CN1793866A - Fast detection method of pine tree wilt disease pathogenic nematode - Google Patents
Fast detection method of pine tree wilt disease pathogenic nematode Download PDFInfo
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Abstract
A method for quickly detecting pathogenic threadworm of pine wilt disease includes designing each pair of specific primer for PCR detection of pine threadworm and pine imitate-threadworm, obtaining single threadworm DNA as template by mechanical grinding and filtering paper absorption, carrying out specific PCR amplification of pine threadworm and pine imitate-threadworm as well as detecting PCR product.
Description
Technical field:
The invention belongs to forest disease detection technique field, the method that pine wood nematode that specifically a kind of energy is right and plan pine wood nematode detect.
Background technology:
The pine tree wilt disease that is caused by pine wood nematode (Bursaphelenchus xylophilus) is a kind of destructive forest disease, have that harm is extremely serious, rate of propagation is fast, prevent and treat characteristics such as difficulty, in case take place and to cause great loss to national economy and ecologic environment, now become the No.1 biological disaster in China's production of forestry, its cause of disease pine wood nematode is the internal and external important quarantine object of China.Owing to lack the counter-measure after pine tree wilt disease takes place preferably, improvement for pine tree wilt disease mainly is by removing the infection sources and cutting off the speed that the routes of infection are controlled its its spread in china at present, specific practice is to cut down to remove sick tree and destroy in the pine tree wilt disease generating region to prevent diffusion, pine tree wilt disease not generating region then carry out internal and external quarantine and prevent to import into.The key problem in technology that above-mentioned two work relate to is the detection of pathogenic nematode in sick diagnosis of setting of woodland and the pine goods.
The most direct for the diagnosis of pine tree wilt disease and quarantine, effective method is the pathogenic nematode kind that identifies fast and accurately in diseased wood or the pine goods, the evaluation of pine wood nematode for a long time mainly relies on morphology, especially to observation female, some feature of male imago.The advantage of this method is easy, directly perceived, the drawback that exists is: 1. numerous nematodes is arranged in the pine material, the nematode morphological feature of some kind is similar to pine wood nematode, the more weak sibling species of the pathogenicity of special pine wood nematode is intended pine wood nematode (Bursaphelenchus mucronatus), its form and pine wood nematode are easy to obscure, and make the accuracy of identifying rely on operator's experience fully; 2. pine wood nematode has the situation of variation to exist on form, needs at least when practical operation that each is observed just more than 10 and can make a definite diagnosis to female, male imago, takes a lot of work, time-consuming; 3. morphology is identified and only to be applicable to adult, is not suitable for larva, and the pine wood nematode in the diseased wood to be form with larva under many circumstances exist, this method that makes is restricted in actual applications..
In recent years along with the development of Protocols in Molecular Biology, adopt round pcr to pine wood nematode carry out fast, accurate evaluation become trend.Pine beam thread insect PCR is identified and is comprised two gordian techniquies, i.e. pcr amplification technology and nematode DNA technology of preparing.After identifying and comprise the RAPD-PCR technology that adopts the random primer amplification and adopt the amplification of nematode universal primer, early stage PCR again amplified production is carried out the PCR-RFLP technology of restriction enzyme digestion, the preparation of nematode DNA adopts phenol to imitate extraction process more, their common drawback is owing to the special primer that uses non-pine wood nematode special use causes the stability of evaluation and accuracy not high, and needing a large amount of nematodes to prepare DNA, preparation time is long." the pine wood nematode detection kit and the detection method thereof " of inventions such as the Lin Maosong (patent No.: the ZL2003l0106109.5) new method that provides a kind of pine tree wilt disease to diagnose, this method adopts three special primers that the wall scroll nematode that separates from the pine tree diseased wood is carried out the PCR detection, evaluation accuracy rate height, after obtaining the nematode parting liquid, can finish evaluation in 8 hours, improve the diagnosis efficiency of pine tree wilt disease greatly.But owing to adopted the protease digestion method to extract the DNA of wall scroll nematode, make extraction time prolong, easily lose in transfer process at the nematode sample simultaneously, increased the difficulty of operation.Adopt the real-time fluorescence PCR that adds fluorescence probe in the PCR reaction to take turns circulation time and detect fluorescence signal intensity, and needn't wait until and to detect amplification after the PCR reaction is all over, therefore can shorten the time of pcr amplification greatly at each.The pine wood nematode real-time fluorescence PCR detection technique that Cao etc. (Phytopathology 200595:566-571) set up can foreshorten to the detection time behind the acquisition nematode DNA sample in 1 hour.But finishing of this method must rely on cost an arm and a leg fluorescent real time PCR instrument, checkout equipment and reagent, thereby its application is restricted.
Summary of the invention:
The objective of the invention is to overcome the deficiencies in the prior art, provide a kind of quick, accurate, and the PCR detection method of economical and practical pine tree wilt disease pathogenic nematode.
The present invention includes following steps:
(1) the rDNA sequences Design according to pine wood nematode and plan pine wood nematode is used for a pair of primer BX1:5 '-CGATGATGCGATTGGTGACTT-3 ' and the BX2:5 '-ATGCGAGACGACTGTCACAACG-3 ' that pine beam thread insect PCR detects, and is used to intend a pair of primer BM1:5 '-TTCTCAAGTTTCTGCATTTGTAAG-3 ' and the BM2:5 '-GGCGCGGTCGCACAATCGAG-3 ' that pine beam thread insect PCR detects.
(2) be used for the wall scroll nematode dna profiling preparation that PCR detects: one through autoclaving and be cut into drip on the square quantitative filter paper of 2～4mm 2 μ l contain 1.25mM dNTP and each 15 μ M special primer BX1, BX2, BM1 and BM2 and nematode detect liquid, dry stand-by; After cleaning during the distilled water that nematode of picking goes into to drop in advance microslide from the nematode parting liquid under anatomical lens drips, move to outside the water droplet, when treating nematode polypide water on every side near bone dry, getting a dry filter paper that contains the nematode detectable with tweezers is covered on the nematode polypide, tweezers flicking filter paper crushes nematode, filter paper is moved into 95 ℃ of heating 10min in the 0.2ml thin walled tube that 15 μ l distilled waters are housed, be cooled to room temperature, the centrifugal 30s of 8000rpm obtains wall scroll nematode dna profiling and prepares liquid.
(3) pcr amplification: the wall scroll nematode dna profiling that obtains in step (2) prepares and adds 2.5 μ l in the liquid and contain 1.5～2.0mM MgCl
210 * PCR buffer, 1U Taq enzyme is mended distilled water to 25 μ l; Place in the PCR instrument and increase, the amplification condition through optimizing is: pre-94 ℃ of 2min of sex change; 30 circulations, each circulation comprises: 94 ℃ of 45s, 54～55 ℃ of 60s, 72 ℃ of 60s; At last again 72 ℃ extend 10min.
(4) the PCR product detects: get pcr amplification product that 5～10 μ l steps (3) obtain and carry out 1.5% agarose gel electrophoresis, Ago-Gel finishes through observing under ultraviolet transilluminator behind the ethidium bromide staining at electrophoresis, as the electrophoretic band that to detect a molecular weight be 501bp shows that being examined nematode is pine wood nematode, as the electrophoretic band that to detect a molecular weight be 160bp shows and examined nematode for intending pine wood nematode.
The present invention compared with prior art, its advantage and good effect show:
1. the present invention has adopted the method that Mechanical Crushing and filter paper directly adsorb to prepare the wall scroll nematode dna profiling that is used for pcr amplification, need not the imitative extracting of protease digestion and phenol, the preparation time of nematode DNA has shortened within the 15min, finish in 4 hours thereby make from obtaining the whole qualification process that the nematode parting liquid begins, be convenient to after simultaneously under anatomical lens, being adsorbed in nematode DNA on the filter paper observe, be difficult for taking place the situation that nematode DNA loses in transfer, make that operation is more convenient.
2. the present invention is used for the primer special of primer for designing according to pine wood nematode and the distinguished sequence of intending pine wood nematode rDNA district that PCR detects, the good stability of evaluation, accuracy height.
3. the present invention adopts regular-PCR instrument and conventional reagent, detects cost, is beneficial to and applies.
4. that detects is highly sensitive, and the DNA of wall scroll nematode preparation is enough to finish detection, can detect adult and larva.
Description of drawings
The amplification of Fig. 1 pine wood nematode population adult and larva
Fig. 2 intends the amplification of pine wood nematode population adult and larva.
Fig. 3 is to the amplification of the pine wood nematode and the plan pine wood nematode population of separate sources
Embodiment
Embodiment one
1. be used for the design of pine wood nematode and the special primer of intending the pine wood nematode detection: adopt 2 pine wood nematodes and 5 plan pine wood nematode populations of nematode ITS universal primer amplification separate sources, obtain to comprise ITS1, the 5.8S of rDNA, the amplified fragments in ITS2 district; Two deoxidation cessation method are measured the dna sequence dna of each amplified fragments; The sequence information of gained DNASTAR 5.0 software analysis, find out the diversity sequence between pine wood nematode and plan pine wood nematode population, utilize special primer BX1:5 '-CGATGATGCGATTGGTGACTT-3 ' and the BX25 '-ATGCGAGACGACTGTCACAACG-3 ' of primer Premier 5.0 software design pine wood nematodes according to diversity sequence, intend special primer BM1:5 '-TTCTCAAGTTTCTGCATTTGTAAG-3 ' and the BM25 '-GGCGCGGTCGCACAATCGAG-3 ' of pine wood nematode, wherein BX1 is positioned the ITS1 district of rDNA, BX2 is positioned at the ITS2 district of rDNA, and the expection amplified fragments is 501bp; BM1 and BM2 are positioned the ITS2 district of rDNA, and the expection amplified fragments is 160bp; Dna synthesizer synthesizes above-mentioned 4 oligonucleotide primers.
2. be used for the dna profiling preparation that wall scroll nematode PCR identifies: one through autoclaving and be cut into and drip the nematode that 2 μ l contain special primer BX1, BX2, M1 and the BM2 of 1.25mM dNTP and each 15 μ M on the square quantitative filter paper of 4mm and detect liquid, dry stand-by; After cleaning during the distilled water that typical adult of picking goes into to drop in advance microslide from the pine wood nematode parting liquid that derives from Guangdong under anatomical lens drips, move to outside the water droplet, treat that nematode polypide water on every side is near bone dry; Getting the dry filter paper that contains the nematode detectable with tweezers is covered on the nematode polypide, tweezers flicking filter paper crushes nematode, filter paper is moved into 95 ℃ of heating 10min in the 0.2ml thin walled tube that 15 μ l distilled waters are housed, be cooled to room temperature, the centrifugal 30s of 8000rpm obtains wall scroll nematode dna profiling and prepares liquid.
3. the PCR specific amplified of pathogenic nematode: prepare at wall scroll nematode dna profiling and to add 2.5 μ l in the liquid and contain 1.5mM MgCl
210 * PCR buffer, 1U Taq enzyme is mended distilled water to 25 μ l; Place in the PCR instrument and increase, amplification condition is: pre-94 ℃ of 2min of sex change; 30 circulations, each circulation comprises: 94 ℃ of 45s, 55 ℃ of 60s, 72 ℃ of 60s; At last again 72 ℃ extend 10min.
4.PCR product detects; The pcr amplification product of getting 5 μ l adds 2 μ l, 6 * electrophoresis sample-loading buffer, carries out 1.5% agarose gel electrophoresis; Electrophoresis finishes the back gel piece to be observed under ultraviolet transilluminator after with ethidium bromide staining, and the electrophoretic band that to detect a molecular weight be 501bp shows that being examined nematode is pine wood nematode (Fig. 1, Fig. 3).
Embodiment two
1. be used for the design of pine wood nematode and the special primer of intending the pine wood nematode detection: adopt 2 pine wood nematodes and 5 plan pine wood nematode populations of nematode ITS universal primer amplification separate sources, obtain to comprise ITS1, the 5.8S of rDNA, the amplified fragments in ITS2 district; Two deoxidation cessation method are measured the dna sequence dna of each amplified fragments; The sequence information of gained DNASTAR 5.0 software analysis, find out the diversity sequence between pine wood nematode and plan pine wood nematode population, utilize special primer BX1:5 '-CGATGATGCGATTGGTGACTT-3 ' and the BX25 '-ATGCGAGACGACTGTCACAACG-3 ' of primer Premier 5.0 software design pine wood nematodes according to diversity sequence, intend special primer BM1:5 '-TTCTCAAGTTTCTGCATTTGTAAG-3 ' and the BM25 '-GGCGCGGTCGCACAATCGAG-3 ' of pine wood nematode, wherein BX1 is positioned the ITS1 district of rDNA, BX2 is positioned at the ITS2 district of rDNA, and the expection amplified fragments is 501bp; BM1 and BM2 are positioned the ITS2 district of rDNA, and the expection amplified fragments is 160bp; Dna synthesizer synthesizes above-mentioned 4 oligonucleotide primers.
2. be used for the dna profiling preparation that wall scroll nematode PCR identifies: one through autoclaving and be cut into and drip the nematode that 2 μ l contain special primer BX1, BX2, M1 and the BM2 of 1.25mM dNTP and each 15 μ M on the square quantitative filter paper of 2mm and detect liquid, dry stand-by; After cleaning during the distilled water that typical larva of picking goes into to drop in advance microslide from the pine wood nematode parting liquid that derives from Jiangsu under anatomical lens drips, move to outside the water droplet, treat that nematode polypide water on every side is near bone dry; Getting the dry filter paper that contains the nematode detectable with tweezers is covered on the nematode polypide, tweezers flicking filter paper crushes nematode, filter paper is moved into 95 ℃ of heating 10min in the 0.2ml thin walled tube that 15 μ l distilled waters are housed, be cooled to room temperature, the centrifugal 30s of 8000rpm obtains wall scroll nematode dna profiling and prepares liquid.
3. the PCR specific amplified of pathogenic nematode: prepare at wall scroll nematode dna profiling and to add 2.5 μ l in the liquid and contain 2.0mM MgCl
210 * PCR buffer, 1U Taq enzyme is mended distilled water to 25 μ l; Place in the PCR instrument and increase, amplification condition is: pre-94 ℃ of 2min of sex change; 30 circulations, each circulation comprises: 94 ℃ of 45s, 54 ℃ of 60s, 72 ℃ of 60s; At last again 72 ℃ extend 10min.
4.PCR product detects: the pcr amplification product of getting 10 μ l adds 2 μ l, 6 * electrophoresis sample-loading buffer, carries out 1.5% agarose gel electrophoresis; Electrophoresis finishes the back gel piece to be observed under ultraviolet transilluminator after with ethidium bromide staining, and the electrophoretic band that to detect a molecular weight be 501bp shows that being examined nematode is pine wood nematode (Fig. 1, Fig. 3).
Embodiment three
1. be used for the design of pine wood nematode and the special primer of intending the pine wood nematode detection: adopt 2 pine wood nematodes and 5 plan pine wood nematode populations of nematode ITS universal primer amplification separate sources, obtain to comprise ITS1, the 5.8S of rDNA, the amplified fragments in ITS2 district; Two deoxidation cessation method are measured the dna sequence dna of each amplified fragments; The sequence information of gained DNASTAR 5.0 software analysis, find out the diversity sequence between pine wood nematode and plan pine wood nematode population, utilize special primer BX1:5 '-CGATGATGCGATTGGTGACTT-3 ' and the BX25 '-ATGCGAGACGACTGTCACAACG-3 ' of primer Premier 5.0 software design pine wood nematodes according to diversity sequence, intend special primer BM1:5 '-TTCTCAAGTTTCTGCATTTGTAAG-3 ' and the BM2:5 '-GGCGCGGTCGCACAATCGAG-3 ' of pine wood nematode, wherein BX1 is positioned the ITS1 district of rDNA, BX2 is positioned at the ITS2 district of rDNA, and the expection amplified fragments is 501bp; BM1 and BM2 are positioned the ITS2 district of rDNA, and the expection amplified fragments is 160bp; Dna synthesizer synthesizes above-mentioned 4 oligonucleotide primers.
2. be used for the dna profiling preparation that wall scroll nematode PCR identifies: one through autoclaving and be cut into and drip the nematode that 2 μ l contain special primer BX1, BX2, M1 and the BM2 of 1.25mM dNTP and each 15 μ M on the square quantitative filter paper of 4mm and detect liquid, dry stand-by; After cleaning during the distilled water that typical adult of picking goes into to drop in advance microslide from the plan pine wood nematode parting liquid that derives from Yunnan under anatomical lens drips, move to outside the water droplet, treat that nematode polypide water on every side is near bone dry; Getting the dry filter paper that contains the nematode detectable with tweezers is covered on the nematode polypide, tweezers flicking filter paper crushes nematode, filter paper is moved into 95 ℃ of heating 10min in the 0.2ml thin walled tube that 15 μ l distilled waters are housed, be cooled to room temperature, the centrifugal 30s of 8000rpm obtains wall scroll nematode dna profiling and prepares liquid.
3. the PCR specific amplified of pathogenic nematode: prepare at wall scroll nematode dna profiling and to add 2.5 μ l in the liquid and contain 1.5mM MgCl
210 * PCR buffer, 1U Taq enzyme is mended distilled water to 25 μ l; Place in the PCR instrument and increase, amplification condition is: pre-94 ℃ of 2min of sex change; 30 circulations, each circulation comprises: 94 ℃ of 45s, 54 ℃ of 60s, 72 ℃ of 60s; At last again 72 ℃ extend 10min.
4.PCR product detects: the pcr amplification product of getting 5 μ l adds 2 μ l, 6 * electrophoresis sample-loading buffer, carries out 1.5% agarose gel electrophoresis; Electrophoresis finishes the back gel piece to be observed under ultraviolet transilluminator after with ethidium bromide staining, and the electrophoretic band that to detect a molecular weight be 160bp shows and examined nematode for intending pine wood nematode (Fig. 2, Fig. 3).
Embodiment four
1. be used for the design of pine wood nematode and the special primer of intending the pine wood nematode detection: adopt 2 pine wood nematodes and 5 plan pine wood nematode populations of nematode ITS universal primer amplification separate sources, obtain to comprise ITS1, the 5.8S of rDNA, the amplified fragments in ITS2 district; Two deoxidation cessation method are measured the dna sequence dna of each amplified fragments; The sequence information of gained DNASTAR 5.0 software analysis, find out the diversity sequence between pine wood nematode and plan pine wood nematode population, utilize special primer BX1:5 '-CGATGATGCGATTGGTGACTT-3 ' and the BX25 '-ATGCGAGACGACTGTCACAACG-3 ' of primer Premier 5.0 software design pine wood nematodes according to diversity sequence, intend special primer BM1:5 '-TTCTCAAGTTTCTGCATTTGTAAG-3 ' and the BM25 '-GGCGCGGTCGCACAATCGAG-3 ' of pine wood nematode, wherein BX1 is positioned the ITS1 district of rDNA, BX2 is positioned at the ITS2 district of rDNA, and the expection amplified fragments is 501bp; BM1 and BM2 are positioned the ITS2 district of rDNA, and the expection amplified fragments is 160bp; Dna synthesizer synthesizes above-mentioned 4 oligonucleotide primers.
2. be used for the dna profiling preparation that wall scroll nematode PCR identifies: one through autoclaving and be cut into and drip the nematode that 2 μ l contain special primer BX1, BX2, M1 and the BM2 of 1.25mM dNTP and each 15 μ M on the square quantitative filter paper of 2mm and detect liquid, dry stand-by; After cleaning during the distilled water that typical larva of picking goes into to drop in advance microslide from the plan pine wood nematode parting liquid that derives from the Hunan under anatomical lens drips, move to outside the water droplet, treat that nematode polypide water on every side is near bone dry; Getting the dry filter paper that contains the nematode detectable with tweezers is covered on the nematode polypide, tweezers flicking filter paper crushes nematode, filter paper is moved into 95 ℃ of heating 10min in the 0.2ml thin walled tube that 15 μ l distilled waters are housed, be cooled to room temperature, the centrifugal 30s of 8000rpm obtains wall scroll nematode dna profiling and prepares liquid.
3. the PCR specific amplified of pathogenic nematode: prepare at wall scroll nematode dna profiling and to add 2.5 μ l in the liquid and contain 2.0mM MgCl
210 * PCR buffer, 1U Taq enzyme is mended distilled water to 25 μ l; Place in the PCR instrument and increase, amplification condition is: pre-94 ℃ of 2min of sex change; 30 circulations, each circulation comprises: 94 ℃ of 45s, 55 ℃ of 60s, 72 ℃ of 60s; At last again 72 ℃ extend 10min.
4.PCR product detects: the pcr amplification product of getting 10 μ l adds 2 μ l, 6 * electrophoresis sample-loading buffer, carries out 1.5% agarose gel electrophoresis; Electrophoresis finishes the back gel piece to be observed under ultraviolet transilluminator after with ethidium bromide staining, and the electrophoretic band that to detect a molecular weight be 160bp shows and examined nematode for intending pine wood nematode (Fig. 2, Fig. 3).
Claims (3)
1. the method for quick of a pine tree wilt disease pathogenic nematode may further comprise the steps:
(1) the rDNA sequences Design according to pine wood nematode is used for a pair of primer that pine beam thread insect PCR detects:
BX1：5’-CGATGATGCGATTGGTGACTT-3’
BX2：5’-ATGCGAGACGACTGTCACAACG-3’
Be used to intend a pair of primer that pine beam thread insect PCR detects according to the rDNA sequences Design of intending pine wood nematode:
BM1：5’-TTCTCAAGTTTCTGCATTTGTAAG-3’
BM2：5’-GGCGCGGTCGCACAATCGAG-3’
(2) be used for the wall scroll nematode dna profiling preparation that PCR detects: after cleaning the distilled water of going into to drop in advance microslide from nematode of nematode parting liquid picking under anatomical lens drips, move to outside the water droplet, when treating nematode polypide water on every side near bone dry, getting one with tweezers drips in advance and has 2 μ l to contain the special primer BX1 of 1.25mM dNTP and each 15 μ M, BX2, the nematode detectable of BM1 and BM2 and dry filter paper are covered on the nematode polypide, tweezers flicking filter paper crushes nematode, filter paper is moved into 95 ℃ of heating 10min in the 0.2ml thin walled tube that 15 μ l distilled waters are housed, be cooled to room temperature, the centrifugal 30s of 8000rpm obtains wall scroll nematode dna profiling and prepares liquid
(3) pcr amplification: the wall scroll nematode dna profiling that obtains in step (2) prepares and adds 2.5 μ l in the liquid and contain 1.5～2.0mM MgCl
210 * PCR buffer, 1U Taq enzyme is mended distilled water to 25 μ l; Place in the PCR instrument and increase
(4) the PCR product detects: get pcr amplification product that 5～10 μ l steps (3) obtain and carry out 1.5% agarose gel electrophoresis, Ago-Gel finishes through observing under ultraviolet transilluminator behind the ethidium bromide staining at electrophoresis, as the electrophoretic band that to detect a molecular weight be 501bp shows that being examined nematode is pine wood nematode, as the electrophoretic band that to detect a molecular weight be 160bp shows and examined nematode for intending pine wood nematode.
2. the method for quick of pine tree wilt disease pathogenic nematode according to claim 1 is characterized in that the filter paper of the wall scroll nematode dna profiling preparation that the described PCR of being used for detects is to handle through autoclaving, and is cut into the square quantitative filter paper of the length of side 2～4mm.
3. the method for quick of pine tree wilt disease pathogenic nematode according to claim 1 is characterized in that described pcr amplification condition is: pre-94 ℃ of 2min of sex change; 30 circulations, each circulation comprises: 94 ℃ of 45s, 54～55 ℃ of 60s, 72 ℃ of 60s; At last again 72 ℃ extend 10min.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100429513C (en) * | 2006-07-31 | 2008-10-29 | 西北农林科技大学 | Wheat stripe rust bacteria molecalar detecting method |
| 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 |
| CN101849534A (en) * | 2010-06-22 | 2010-10-06 | 中国科学院东北地理与农业生态研究所 | Separation and detection device of entomopathogenic nematode, separation method of entomopathogenic nematode in soil and measuring method of population density |
| CN105675375A (en) * | 2016-01-13 | 2016-06-15 | 华南农业大学 | Separating medium of inactivated nematode in dead masson pine wood and separation method and kit |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5741657A (en) * | 1995-03-20 | 1998-04-21 | The Regents Of The University Of California | Fluorogenic substrates for β-lactamase and methods of use |
| JP2005073609A (en) * | 2003-09-01 | 2005-03-24 | Shimadzu Corp | Method for amplifying template dna and method for synthesizing cell-free protein by using amplified template dna |
-
2005
- 2005-12-22 CN CNB2005100487225A patent/CN100487435C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100429513C (en) * | 2006-07-31 | 2008-10-29 | 西北农林科技大学 | Wheat stripe rust bacteria molecalar detecting method |
| 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 |
| 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 |
| CN101849534A (en) * | 2010-06-22 | 2010-10-06 | 中国科学院东北地理与农业生态研究所 | Separation and detection device of entomopathogenic nematode, separation method of entomopathogenic nematode in soil and measuring method of population density |
| CN105675375A (en) * | 2016-01-13 | 2016-06-15 | 华南农业大学 | Separating medium of inactivated nematode in dead masson pine wood and separation method and kit |
| CN105675375B (en) * | 2016-01-13 | 2018-09-07 | 华南农业大学 | The separating liquid of inactivation nematode and its separation method and kit in masson pine Deceased wood |
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| CN100487435C (en) | 2009-05-13 |
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