeling probe of specific detection citrus ulcer bacteria, its probe sequence be 5 '-CCA GAC AGT GTC TCG GAA ATTCGA CCT CTC CGA AC-3 ' two ends upstream or 3-6 base of downstream displacement the zone in nucleotide sequence.
3, the oligonucleotide probe that is used for the detection of citrus ulcer bacteria real-time fluorescence PCR according to claim 2 is characterized in that probe sequence is 5 '-AGT GTC TCG GAA ATT CGA CCT CTC CGA AC-3 '.
4, a kind of oligonucleotide probe that is used for the detection of citrus ulcer bacteria real-time fluorescence PCR, it is characterized in that, the fluorescence labeling probe of specific detection citrus ulcer bacteria, its probe sequence be 5 '-AGC ATG CGA CGT TTC TAC CTCTCA TAC TCC CAG CC-3 ' two ends upstream or 3-6 base of downstream displacement the zone in nucleotide sequence.
5, the oligonucleotide probe that is used for the detection of citrus ulcer bacteria real-time fluorescence PCR according to claim 4 is characterized in that probe sequence is 5 '-CGA CGT TTC TAC CTC TCA TAC TCC CAG CC-3 '.
6, according to any fluorescent mark oligonucleotide probe that is used for the detection of citrus ulcer bacteria real-time fluorescence PCR in the claim 2 to 5, it is characterized in that: the fluorescence report group is marked at 5 ' end, and the fluorescent quenching group is marked at 3 ' end.
7, a kind of citrus ulcer bacteria SYBR Green real-time fluorescence PCR immobilization test kit that is used for, comprise sample extraction reagent, nucleic acid amplification immobilization reagent, innoxious quantitative criterion product, it is characterized in that, described nucleic acid amplification immobilization reagent is PCR immobilization mixture freeze-drying glue pearl, contains following reagent in its composition:
The component final concentration
PCR damping fluid 1X;
MgCl
2 3mmol/L；
dNTP 0.2mmol/L；
Taq polysaccharase 1Unit/25uL;
Primer is to each 0.5umol/Lol/L;
SYBR Green I fluorescence dye 10umol/Lol/L;
The biomacromolecule stablizer adds to 23uL;
Employed primer is that NO.1 or the NO.2 Oligonucleolide primers in the claim 1 is right.
8, a kind of citrus ulcer bacteria fluorescence labeling probe real-time fluorescence PCR immobilization test kit that is used for, comprise sample extraction reagent, nucleic acid amplification immobilization reagent, innoxious quantitative criterion product, it is characterized in that, nucleic acid increases and expands immobilization reagent is PCR immobilization mixture freeze-drying glue pearl, contains following reagent in its composition:
The component final concentration
PCR damping fluid 1X;
MgCl
2 2.5mmol/L；
dNTPs 0.3mmol/L；
Taq polysaccharase 1Unit/25uL;
Primer is to each 0.5umol/Lol/L;
Fluorescence labeling probe 0.2umol/Lol/L;
The biomacromolecule stablizer adds to 23uL;
Employed primer is that NO.3 and NO.4 Oligonucleolide primers are right in the claim 1,
Employed probe is the fluorescence labeling probe in claim 2 or 4.
9, a kind of citrus ulcer bacteria real-time fluorescence PCR detection method is characterized in that carrying out according to the following steps:
(1) earlier citrus ulcer bacteria and allied species thereof or kind of the DNA that is correlated with are carried out sequence comparing analysis, find out the distinctive base sequence that citrus ulcer bacteria is different from allied species;
(2) according to the principle of design and the method for design of primer and probe in SYBR Green fluorescence dye method and the fluorescence labeling probe method, design citrus ulcer bacteria real-time fluorescence PCR detecting character primer and probe according to the citrus ulcer bacteria specific DNA sequences;
(3) designed probe is screened and the optimization of reaction system and reaction conditions, filter out specific probe, and suitable reaction system and the reaction conditions of optimization;
(4) use the sample extraction reagent that provides in the test kit from plant sample, to extract the sample total DNA of purifying;
(5) SYBR Green fluorescence dye method adds the primer in the claim 1, and primer in the fluorescence labeling probe method adding claim 1 and the positive control template of the probe described in claim 2 or 4 and (4) gained are carried out the reaction of citrus ulcer bacteria real-time fluorescence PCR.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100574774A CN100404689C (en) | 2005-12-29 | 2005-12-29 | Real-time fluorescent PCR testing primer, probe and immobilized kit for citrus ulcer bacteria and testing process thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100574774A CN100404689C (en) | 2005-12-29 | 2005-12-29 | Real-time fluorescent PCR testing primer, probe and immobilized kit for citrus ulcer bacteria and testing process thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1824801A true CN1824801A (en) | 2006-08-30 |
| CN100404689C CN100404689C (en) | 2008-07-23 |
Family
ID=36935642
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2005100574774A Expired - Fee Related CN100404689C (en) | 2005-12-29 | 2005-12-29 | Real-time fluorescent PCR testing primer, probe and immobilized kit for citrus ulcer bacteria and testing process thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100404689C (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101260399B (en) * | 2008-03-13 | 2011-08-10 | 湖南农业大学 | Orange canker resistant pthA-nls gene and its construction method and application |
| CN102243239A (en) * | 2011-04-25 | 2011-11-16 | 重庆大学 | Quick colloidal gold detection test strip for xanthomonas citri |
| CN101928779B (en) * | 2010-02-08 | 2012-11-14 | 江苏出入境检验检疫局动植物与食品检测中心 | Real-time fluorescent RCR molecular detection kit for leptosphaeria maculans and detection method thereof |
| CN113186318A (en) * | 2021-06-07 | 2021-07-30 | 中国海关科学技术研究中心 | Primer group, kit and detection method for PCR detection of citrus canker pathogen |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101712983B (en) * | 2009-11-04 | 2012-08-01 | 中华人民共和国湖南出入境检验检疫局 | Primer pair for detecting citrus canker pathogenic bacteria and detection method thereof |
| CN102634603B (en) * | 2012-05-10 | 2013-04-10 | 湖南农业大学 | PCR detection method for citrus canker |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5420009A (en) * | 1992-03-23 | 1995-05-30 | The United States Of America As Represented By The Secretary Of Agriculture | Detection of Xanthomonas campestris pv. citri by hybridization and polymerase chain reaction assays |
-
2005
- 2005-12-29 CN CNB2005100574774A patent/CN100404689C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101260399B (en) * | 2008-03-13 | 2011-08-10 | 湖南农业大学 | Orange canker resistant pthA-nls gene and its construction method and application |
| CN101928779B (en) * | 2010-02-08 | 2012-11-14 | 江苏出入境检验检疫局动植物与食品检测中心 | Real-time fluorescent RCR molecular detection kit for leptosphaeria maculans and detection method thereof |
| CN102243239A (en) * | 2011-04-25 | 2011-11-16 | 重庆大学 | Quick colloidal gold detection test strip for xanthomonas citri |
| CN113186318A (en) * | 2021-06-07 | 2021-07-30 | 中国海关科学技术研究中心 | Primer group, kit and detection method for PCR detection of citrus canker pathogen |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100404689C (en) | 2008-07-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1286989C (en) | Biochip for detecting pathogenesis fungus | |
| CN1814805A (en) | H5, H7, H9 subtype auian flu virus real-time fluo rescent quantixative PCR detecting method | |
| CN1940087A (en) | Method for inspecting hepatitis and AIDS virus nucleic acid by synchronous amplification and its reagent kit | |
| CN1582341A (en) | Method of isolating cells and uses thereof | |
| CN1824801A (en) | Real-time fluorescent PCR testing primer, probe and immobilized kit for citrus ulcer bacteria and testing process thereof | |
| CN101045943A (en) | Process of PCR detecting salmonella with added amplifying internal standard | |
| CN1269968C (en) | Analytic method and use for reducing real-time fluorescent PCR instrument quantitative analytic system error | |
| CN1854287A (en) | Assay systems, kits and methods for detecting microorganisms | |
| CN101041864A (en) | Chlamydi trachomatis and Neisseria gonorrhoeae dual real-time fluorescence PCR detection method | |
| CN1955310A (en) | Nucleotide sequential, testing kit and method for detecting swine streptococcus II | |
| CN1873019A (en) | Quick method for testing chain reaction of multiple reverse transcription polymerase of lily virus | |
| CN1824802A (en) | Real-time fluorescent PCR testing primer, probe and immobilized kit for citrus candidatus liberobacter asiaticum Jagoueix and testing process thereof | |
| CN1930302A (en) | Method for detecting pathogenic mycobacteria in clinIcal specimens | |
| CN1724686A (en) | Target sequence used for detecting mycoplasma pnoumoniae and reagent box | |
| CN1506468A (en) | PCR test kit for hygrophilous aeromonad and its test method | |
| CN1900314A (en) | Fluorescence labelling ABO gene typing method and its reagent kit | |
| CN1289692C (en) | Magnetic capture of SARS coronary virus and PCR detection therewith | |
| CN1215177C (en) | Gene chip used for identifying pathogenic bacteria in blood and its making method | |
| CN1188530C (en) | Gene detection reagent kit for SARS virus and its detection method | |
| CN100351395C (en) | Norwalk virus expression detecting kit and its special primer and probe | |
| CN1357636A (en) | CpG insular methylation test reagent kit and its application | |
| CN1772922A (en) | Method of identifying invasion of south American glim ant and its nucleic acid sequence, probe and reagent kit | |
| CN1837364A (en) | Real-time fluorescence PCR immobilization kit of wheat dwarf bunt germ (Tilletia controversa kuhn) and its detection method | |
| CN101045942A (en) | Transgenic papaya and detection of transgenic component in transgenic papaya product | |
| CN1203188C (en) | Nano-class amplifying detection method for biochip |
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 | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20080723 Termination date: 20131229 |