CN1614396A - Special primer probe and detection for nematode of pine - Google Patents
Special primer probe and detection for nematode of pine Download PDFInfo
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Abstract
A special primer is composed of forward primer and backward primer as forward primer is oligonucleotide order of 16-30 pieces of nucleotides with 3' tail end order of 3'-TCAGT and back ward primer is oligonucleotide order of 16-30 pieces of nucleotides with 3' tail end order of 3'-CCAAG. The probe is oligonucleotide order of 16-25 pieces of nucleotides with 3' tail end order of 3'-GGTAGTACGCGCC. The method utilizing the primer and the probe to detect pin timber threadworm is also disclosed.
Description
Technical field
The present invention relates to a kind of method and primer special and probe that detects pine wood nematode in biological control, molecular diagnosis and the nematology field, particularly a kind of detection kit that detects method and primer special and the probe of pine wood nematode and comprise this primer and probe.
Background technology
Pine nematode claims loose droop again, is to have one of dangerous pine forest disease, can be dead after plant caught an illness 40 days, only need the 3-5 year from falling ill to the destruction of full wafer pine forest, and therefore be also referred to as " cancer " of pine tree.Pine nematode is mainly propagated by Monochamus alternatus, timber and three kinds of channels of wood package materials.This disease is found as far back as the North America, China finds first in the Zhongshan Tomb, Nanjing nineteen eighty-two, later in Anhui, also popular causing disaster take place in some areas of province such as Shandong, Zhejiang, Guangdong, nearly 1,300,000 mu of the woods area of being injured now, 2,500,000,000 yuan of direct economic losses, indirect loss is up to 25,000,000,000 yuan, and this disease now directly threatens the pine forest resource of more than 500,000,000 mu in China.
Prophylactico-therapeutic measures to pine wood nematode mainly is to completely cut off by quarantine, and trees or wood materials once finding to have infected pine wood nematode will burn destruction to it immediately, therefore works out pine wood nematode accurate detection method most important.Present detection means mainly is to judge according to the morphological feature of pine wood nematode.Pine wood nematode belongs to umbrella Aphelenchoides (Bursaphelenchus), kind surplus the existing report of this genus has 60, and wherein pine wood nematode is very similar to the form of intending pine wood nematode, especially more is difficult to distinguish in larval stage.In the Asia (especially China and Japan), intending pine wood nematode is dominant population, but its pathogenic a little less than, be difficult to the two is differentiated by morphologic observation merely.People such as Tares (1993) and Harmey (1994) study microsatellite DNA and the nuclear DNA of pine wood nematode, and people such as Beckenbach (1992) and Harmey (1993) utilize PCR and dna fingerprinting technology to carry out researchs such as the detection of pine wood nematode, evaluation.But common molecular Biological Detection method generally all needs electrophoresis or enzyme operation such as to cut, and detection speed is slow (at least 6 hours).
Real-time fluorescence quantitative PCR (Realtime fluorence quantative PCR) technology is released in 1996 by U.S. AppliedBiosystems company, be meant in the PCR reaction system and add fluorophor, utilize the fluorescence signal accumulation whole PCR process of monitoring in real time, the method for by typical curve unknown template being carried out quantitative test at last.The employed fluorescence chemical of real-time fluorescence quantitative PCR can be divided into two kinds: TaqMan fluorescence probe and SYBR fluorescent dye.Wherein, add a specific fluorescence probe when TaqMan fluorescence probe is meant pcr amplification when adding a pair of primer, this probe is an oligonucleotides, and two ends are mark report fluorophor and a cancellation fluorophor respectively.When probe was complete, the reporter group fluorescent signal emitted was absorbed by quenching group; During pcr amplification, 5 '-3 ' 5 prime excision enzyme activity of Taq enzyme is cut degraded with the probe enzyme, the report fluorophor is separated with the cancellation fluorophor, thereby the fluorescence monitoring system can receive fluorescence signal, it is DNA chain of every amplification, just there is a fluorescence molecule to form, realized that the accumulation of fluorescence signal and PCR product form fully synchronously.This technology has realized the leap of PCR from qualitative to quantitative, it has, and specificity is stronger, pollution-free, the automaticity advantages of higher, now be applied in the detection to some important quarantine objects, especially to the detection of virus, as apple chlorotic leaf spot virus, the tomato spot virus (TSWV) etc. of here withering; In addition, with this method bacterial detection (as Escherichia coli) and fungi (as sickle-like bacteria) reported in literature is also arranged.
Summary of the invention
The purpose of this invention is to provide a kind of primer special and probe that detects pine wood nematode.
The primer special of detection pine wood nematode provided by the present invention is made up of forward primer and reverse primer, forward primer is that 3 ' end sequence is the oligonucleotide sequence of 16-30 the nucleotide of 3 '-TCAGT, and reverse primer is that 3 ' end sequence is the oligonucleotide sequence of 16-30 the nucleotide of 3 '-CCAAG; Probe is that 3 ' end sequence is the oligonucleotide sequence of 16-25 the nucleotide of 3 '-GGTAGTACGCGCC.
3 ' end of described probe also is marked with a cancellation fluorophor (TAMRA), and 5 ' end is marked with a report fluorophor (FAM).
Described forward primer is preferably the nucleotide sequence with sequence 1 in the sequence table, and described reverse primer is preferably the nucleotide sequence with sequence 2 in the sequence table.Described probe has the nucleotide sequence of sequence 3 in the sequence table.
Second purpose of the present invention provides a kind of method that can detect pine wood nematode quickly and accurately.
The method of detection provided by the present invention pine wood nematode is that the genomic DNA with the nematode sample is a template, carries out real-time fluorescence quantitative PCR, detection amplified production fluorescence signal with primer and the probe of above-mentioned detection pine wood nematode.
Described nematode sample can be with pure sample product of a kind of nematode or the biased sample of multiple nematode, and its worm attitude can be larva and/or worm attitudes such as adult and/or ovum.
In the said method, as from amplified production, detecting fluorescence signal, illustrate that described test sample is pine wood nematode or contains pine wood nematode, and can determine the amount of the starting template in the sample according to the size of Ct (circulation thresholding).
Described nematode sample is got supernatant through extract and Proteinase K processing, centrifugal, promptly obtains the genomic DNA of described nematode sample; Described extract is 25mM Tris-HCl, 125mM KCl, 0.3mM Mg
2+, 0.25mMDTT, 1.2%Tween-20, pH9-10.
The reaction system of described real-time fluorescence quantitative PCR also contains the PCR premixed liquid except that the genomic DNA that contains described nematode sample: 2.5-3.5mM Mg
2+, 0.2-0.3mM dNTP, each 300-500nM of forward and reverse primer of detection pine wood nematode, probe 150-250nM, Taq archaeal dna polymerase 3-3.5U/50 μ L; The response procedures of described real-time fluorescence quantitative PCR is: 95 ℃ of pre-sex change 3min of elder generation, carry out 40 circulations subsequently: 95 ℃ sex change 45sec, 55-61 ℃ is extended 15sec.
The present invention also provides a kind of kit that detects pine wood nematode.
The kit of detection pine wood nematode provided by the present invention comprises primer and the probe of above-mentioned detection pine wood nematode.
The kit of detection pine wood nematode provided by the present invention also comprises the nematode sample extracting solution, PCR reaction buffer, Mg
2+, dNTP, Taq archaeal dna polymerase.
Described nematode sample extracting solution can be 25mM Tris-HCl, 125mM KCl, 0.3mM Mg
2+, 0.25mMDTT, 1.2%Tween-20, pH9-10.
The detection method that the present invention is directed to existing pine wood nematode is time-consuming, the shortcoming of effort, provides a kind of energy quick, simple and easy and detect method and the kit of pine wood nematode exactly.Method of the present invention and primer special thereof and kit have very strong specialization to pine wood nematode, and by the optimization to reaction system, can not only increase substantially efficient and accuracy that pine wood nematode detects, in addition, can also carry out quantitative test to being subjected to the sample product.Use pine wood nematode detection kit provided by the present invention, normal operations people personnel (but not pine wood nematode expert) just can detect this kind nematode rapidly and accurately.
The present invention has the following advantages: A. is simple fast: only need a spot of polypide DNA, with the fluorescent PCR instrument only need can draw assay in 30 minutes, avoided electrophoresis, enzyme in the common molecular detection technology treatment process such as to cut; B. highly sensitive: collect fluorescence signal automatically by the fluorescent PCR instrument, avoid the interference from human factor in the conventional PCR end point analysis method, further improve sensitivity, probe sensitivity can reach 8pg DNA; C. high specificity: adopting the special primer of a pair of pine wood nematode to carry out the amplification of target gene fragment and one can detect with the fluorescence probe of template (genomic DNA of pine wood nematode) complementary pairing, improve specificity, avoided false positive and false negative effectively; D. interference is polluted low: adopt totally-enclosed reaction system, and online real-time fluorescence monitoring, the PCR product need not carry out gel electrophoresis, can prevent the pollution of PCR product effectively.E. use method of the present invention and can not only detect the pine wood nematode adult, can also detect its larva, solved simple dependence form and be difficult to problem that larva is identified, and testing not necessarily needs expert's intervention, the ordinary person can finish.Method of the present invention also provides new approaches for the detection of other quarantine nematodes.
Embodiment
Method therefor is conventional method if no special instructions among the following embodiment.
Embodiment 1, the primer special that detects pine wood nematode and the design of probe
Detailed process may further comprise the steps:
1, the separation of pine wood nematode
To separate with the graceful funnel method of shellfish from the pine wood nematode (Bursaphelenchus xylophilus) of the different population in Japan, Germany, Italy, the U.S. and Chinese Jiangsu, Shandong, Zhejiang, Anhui, Guangdong and relevant kind.Concrete steps are: 1) pine tree that is injured is cut into segment, rises with 8 layers of bundle, put into the funnel that has sebific duct, add sterilized water in funnel, leave standstill 12-24h, collect nematode; 2) adding 200mL concentration is that the tetracycline salt of 3mg/100ml and the streptomycin sulphate of 0.5g/100ml carry out disinfection to it, again the nematode suspension is placed centrifuge tube, the centrifugal 3min of 2000rpm, abandon supernatant, add 0.9%NaCl solution then, the centrifugal 3min of 2000rpm abandons supernatant, at last with sterilized water again with nematode centrifuge washing 3 times.At last, picking nematode under inverted microscope ,-20 ℃ of preservations are standby.
2, the extraction of pine wood nematode genomic DNA
Wall scroll nematode in the picking step 1 is cut into the 2-3 section, adds the nematode extract and (contains 125mM KCl, 25mMTris-Cl, 0.3mM Mg
2+, 0.25mM DTT, 1.2%Tween-20) and Proteinase K (24 μ g/mL) (Merck), extract the pine wood nematode genomic DNA.
3, the dna fragmentation in pcr amplification pine wood nematode ribosomes ITS district
Select the universal primer in ITS district for use,
Primer 1 (upstream primer): 5 '-CGTAACAAGGTAGCTGTAG-3 '
Primer 2 (downstream primer): 5 '-TTTCACTCGCCGTTACTAAGG-3 '
The nematode gene group DNA that extracts with step 2 is a template, under the guiding of primer 1 and primer 2, and the dna fragmentation (about 900bp) in pcr amplification pine wood nematode ribosomes ITS district, 50 μ L PCR reaction systems are: 5 μ L10 * PCR reaction buffer, 4 μ L MgCl
2(25mM), 4 μ L dNTP (2.5mM), each 4 μ L of primer 1 and primer 2 (10 μ M), 0.4 μ L Taq archaeal dna polymerase (5U/ μ L), 2 μ L template DNAs (the about 0.2ng/ μ of final concentration L) add to 50 μ L with distilled water at last; The PCR reaction conditions is: 94 ℃ of sex change 1min of elder generation, and 57 ℃ of annealing 1min, 72 ℃ are extended 2min, circulate 35 times; 72 ℃ are incubated 10min again.
4, the clone and the order-checking in collected nematode ribosomes ITS district
Concrete steps are: 1) the PCR product with step 3 carries out 1% agarose gel electrophoresis, reclaims the dna fragmentation that kit (QIAquick Gel Extraction Kit, QIAGEN company) reclaims the about 900bp of this length with DNA; 2) with the dna fragmentation and carrier pMD 18-T (Takara) the preparation recombinant vector ITS/pMD 18-T that reclaim; 3) with recombinant vector ITS/pMD18-T CaCl
2Method is transformed into bacillus coli DH 5 alpha, and the reorganization bacterium is coated with on the LB resistant panel of 20mg/mlX-gal and 0.8M IPTG, cultivates 12-24h for 37 ℃ and carries out blue hickie screening; 4) the single colony inoculation of picking white is in the LB fluid nutrient medium, 37 ℃ cultivate 12-24h after, plasmid directly send company's check order (going up the sea base health).
5, detect the primer special of pine wood nematode and the design and the screening of probe
With Clustalx1.8.1 software the sequencing result in the above-mentioned steps 4 is carried out sequential analysis, according to the rule of primer and probe design,, finally determine a pair of primer and a probe again by screening with primer express software design probe and primer.Wherein upstream primer (forward primer) binding site is between pine wood nematode ITS1 district 30-60bp, and its sequence is 5 '-GATGATGCGATTGGTGACT-3 ' (sequence 1), primer length 19nt; Downstream primer (reverse primer) binding site is between ITS1 district 70-100bp, and its sequence is 5 '-AACGACGCGAATCGAACC-3 ' (sequence 2), primer length 18nt; The probe sequence of determining is 5 ' FAM-CGGTTGCCGCGCATGATGG-TAMRA 3 ' (sequence 3), and probe length is 19nt.
The optimization of embodiment 2, real-time fluorescence quantitative PCR detection architecture
The pine wood nematode genomic DNA that obtains with step 2 among the embodiment 1 is a template, respectively the magnesium ion concentration in the real-time fluorescence quantitative PCR detection architecture, dNTP concentration, Taq archaeal dna polymerase concentration, the primer that detects pine wood nematode and the concentration of probe and the elongating temperature in the quantitative fluorescent PCR cycling condition are optimized, concrete steps are as follows:
One, the optimization of magnesium ion concentration
By following real-time fluorescence quantitative PCR reaction system and cycling condition magnesium ion concentration is optimized, 50 μ L reaction systems are: 5 μ L, 10 * PCR reaction buffer, (selective body is long-pending to be respectively 2 to magnesium ion (25mM), 3,4,5,6,7,8,9 μ L), 4 μ L dNTP (2.5mM), 4 μ L detect the forward primer (sequence 1) (10 μ M) of pine wood nematode, 4 μ L detect the reverse primer (sequence 2) (10 μ M) of pine wood nematode, 2 μ L probes (sequence 3) (10 μ M), 0.4 μ L Taq archaeal dna polymerase (5U/ μ L), 2 μ L template DNAs (the about 0.2ng/ μ of final concentration L) add to 50 μ L with distilled water at last; Reaction conditions is: phase one 95 ℃ of 3min; 95 ℃ of 45s of subordinate phase, 60 ℃ of 15s, 40 circulations.The result shows that the best final concentration of magnesium ion is 3mM.
Two, the optimization of Taq archaeal dna polymerase concentration
By following real-time fluorescence quantitative PCR reaction system and cycling condition Taq archaeal dna polymerase concentration is optimized, 50 μ L reaction systems are: 5 μ L, 10 * PCR reaction buffer, 4 μ L MgCl
2(25mM), 4 μ L dNTP (2.5mM), the forward primer (sequence 1) that detects pine wood nematode, each 4 μ L (10 μ M) of reverse primer (sequence 2), 2 μ L probes (sequence 3) (10 μ M), 2 μ L template DNAs (the about 0.2ng/ μ of final concentration L), Taq archaeal dna polymerase (5U/ μ L) (selective body amasss and is respectively 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8 μ L), add to 50 μ L with distilled water at last; Reaction conditions is: phase one 95 ℃ of 3min; 95 ℃ of 45s of subordinate phase, 60 ℃ of 15s, 40 circulations.The result shows that Taq archaeal dna polymerase optium concentration is 3U/50 μ L.
Three, the optimization of dNTP concentration
By following real-time fluorescence quantitative PCR reaction system and cycling condition dNTP concentration is optimized, 50 μ L reaction systems are: 5 μ L, 10 * PCR reaction buffer, 4 μ L MgCl
2(25mM), dNTP (2.5mM) (select add volume be respectively 0.4,1,2,4,6,8,10 μ L), the forward primer (sequence 1) that detects pine wood nematode, each 4 μ L (10 μ M) of reverse primer (sequence 2), 2 μ L probes (sequence 3) (10 μ M), 2 μ L template DNAs (the about 0.2ng/ μ of final concentration L), 0.4 μ L Taq archaeal dna polymerase (5U/ μ L), add to 50 μ L with distilled water at last; Reaction conditions is: phase one 95 ℃ of 3min; 95 ℃ of 45s of subordinate phase, 60 ℃ of 15s, 40 circulations.The result shows that the dNTP optium concentration is 0.2mM.
Four, detect the optimization of the primer concentration of pine wood nematode
By following real-time fluorescence quantitative PCR reaction system and cycling condition primer concentration is optimized, 50 μ L reaction systems are: 5 μ L, 10 * PCR reaction buffer, 4 μ L MgCl
2(25mM), 4 μ L dNTP (2.5mM), the forward primer (sequence 1) (10 μ M) that detects pine wood nematode, reverse primer (sequence 2) (10 μ M) (add volume and be respectively 0.25,0.5,1,1.5,2,2.5,3,3.5,4 μ L), 2 μ L probes (sequence 3) (10 μ M), 0.4 μ L Taq archaeal dna polymerase (5U/ μ L), 2 μ L template DNAs (the about 0.2ng/ μ of final concentration L), add to 50 μ L with distilled water at last; Reaction conditions is: phase one 95 ℃ of 3min; 95 ℃ of 45s of subordinate phase, 60 ℃ of 15s, 40 circulations.The result shows that the optium concentration of forward, reverse primer is 400nM.
Five, detect the optimization of the concentration and probe concentration of pine wood nematode
By following real-time fluorescence quantitative PCR reaction system and cycling condition concentration and probe concentration is optimized, 50 μ L reaction systems are: 5 μ L, 10 * PCR reaction buffer, 4 μ L MgCl
2(25mM), 4 μ L dNTP (2.5mM), the forward primer (sequence 1) that detects pine wood nematode, each 4 μ L (10 μ M) of reverse primer (sequence 2), 0.4 μ L TaqDNA polymerase (5U/ μ L), probe (sequence 3) (10 μ M) (volume is respectively 0.125,0.25,0.5,0.75,1,1.25,1.5,1.75,2 μ L), 2 μ L template DNAs (the about 0.2ng/ μ of final concentration L) add to 50 μ L with distilled water at last; Reaction conditions is: phase one 95 ℃ of 3min; 95 ℃ of 45s of subordinate phase, 60 ℃ of 15s, 40 circulations.The result shows that the probe optium concentration is 200nM.
Six, the optimization of elongating temperature in the real-time fluorescence quantitative PCR cycling condition
By following real-time fluorescence quantitative PCR reaction system and cycling condition elongating temperature in the real-time fluorescence quantitative PCR cycling condition is optimized, 50 μ L reaction systems are: 5 μ L, 10 * PCR reaction buffer, 4 μ L MgCl
2(25mM), 4 μ L dNTP (2.5mM), the forward primer (sequence 1) that detects pine wood nematode, each 4 μ L (10 μ M) of reverse primer (sequence 2), 2 μ L probes (sequence 3) (10 μ M), 0.4 μ L Taq archaeal dna polymerase (5U/ μ L), 2 μ L template DNAs (the about 0.2ng/ μ of final concentration L), add to 50 μ L with distilled water at last; Reaction conditions is: phase one 95 ℃ of 3min, and 95 ℃ of 45s of subordinate phase extend 15s, 40 circulations, elongating temperature is respectively 55 ℃, 58 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃.The result shows that best elongating temperature is 60 ℃ in the cycling condition.
Wherein, preferred real-time fluorescence PCR detection architecture is: 3mM Mg
2+, 0.2mM dNTP, forward primer (sequence 1), each 400nM of reverse primer (sequence 2) of detection pine wood nematode, probe (sequence 3) 200nM of detection pine wood nematode, Taq archaeal dna polymerase 3U/50 μ L, 60 ℃ of elongating temperatures.
The sensitivity of the probe of embodiment 3, detection pine wood nematode detects
Measure the concentration of the pine wood nematode genomic DNA that extracts among the embodiment 1 with ultraviolet spectrophotometer, and be to contain 800pg, 80pg, 8pg, 0.8pg, 0.08pg, 0.008pg and 0.0008pg nucleic acid among the 50 μ L respectively with its dilution, pine wood nematode genomic DNA with above-mentioned 7 variable concentrations is a template, sensitivity with embodiment 2 preferred real-time fluorescence PCR detection architecture detector probe, reaction conditions is: phase one 95 ℃ of 3min, 95 ℃ of 45s of subordinate phase, 60 ℃ of 15s, 40 circulations.The result shows that this probe can detected minimum dna content be 8pg.
The specialization experiment of embodiment 4, usefulness real-time fluorescence quantitative PCR technology for detection pine wood nematode
Respectively from the pine wood nematode (Bursaphelenchus xylophilus) of ground different populations such as Japan, Germany, Italy, the U.S. and Chinese Jiangsu, Shandong, Zhejiang, Anhui, Guangzhou, intend pine wood nematode (B.mucronatus), B.fraudulentus, B.tusciae, and totally 17 of type species Caenorhabditis elegans, 8 different populations of pine wood nematode wherein, intend 6 different populations of pine wood nematode, each 1 of B.fraudulentus, B.tusciae, 1 of pattern nematode.
By following real-time fluorescence quantitative PCR reaction system and cycling condition elongating temperature in the real-time fluorescence quantitative PCR cycling condition is optimized, 50 μ L reaction systems are: 5 μ L, 10 * PCR reaction buffer, 3mM Mg
2+, 0.2mM dNTP, 2 μ L template DNAs (the about 0.2ng/ μ of final concentration L), the forward primer (sequence 1) that detects pine wood nematode, each 400nM of reverse primer (sequence 2), detect probe (sequence 3) 200nM, the Taq archaeal dna polymerase 3U/50 μ L of pine wood nematode; Reaction conditions is: phase one 95 ℃ of 3min; 95 ℃ of 45s of subordinate phase extend 15s, 40 circulations, and elongating temperature is 60 ℃.8 pine wood nematode populations can both detect, and other nematodes do not have signal.
Sequence table
<160>3
<210>1
<211>19
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
gatgatgcga?ttggtgact 19
<210>2
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
aacgacgcga?atcgaacc 18
<210>3
<211>19
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
cggttgccgc?gcatgatgg 19
Claims (10)
1, a kind of primer special and probe that detects pine wood nematode, described primer special is made up of forward primer and reverse primer, forward primer is that 3 ' end sequence is the oligonucleotide sequence of 16-30 the nucleotide of 3 '-TCAGT, and reverse primer is that 3 ' end sequence is the oligonucleotide sequence of 16-30 the nucleotide of 3 '-CCAAG; Described probe is that 3 ' end sequence is the oligonucleotide sequence of 16-25 the nucleotide of 3 '-GGTAGTACGCGCC.
2, primer special according to claim 1 and probe is characterized in that: 3 ' end of described probe is marked with a cancellation fluorophor, and 5 ' end is marked with a report fluorophor.
3, primer special according to claim 1 and 2 and probe is characterized in that: described forward primer has the nucleotide sequence of sequence 1 in the sequence table, and described reverse primer has the nucleotide sequence of sequence 2 in the sequence table; Described probe has the nucleotide sequence of sequence 3 in the sequence table.
4, a kind of method of utilizing claim 1 or 2 or 3 described primers and probe in detecting pine wood nematode, be that genomic DNA with the nematode sample is a template, primer and probe with described detection pine wood nematode carry out real-time fluorescence quantitative PCR, detect the fluorescence signal of amplified production.
5, method according to claim 4 is characterized in that: described nematode sample is that its worm attitude is adult and/or ovum and/or larva with pure sample product of a kind of nematode or the biased sample of multiple nematode.
6, according to claim 4 or 5 described methods, it is characterized in that: described nematode sample is got supernatant through extract and Proteinase K processing, centrifugal, promptly obtains the genomic DNA of described nematode sample; Described extract is 25mMTris-HCl, 125mM KCl, 0.3mM Mg
2+, 0.25mM DTT, 1.2%Tween-20, pH 9-10.
7, according to claim 4 or 5 described methods, it is characterized in that: the reaction system of described real-time fluorescence quantitative PCR also contains the PCR premixed liquid except that the genomic DNA that contains described nematode sample: 2.5-3.5mM Mg
2+, 0.2-0.3mM dNTP, Taq archaeal dna polymerase 3-3.5U/50 μ L, each 300-500nM of forward and reverse primer of detection pine wood nematode, the probe 150-250nM of detection pine wood nematode.
8, according to claim 4 or 5 described methods, it is characterized in that: the response procedures of described real-time fluorescence quantitative PCR is: 95 ℃ of pre-sex change 3min of elder generation, carry out 40 circulations subsequently: 95 ℃ sex change 45sec, 55-61 ℃ is extended 15sec.
9, a kind of kit that detects pine wood nematode, it comprises claim 1 or 2 or 3 described primer special and probes.
10, kit according to claim 9 is characterized in that: described kit also comprises the nematode sample extracting solution, PCR reaction buffer, Mg
2+, dNTP and Taq archaeal dna polymerase.
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| CN100392100C (en) * | 2005-06-13 | 2008-06-04 | 上海市林业病虫防治检疫站 | Detection kit for pine wood nematode and detection method therefor |
| WO2009136456A1 (en) * | 2008-05-07 | 2009-11-12 | 独立行政法人森林総合研究所 | Dna extraction method for bursaphelenchus xylophilus from wood chips, lamp primer set for bursaphelenchus xylophilus, and detection method for bursaphelenchus xylophilus from wood chips |
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| CN1244703C (en) * | 2003-12-12 | 2006-03-08 | 中国科学院上海生命科学研究院 | Pine wireworm microsatellite amplifying primer, detection method and detection kit |
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| CN100392100C (en) * | 2005-06-13 | 2008-06-04 | 上海市林业病虫防治检疫站 | Detection kit for pine wood nematode and detection method therefor |
| WO2009136456A1 (en) * | 2008-05-07 | 2009-11-12 | 独立行政法人森林総合研究所 | Dna extraction method for bursaphelenchus xylophilus from wood chips, lamp primer set for bursaphelenchus xylophilus, and detection method for bursaphelenchus xylophilus from wood chips |
| JP2009268404A (en) * | 2008-05-07 | 2009-11-19 | Forestry & Forest Products Research Institute | Method for extracting dna of bursaphelenchus xylophilus from wood chip, lamp primer set of bursaphelenchus xylophilus, and method for detecting bursaphelenchus xylophilus from wood chip |
| US8318435B2 (en) | 2008-05-07 | 2012-11-27 | Forestry And Forest Products Research Institute | DNA extraction method for bursaphelenchus xylophilus from wood chips, lamp primer set for bursaphelenchus xylophilus, and detection method for bursaphelenchus xylophilus from wood chips |
| CN101545008B (en) * | 2009-03-09 | 2011-12-07 | 中国检验检疫科学研究院 | Primer and probe for detecting globodera rostochiensis |
| CN101875965B (en) * | 2009-04-28 | 2012-12-26 | 周卫川 | TaqMan probe real-time fluorescence PCR (Polymerase Chain Reaction) detection of angiostrongylus cantonensis infected snails |
| CN106755554A (en) * | 2017-03-21 | 2017-05-31 | 四川农业大学 | Pine wood nematode SSR label primer is used for the purposes of pine wood nematode detection and the PCR detection method of pine wood nematode |
| CN107287316A (en) * | 2017-07-09 | 2017-10-24 | 中国农业科学院植物保护研究所 | The specific SCAR label of beet cyst roundworm and quick SCAR PCR molecular detecting methods |
| CN107287316B (en) * | 2017-07-09 | 2020-08-11 | 中国农业科学院植物保护研究所 | Specific SCAR marker of beet cyst nematode and rapid SCAR-PCR molecular detection method |
| CN108315441A (en) * | 2018-03-29 | 2018-07-24 | 中国林业科学研究院亚热带林业研究所 | Identify the Primer composition of Bursaphelenchus xylophilus and using its product and its application and detection method and its application |
| CN109682786A (en) * | 2019-01-16 | 2019-04-26 | 四川省畜牧科学研究院 | The discrimination method of nematode and its application |
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