CN102703582A - Method for direct rapid detection and identification of banana-root nematode in soil or other media - Google Patents
Method for direct rapid detection and identification of banana-root nematode in soil or other media Download PDFInfo
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- CN102703582A CN102703582A CN2012100720111A CN201210072011A CN102703582A CN 102703582 A CN102703582 A CN 102703582A CN 2012100720111 A CN2012100720111 A CN 2012100720111A CN 201210072011 A CN201210072011 A CN 201210072011A CN 102703582 A CN102703582 A CN 102703582A
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Abstract
The invention discloses a method for direct rapid detection and identification of banana-root nematode in soil or other media. The method includes extracting DNA (deoxyribonucleic acid) in a sample to be detected, using a Wizard DNA clean-up systems kit of Promega to purify the DNA, using the purified DNA as a template, using SEQs ID NO.1 and NO.2 as primer for PCR (polymerase chain reaction) amplification, and confirming presence of the banana-root nematode in the sample to be detected if an objective band 518bp is obtained by amplification. The method is applicable to detection of banana culture soil or other media, detection results are accurate, sensitivity is high, and the method is widely applicable and has broad application prospect.
Description
Technical field
The present invention relates to molecular Biological Detection authenticate technology field, be specifically related to a kind of directly method of rapid detection evaluation radopholus similes thorne from soil or other matrix.
Background technology
Radopholus similes thorne (
Radopholus similis(Cobb) be a kind of transport property plant endoparasitism nematode Thorne, 1949), the host plant of having reported surpasses 250 kinds (O ' Bannon, 1977), the banana of seriously causing harm (
MusaSpp.), oranges and tangerines (
CitrusSpp.), pepper (
Piper nigrum), ginger (
Zingier officinale) etc. diversified economy plant and ornamental plant (Luc et al, 1990; Richardson et al, 1993; Uchida et al, 2003; Sundararaju et al; 1979); Radopholus similes thorne is distributed widely in the torrid zone and the geographic greenhouse in semi-tropical most of banana producing region and temperate zone; Having reported that the countries and regions that this nematode distributes surpass 90 (Xie Hui, 2006), is the dangerous plant harmful organism of important quarantine (the Cotton J et al of many countries; OEPP/EPPO, 2008), also be that China forbids the Plant Quarantine property harmful organism (The Ministry of Agriculture of the People's Republic of China, MOA, 2007) that enters the territory.Radopholus similes thorne be the earliest by U.S. nematologist Cobb 1891 from pick up from the Fiji banana (
MusaSp.) root is found; Since 19th century along with planting materials such as banana bulb and any of several broadleaf plants seedling are propagated between the banana planting countries and regions; The sixties in 20th century, this nematode propagated in the greenhouse of European countries such as France, Belgium along with ornamental plant; All there are this nematode distribution (Gowen et al, 1990 in most of in the world banana plantings district to 20 end of the centurys; Loof, 1991; Willams et al, 1973; Marin et al, 1998).The fiery crane that TaiWan, China was introduced from Hawaii, America in 1971 is taken and finds radopholus similes thorne (Luo Zongjue etc., 1973); Radopholus similes thorne (Jessica Lynch's power, 1986 are are repeatedly intercepted and captured from enter the territory banana seedlings and ornamental plant since the eighties in 20th century in the port, inland of China; Song Shao etc., 1999; Chen Yong etc., 2002; Clock Guoqiang etc., 1999; Sun Guokun, 2004; Zhang Shaosheng etc., 2003; Li Yinong etc., 2004; Zhao Lirong etc., 2004; Clock Guoqiang etc., 2005; Clock Guoqiang etc., 2004; Huang Fayu etc., 2001a; Huang Fayu etc., 2001b; Huang Fayu etc., 2002; Jiang Han etc., 2004).It is thus clear that; Along with economy and agriculture prodn and Agricultural Products Trade develop rapidly; The quantity that planting materials such as plant seedling are allocated and transported between countries and regions constantly increases, and wherein a lot of plant is the host of radopholus similes thorne, thereby has impelled radopholus similes thorne to propagate widely in the world.Because radopholus similes thorne is very big to the cause harm loss that caused of crop; To effectively stop propagation and the diffusion of radopholus similes thorne between countries and regions; Need to strengthen introducing and the radopholus similes thorne host plant of allocation and transportation and carry soil or matrix detects, and detect quickly and accurately and identify the radopholus similes thorne that possibly carry.
Banana seedlings and ornamental plant are to carry at present to propagate the maximum host plant of radopholus similes thorne risk, therefore strengthen the detection of the plant growing material that possibly carry this nematode and adherent soil or other culture substratees most important.But soil or matrix that the conventional sense method will at first be carried suspicious plant are separated; Under stereoscopic microscope, the nematode that is separated to is carried out morphologic observation; Choose doubtful radopholus similes thorne then, carry out concrete morphology again and identify that perhaps carrying out Molecular Detection identifies.The time that this detection authentication method needs is longer relatively, and operator need possess special Plant nematode and identify that skills and experience could detect evaluation and choose doubtful radopholus similes thorne under stereoscopic microscope.This seminar successfully set up from plant tissue directly detect the Protocols in Molecular Biology of identifying radopholus similes thorne (Liu Yifan etc. the direct ITS-PCR method of detection radopholus similes thorne from mix nematode sample and plant tissue. Scientia Agricultura Sinica; 2011; 44 (19): 3991-3998), and the technology that the living method of utilization molecule directly detects radopholus similes thorne in evaluation soil or the matrix does not have report as yet.
The report that utilizes molecular biology method directly to detect to identify nematode in the soil at present seldom.Qiu etc. (2006) with soil DNA extract test kit with universal primer detection identify the Meloidogyne incognita that is mixed in the soil (
Meloidogyne incognita), peanut root-knot nematode (
M. arenaria) and javanese root knot nematode (
M. javanica); Yan and Smiley (Guiping Yan etc., Detection and Discrimination of
Pratylenchus neglectusAnd
P. thorneiIn DNA Extracts from Soil, plant disease, November 2008, Volume 92, Number 11:1480-1487) utilize homemade DNA extraction buffer to extract to contain unelected Pratylenchidae (
Pratylenchus neglectus) and the Sohne Pratylenchidae (
P. thornei) soil DNA, and with its special primer established counted and the DNA extraction and the purification process of foundation, realized that direct detection identifies the purpose of these the 2 kinds of nematodes in the soil.
Summary of the invention
The objective of the invention is on the basis of Yan and Smiley research, to be optimized, draw a kind of directly method of rapid detection evaluation radopholus similes thorne from soil or other matrix.
The present invention realizes above-mentioned purpose through following technical scheme:
A kind of directly method of rapid detection evaluation radopholus similes thorne from soil or other matrix, step is following:
(1) DNA extraction in the sample to be checked:
Take by weighing sample to be checked, with etc. heavy diameter 0.5 mm granulated glass sphere move in the conical centrifuge tube (preferred specification be the conical centrifuge tube of 15mL), adding volume ratio is phosphoric acid buffer and the SDS damping fluid of 1:1, overturns 3 ~ 4 times;
Add chloroform and primary isoamyl alcohol that volume ratio is 24:1 again, the vibration back is centrifugal with the speed of 3 000 rpm, and supernatant is transferred to one 1.5 mL centrifuge tubes;
The sodium-acetate that adds precooling, mixing ,-20 ℃ leave standstill 5 min, and normal temperature is centrifugal, and supernatant is transferred to another new 1.5 mL centrifuge tubes;
Add cold isopropanol, leave standstill 20min, centrifugal, deposition adding volumn concentration is 70% washing with alcohol, and is centrifugal, abandons supernatant, and repeated washing once, and is centrifugal;
The deposition natural air drying adds ultrapure water and leaves standstill 10min, obtains the DNA crude extract.
(2) the DNA extraction liquid of the Wizard DNA Clean-up Systems test kit purification step (1) of use Promega company.
(3) PCR detects: the DNA with purifying is a template, and nucleotides sequence is classified primer as shown in SEQ ID NO:1 ~ 2, carries out pcr amplification, if amplify the nucleotide sequence of 518bp, then proving in the sample to be checked has radopholus similes thorne.
Above-mentioned steps (1) main reference literature (Yan G, Smiley R W. Detection and discrimination of
Pratylenchus neglectusAnd
P. thorneiIn DNA extracts from soil. Plant Disease, 2008,92 (11): method 1180-1487) is carried out, but the center separating method is revised: 1) use with add the granulated glass sphere that heavy and diameter such as sample to be checked is 0.5 mm (
Vs1 mm); 2) with sample to be checked and granulated glass sphere put into 15 mL conical centrifuge tubes (
Vs2 mL nut centrifuge tubes); When 3) centrifugal, rotating speed be set to 3 000 rpm (
Vs16 000 g).
Above-mentioned detection method step (2) is direct commodity in use test kit, presses the operation of test kit specification sheets and gets final product.Compare with the method for Smiley (2008) with Yan, use the test kit working specification, be prone to promote, the DNA purification effect is good, to be detected highly sensitive.
As a kind of preferred version, the said pcr amplification reaction of above-mentioned detection method step (3) uses the KOD FX enzyme of (Japan is spun) TOYOBO company to carry out pcr amplification, and this enzyme has characteristics such as high amplification rate and hi-fi, and amplified band is clear.
The said pcr amplification reaction preferred reaction of step in the above-mentioned detection method (3) system is 25 μ L:DNA templates, 5 μ L; 2 * PCR buffer, 12.5 μ L; The dNTP 5 μ L of 2mmol/L, the SEQ ID NO:1 primer 0.75 μ L of 10 μ mol/L, the SEQ ID NO:2 primer 0.75 μ L of 10 μ mol/L; KOD FX enzyme 0.5 μ L supplies volume with tri-distilled water; Response procedures is: 94 ℃ of preparatory sex change 2 min, and 98 ℃ of sex change 10 s, 50 ℃ of annealing 30 s, 68 ℃ are extended 1 min, from the sex change to the extension, carry out 35 circulations, and 72 ℃ are fully extended 5 min more then.
Compared with prior art, the present invention has following beneficial effect:
The technology of the radopholus similes thorne in soil or the matrix is identified in the direct detection that the present invention sets up; Adopted the DNA extraction method of Yan and Smiley (2008); But the DNA purifying does not adopt the method for Yan and Smiley (2008); But adopt the Wizard DNA Clean-up Systems test kit of Promega company; Make the purification effect of DNA better, sensitivity to be detected is higher; PCR reaction system and the program of using the present invention to set up are carried out pcr amplification with the Auele Specific Primer that this seminar designs, and have realized that direct detection identifies the radopholus similes thorne that is mixed in soil or the matrix, and sensitivity reaches and from 0.5 g soil or matrix, detects 1 radopholus similes thorne.
The detection method that the present invention sets up is the technology of rapid detection evaluation radopholus similes thorne from soil or other matrix directly; Do not need the detected sample of being gathered is carried out that nematode separates and carries out preliminary identification of morphology and choose process such as worm at stereoscopic microscope; Directly soil or other matrix are carried out extraction and the purifying of total DNA; Use radopholus similes thorne ITS district Auele Specific Primer to carry out conventional pcr amplification; If have a radopholus similes thorne can amplify the specific fragment of about 500bp in 0.5g soil or other matrix, therefore detection authentication method of the present invention has easy quick, saving of labor and sensitive advantage.
Method of the present invention is used conventional PCR appearance and conventional PCR method, be the most basic at present molecular biology instruments and method commonly used, so practical being easy to is promoted.
Method of the present invention can realize the early diagnosis that radopholus similes thorne is sick; Prevent that the sick burst of radopholus similes thorne from causing disaster; Can accelerate Check and Examination of Port quarantine office to importing and exporting the detection level and the detection speed of plant and matrix; Avoid to have the plant and the matrix input China of radopholus similes thorne, accelerate the port watercraft velocity simultaneously, reduce goods and deposit time and the expense that deposit the field at goods; Improve my standing and image of inspection and quarantine office in international trade.
Description of drawings
Fig. 1. the dna profiling with radopholus similes thorne and mixing with soil sample carries out the Auele Specific Primer pcr amplification, and wherein a:1-3. 0.5 g adds the pedotheque of 50 radopholus similes thornes, 4-6, pedotheque; B:1-3. add 5 radopholus similes thornes in the 0.5 g soil, add 4 radopholus similes thornes in the 4-6. 0.5 g soil; C:1-3. add 3 radopholus similes thornes in the 0.5 g soil, add 2 radopholus similes thornes in the 4-6. 0.5 g soil, add 1 radopholus similes thorne in the 7-9. 0.5 g soil; M:Marker DL2000.
Fig. 2. from the Brazilian any of several broadleaf plants rhizosphere soil dna profiling of falling ill, detect radopholus similes thorne, 1-1 ~ 5-2: the Brazilian any of several broadleaf plants rhizosphere soil of falling ill with the Auele Specific Primer pcr amplification; 6-1,6-2: sterilization soil; 7: tri-distilled water; M:Marker DL2000.
Fig. 3. the dna profiling with radopholus similes thorne and matrix biased sample carries out Auele Specific Primer pcr amplification result, and a:1-3. adds 0.5 g matrix sample of 50 radopholus similes thornes, 4-6, pedotheque; B:1-3. add 5 radopholus similes thornes in the 0.5 g matrix, add 4 radopholus similes thornes in the 4-6. 0.5 g matrix; C:1-3. add 3 radopholus similes thornes in the 0.5 g matrix, add 2 radopholus similes thornes in the 4-6. 0.5 g matrix, add 1 radopholus similes thorne in the 7-9. 0.5 g matrix; M:Marker DL2000.
Fig. 4. from morbidity calathea makoyana rhizosphere matrix dna profiling, detect radopholus similes thorne, 1-1 ~ 5-2. morbidity calathea makoyana rhizosphere matrix with the Auele Specific Primer pcr amplification; 6-1,6-2. sterilize native; 7. tri-distilled water; M:Marker DL2000.
Embodiment
Embodiment 1 rapid detection from the pedotheque that contains radopholus similes thorne is identified radopholus similes thorne
Test materials: radopholus similes thorne picks up from the population of red palm rhizosphere soil, and this laboratory is cultivated in 25 ℃ of incubators and is kept in the carrot callus; Soil is for picking up from land for growing field crops banana rhizosphere soil.
The practical implementation step is following:
1. the extraction of radopholus similes thorne and mixing with soil sample total DNA
1.1 take by weighing the above-mentioned field soil sample of 0.5 g, in 15 mL that pack into the sterilization conical centrifuge tube, the diameter of weight such as adding is the little granulated glass sphere of 0.5 mm;
1.2 separate the acquisition radopholus similes thorne from the Radix Dauci Sativae callus group of cultivating radopholus similes thorne; Microscopically respectively 50,5,4,3,2 and 1 radopholus similes thornes of picking put into respectively in the different 15 mL conical centrifuge tubes that 0.5 g soil is housed, add phosphate buffered saline buffer (100 mM NaH
2PO
4, pH 8.0) and each 300 μ L of SDS damping fluid (100 mM NaCl, 500 mM Tris, pH 8.0,10% SDS), overturn 3-4 time;
1.3 adding chloroform: primary isoamyl alcohol (volume ratio 24:1) 400 μ L, with the vortex oscillation device with middling speed 30 s that vibrate, centrifugal 5 min of 3 000 rpm, (less than 600 μ L) are transferred in the 1.5 mL centrifuge tubes with supernatant;
1.4 add 300 μ L precoolings NaOAc (3M, pH5.2), the mixing that turns upside down places-20 ℃ of refrigerated tanks to place 5 min, takes out the back with centrifugal 5 min of 12 000rpm, and supernatant (being no more than 650 μ L) is transferred in the new 1.5 mL conical centrifuge tubes;
1.5 add the cold Virahol of 600 μ L, room temperature is placed 20 min and is used for deposit D NA, then centrifugal 5 min of 12 000rpm; Abandon supernatant, add 700 mL volumn concentrations and be 70% absolute ethyl alcohol, gently rotation; Thorough washing DNA deposition; Centrifugal 5 min of 12 000 rpm abandon supernatant again, and repeated washing once;
1.6 DNA deposition is placed room temperature, treat its natural air drying after, add 100 μ L ultrapure waters, room temperature is placed 10 min, makes the abundant acquisition DNA crude extract soluble in water of DNA, can directly carry out next step experiment, it is subsequent use also can to put-20 ℃ of preservations.
2. the purifying of radopholus similes thorne and soil DNA crude extract:
2.1 (Wizard DNA Clean-up Systems is equipped with the bottle (if deposition, 37 ℃ of water-bath 10 min are arranged) of solution Resin in Promega) to first mixing purification kit, gets 1 mL Resin in the 100 μ L DNA crude extracts of preserving, and puts upside down mixing;
2.2 remove 10 mL syringe needles, the purification column in test kit is tightened in, the mixed solution that adds step (1) filters mixed solution in syringe tube;
2.3 add 2 mL, 80% Virahol in syringe tube, filter once more;
2.4 purification column is put into 1.5 mL centrifuge tubes, centrifugal 2 min of 12 000 rpm;
2.5 purification column is transferred in another new 1.5 mL centrifuge tubes, adds TE damping fluid (10 mM Tris, the 1 mM EDTA of 50 μ L preheating in 65 ℃ of water; PH 8.0); Leave standstill 1 min, centrifugal 20 s of 12 000 rpm collect the DNA in the centrifuge tube; Obtain the DNA of purifying, can directly be used for next step experiment or-20 ℃ of preservations are subsequent use.
3 from the DNA of radopholus similes thorne and mixing with soil sample purifying specific detection radopholus similes thorne dna fragmentation:
3.1 the synthetic radopholus similes thorne ITS district special primer that has designed:
Upstream primer PF (5 '-CTACAAATGTGACGCGAA-3 ' SEQ ID NO:1),
Downstream primer PR (5 '-CAATCTGC ACAA TGAACATAC-3 ' SEQ ID NO:2);
3.2 radopholus similes thorne specific amplification PCR reaction system and program are following:
In 200 μ L PCR pipe; Dispose 25 μ L reaction systems: get step 2.5 dna profiling 5 μ L, 2 * PCR buffer (TOYOBO company), 12.5 μ L, dNTP (2 mM each) 5 μ L (TOYOBO); Upstream primer PF (10 μ M) 0.75 μ L; Downstream primer PR (10 μ M) 0.75 μ L, KOD FX enzyme (1U/μ L) 0.5 μ L (TOYOBO), all the other are supplied with tri-distilled water.
On Bio-rad S1000 PCR appearance, setting response procedures is: 94 ℃ of preparatory sex change 2 min, and 98 ℃ of sex change 10 s, 50 ℃ of annealing 30 s, 68 ℃ are extended 1 min, from the sex change to the extension, carry out 35 circulations, and 72 ℃ are fully extended 5 min more then.
Put into the PCR appearance to each PCR reaction tubes, build lid, carry out pcr amplification by setting program.
3.3 agarose gel electrophoresis detects
After treating that 3.2 PCR reaction finishes; Get 3 μ L products and on 1% sepharose that adds Goldview DNA fuel, carry out electrophoresis; Voltage is 121 V; Electrophoresis finishes the back to be observed on gel imaging system, takes pictures and record, if the (see figure 1) that exists that the specific fragment explanation of 500 bp has radopholus similes thorne occurs.
Test materials: the Brazilian any of several broadleaf plants rhizosphere soil of this laboratory potted plant inoculation radopholus similes thorne and morbidity in warmhouse booth.
The practical implementation step is following:
Get about 500 ~ 1 000g of the Brazilian any of several broadleaf plants rhizosphere soil of morbidity, fully mixing takes by weighing 5 part of 0.5 g soil (numbering 1-5); In addition; Take by weighing 0.5 g sterilization soil (numbering 6) again, put into 15 mL sterilization centrifuge tube respectively, the diameter of weight such as adding is the little granulated glass sphere of 0.5 mm; Other steps are with embodiment 1, and detected result is seen Fig. 2.Can know that by Fig. 2 the Brazilian any of several broadleaf plants rhizosphere soil DNA that infects radopholus similes thorne detects through PCR has the purpose fragment to occur, and does not have the soil DNA of radopholus similes thorne then to detect the purpose fragment.
Test materials: the preparation of radopholus similes thorne and acquisition are with embodiment 1, and matrix is available from gardening plant yard of material, the green source of Guangzhou Fang Cun.
The practical implementation step is following:
Take by weighing 0.5g matrix, in the 15mL that packs into the sterilization centrifuge tube, the diameter of weight such as adding is the little granulated glass sphere of 0.5mm.Other steps are with embodiment 1.Detected result is seen Fig. 3.
Test materials: through identifying the sick calathea makoyana rhizosphere soil of radopholus takes place, pick up from Agricultural University Of South China flower planting chamber.
The practical implementation step is following:
Get morbidity calathea makoyana rhizosphere matrix soil, fully mixing takes by weighing 5 parts, every part of 0.5g matrix soil; Numbering 1-5 in addition, takes by weighing 0.5g sterilization matrix soil again; Numbering 6,6 duplicate samples are put into 15mL sterilization centrifuge tube respectively, and the diameter of weight such as adding is the little granulated glass sphere of 0.5mm.Other subsequent steps are with embodiment 1.The detected result electrophorogram is seen Fig. 4, can be known by this figure, and the calathea makoyana rhizosphere matrix soil DNA that infects radopholus similes thorne detects through PCR has the purpose fragment to occur, and does not have the matrix soil DNA of radopholus similes thorne then to detect the purpose fragment.
Above embodiment is a preferred implementation of the present invention; But protection scope of the present invention does not receive any restriction of the foregoing description; Change, the modification that other any do not deviate under spirit of the present invention and the principle to be done, substitute, combination, simplification etc.; All should be regarded as the substitute mode of the present invention's equivalence, be included within protection scope of the present invention.
SEQUENCE?LISTING
< 110>Agricultural University Of South China
< 120>a kind of directly method of rapid detection evaluation radopholus similes thorne from soil or other matrix
<130>
<160> 2
<170> PatentIn?version?3.3
<210> 1
<211> 18
<212> DNA
< 213>artificial sequence
<400> 1
ctacaaatgt?gacgcgaa 18
<210> 2
<211> 21
<212> DNA
< 213>artificial sequence
<400> 2
caatctgcac?aatgaacata?c 21
Claims (3)
- One kind directly from soil or other matrix rapid detection identify and the method for radopholus similes thorne it is characterized in that step is following:(1) DNA extraction in the sample to be checked:Take by weighing sample to be checked, with etc. the diameter 0.5mm granulated glass sphere of weight move in the conical centrifuge tube, adding volume ratio is phosphoric acid buffer and the SDS damping fluid of 1:1, overturns 3 ~ 4 times;Add chloroform and primary isoamyl alcohol that volume ratio is 24:1 again, the vibration back is centrifugal with the speed of 3 000 rpm, and supernatant is transferred to one 1.5 mL centrifuge tubes;The sodium-acetate that adds precooling, mixing ,-20 ℃ leave standstill 5min, and normal temperature is centrifugal, and supernatant is transferred to another new 1.5 mL centrifuge tubes;Add cold isopropanol, leave standstill 20min, centrifugal, deposition adding volumn concentration is 70% washing with alcohol, and is centrifugal, abandons supernatant, and repeated washing once, and is centrifugal;The deposition natural air drying adds ultrapure water and leaves standstill 10min, obtains the DNA crude extract;(2) the DNA crude extract of the Wizard DNA Clean-up Systems test kit purification step (1) of use Promega company;(3) PCR detects: the DNA with purifying is a template, and nucleotides sequence is classified primer as shown in SEQ ID NO:1 ~ 2, carries out pcr amplification, if amplify the nucleotide sequence of 518bp, then proving in the sample to be checked has radopholus similes thorne.
- According to claim 1 said directly from soil or other matrix rapid detection identify the method for radopholus similes thorne, it is characterized in that enzyme that the said pcr amplification reaction of step (3) uses KOD FX enzyme as TOYOBO company.
- 3. according to the said directly method of rapid detection evaluation radopholus similes thorne from soil or other matrix of claim 2; The reaction system that it is characterized in that the said pcr amplification of step (3) is 25 μ L:DNA templates, 5 μ L, 2 * PCR buffer, 12.5 μ L, the dNTP 5 μ L of 2mmol/L; The SEQ ID NO:1 primer 0.75 μ L of 10 μ mol/L; The SEQ ID NO:2 primer 0.75 μ L of 10 μ mol/L, KOD FX enzyme 0.5 μ L supplies volume with tri-distilled water; Response procedures is: 94 ℃ of preparatory sex change 2 min, and 98 ℃ of sex change 10 s, 50 ℃ of annealing 30 s, 68 ℃ are extended 1 min, from the sex change to the extension, carry out 35 circulations, and 72 ℃ are fully extended 5 min more then.
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| CN103031381A (en) * | 2012-12-17 | 2013-04-10 | 中国检验检疫科学研究院 | Method for detecting LAMP specificity of Radopholus similis |
| CN103898095A (en) * | 2014-03-14 | 2014-07-02 | 西北农林科技大学 | Soil nematode community genome extraction method |
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