CN103602668A - Method for extracting genomic DNA (deoxyribonucleic acid) of single plant nematode by cutting nematode in tube - Google Patents

Method for extracting genomic DNA (deoxyribonucleic acid) of single plant nematode by cutting nematode in tube Download PDF

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CN103602668A
CN103602668A CN201310618805.8A CN201310618805A CN103602668A CN 103602668 A CN103602668 A CN 103602668A CN 201310618805 A CN201310618805 A CN 201310618805A CN 103602668 A CN103602668 A CN 103602668A
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nematode
dna
cutting
tube
pcr amplification
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CN103602668B (en
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王金成
林宇
容万韬
张裕君
黄国明
廖芳
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Tianjin Entry Exit Inspection and Quarantine Bureau of Animals Plants and Food Inspection Center
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Abstract

The invention provides a method for extracting the genomic DNA (deoxyribonucleic acid) of a single plant nematode by cutting the nematode in a tube. The key innovation point of the method lies in that a small-sized dissecting knife is used for cutting the nematode open in the PCR (polymerase chain reaction) tube, the transfer of the nematode to the tube is not needed, the DNA loss caused by cutting the nematode outside the tube and then transferring the nematode to the tube is effectively avoided, the efficiency of DNA extraction is greatly improved, and when the method is used for extracting the DNA of the nematode, the freeze thawing link is not needed, so that the extraction step is simplified. A single nematode genomic DNA template obtained by using the method for extraction meets the requirements of molecular detection technologies such as conventional PCR amplification, real-time fluorescence PCR amplification and isothermal amplification. The method is efficient, stable, low in cost, simple to operate, safe and non-toxic and can be popularized and applied in molecule detection and identification based on nucleic acids.

Description

A kind of method of utilizing pipe inscribe worm to extract wall scroll Plant nematode genomic dna
Technical field
The present invention relates to Plant nematode quarantine evaluation field, a kind of method of utilizing pipe inscribe worm to extract wall scroll Plant nematode genomic dna is provided, particularly, the single line molitor genomic dna that described method obtains by extraction meets the requirement of the molecular detection technology based on nucleic acid such as conventional pcr amplification, real-time fluorescence PCR amplification, constant-temperature amplification, is applicable to the relevant quarantine of port and agroforestry identification experiment chamber application.
Background technology
Plant nematode is carried out to the important foundation that precise Identification is research nematode biological habit and occurrence regularity thereof, significant to the measure of further formulation effectively preventing.Traditional Plant nematode authentication method mainly depends on morphological specificity and morphometry value, requires assessor will have very rich experience and skill.Molecular assay method based on DNA requires relatively lowly to assessor's, is the important supplementary means of identification of morphology, is widely used at present in the classification of nematode is identified.The first step of nematode Molecular Identification is the extraction of DNA, as a rule comprises a large amount of nematode DNA extraction and two kinds of methods of wall scroll nematode DNA extraction.And wherein wall scroll nematode DNA extraction method because of its need nematode amount few, without nematode purifying, the advantage such as simple and direct, cheap, in nematode Molecular Identification, enjoy favor.
About wall scroll nematode DNA trace extracting method, there is more research report.Barstead etc. (1991) and Williams etc. (1992) are used WLB lysate successfully to extract the beautiful rhabditic DNA of wall scroll.Powers etc. (1993), by being directly used in the pcr amplification of gene between mitochondrial COII and 16S rRNA after the fragmentation of root knot nematode second instar larvae, have set up the rapid identification method for the second instar larvae of root knot nematode.It is material that Stanton etc. (1998) be take the second instar larvae of root knot nematode, systematic comparison microwave heating method, water bath heating, protease cracking method, directly grind the effect that method and NaOH method are extracted nematode DNA, find that NaOH method success rate of extracting can reach 81%, directly grinding method success ratio is 50%, and other method efficiency is lower.Kumari etc. (2004) are used Triton X-100 method successfully to extract the DNA of wall scroll sword nematode (Xiphinema vuittenezi), can be used for specific primer PCR amplification, this point is different from the result of Stanton etc., may be that sword nematode individuality is larger compared with root knot nematode, DNA more easily extracts and causes.Shen Xiquan etc. (2005) fixedly extract and obtain genomic dna sea nematode from wall scroll, and for the amplification of 18S rRNA gene PCR, find the pcr amplification that is more suitable for that DNA that Proteinase K method obtains obtains than NaOH cracking process.2011, Wang Jiangling etc., on the basis of Wang Jincheng etc. and Shen Xiquan etc., propose to use liquid nitrogen to substitute-80 ℃ of refrigerator freezing nematodes, have further improved the efficiency of wall scroll nematode DNA extraction, the DNA of the sliding sword class of extraction that can be efficient, stable nematode, but effect is poor while extracting Pratylenchidae.The method of the foundation such as Wang Jincheng etc. (2012) Dui Wangjiang ridge has carried out again improving, and has solved wall scroll Pratylenchidae DNA trace extraction problem.
Yet, although the extracting method of wall scroll nematode DNA is a lot, most methods because of complicated operation, step is various or the not high reason of efficiency is eliminated, what be widely used at present only has enzymatic lysis method.But put into practice, show, the efficiency of enzymatic lysis method and stability still need to optimize raising, to be applicable to the extraction of more various nematode DNA.
It is model that relatively ripe freeze thawing-enzymatic lysis method is take in this research, design wall scroll nematode DNA extraction method, find out the impact on nematode DNA extraction of nematode cutting, frozen-thawed and Proteinase K action time, proposed a kind of efficient, stable, cheap, simple and direct wall scroll nematode DNA extraction method.
Summary of the invention
The technical problem to be solved in the present invention is the ubiquitous complicated operation of extracting method that overcomes wall scroll nematode DNA in prior art, step is various and inefficient defect, a kind of efficient, stable, cheap, simple and direct wall scroll Plant nematode genome DNA extracting method is provided, and described method comprises the steps:
Step 1, pre-treatment: nematode to be extracted is cleaned up with distilled water, and picking wall scroll nematode is put into the PCR pipe containing 8 μ L distilled waters, and palm whizzer is instantaneous centrifugal;
Step 2, cutting cracking: in above-mentioned PCR pipe, with small-sized scalper, nematode is cut, added subsequently 2 μ L lysates, described lysate is that volume ratio is the mixed solution of 10 * PCR Buffer, distilled water and the 20mg/mL Proteinase K of 10:9:1;
Step 3, aftertreatment: the nematode by step 2 through cutting cracking is after 56 ℃ of insulation 1h, and 95 ℃ are heated 10min, and instantaneous centrifugal rear supernatant liquor can be used for follow-up Molecular Detection.
Described distilled water is the sterilized water through twice distillation, and it is generally used for molecular biology experiment.
10 * PCR Buffer in described lysate and the Proteinase K of 20mg/mL are TAKARA company and produce, and goods number is respectively R001B and 9033;
Preferably, the application also relates to a kind of method of utilizing pipe inscribe worm to extract wall scroll Plant nematode genomic dna further, can be after the cutting step of step 2, before cleavage step, increase freeze thawing step, be specially: in above-mentioned PCR pipe, with small-sized scalper, nematode is cut to multigelation 2 times, described freeze thawing adopts frozen-thawed 1min, 65 ℃ of water-bath 2min, add 2 μ L lysates subsequently, then carry out the post-processing step of step 3.
The application's method is applicable to the genomic dna of various plants nematode, especially, be applicable to Cobb root (Pratylenchus penetrans), Meloidogyne incognita (Meloidogyne incognita), pine wood nematode (Bursaphelenchus xylophilus), intend pine wood nematode (B.mucronatus), six the end of a thread Tylenchidas (Cephalenchus hexalineatus) or little loop wire worm (Criconemella sp.).
Compared with prior art, progress of the present invention is: use small-sized scalper in PCR pipe, nematode to be cut, without tube, effectively avoided the DNA that tube causes after the outer line of cut worm of pipe to lose, thereby significantly improved the extraction efficiency of nematode DNA.This method is efficient, stable, cost is low, simple to operate, safety non-toxic, can in the Molecular Detection based on nucleic acid is identified, be applicable.
Accompanying drawing explanation
Fig. 1 adopts cutting, cleavage step to extract the pcr amplification detected result of wall scroll nematode DNA;
Fig. 2 adopts cutting, freeze thawing and cleavage step to extract the pcr amplification detected result of wall scroll nematode DNA;
Fig. 3 adopts freeze thawing and cleavage step to extract the pcr amplification detected result of wall scroll nematode DNA;
Fig. 4 adopts cleavage step to extract the pcr amplification detected result of wall scroll nematode DNA;
In figure, each alphabetical implication is as follows: A: Cobb root; B: Meloidogyne incognita; C: pine wood nematode; D: intend pine wood nematode; E: six the end of a thread Tylenchidas; F: little loop wire worm .M:DNAmarker DL2000; Swimming lane is nematode repetition.
Embodiment
For further understanding summary of the invention of the present invention, Characteristic, hereby lift following examples, and coordinate accompanying drawing to be described in detail as follows:
Specimen origin
This experiment test 6 nematode populations, comprising 1 Cobb root colony, 1 Meloidogyne incognita colony, 1 pine wood nematode colony, 1 Ge Ni pine wood nematode colony, 1 six the end of a thread Tylenchida colony and 1 little ring nematode population, details see the following form:
Figure BSA0000098255950000031
Note: PP, MI, BX, BM, BC, MH are respectively writing a Chinese character in simplified form of 6 kinds of nematode Latin titles.
The effect of single line molitor genomic dna is extracted in embodiment 1, employing " cutting, cleavage step "
Wall scroll nematode DNA extraction method: nematode is cleaned up with distilled water, picking wall scroll nematode is put into the PCR pipe containing 8 μ L distilled waters, instantaneous centrifugal, in PCR pipe, with small-sized scalper, nematode is cut, (described lysate is the mixed solution (volume ratio 10:9:1) of 10 * PCR Buffer, distilled water and 20mg/mL Proteinase K to add subsequently 2 μ L lysates, 10 * PCR Buffer and 20mg/mL Proteinase K are all purchased from TAKARA company), 56 ℃ of insulation 1h, 95 ℃ of heating 10min, instantaneous centrifugal rear supernatant liquor can be used for follow-up Molecular Detection.
Wall scroll nematode DNA extraction effect assessment: wall scroll nematode DNA extraction effect is evaluated by the efficiency of D2D3 gene fragment on pcr amplification nematode rrna 28S rRNA.
1.PCR amplification:
Pcr amplification primer: upstream primer is D2A (5 '-ACAAGTACCGTGAGGGAAAGTTG-3 '), downstream primer is D3B (5 '-TCGGAAGGAACCAGCTACTA-3 ');
Pcr amplification system (50 μ L): 10 * PCR Buffer (Mg 2+) 5 μ L, dNTP (2.5mmol/L) 2 μ L, D2A (10 μ mol/L) 1 μ L, D3B (10 μ mol/L) 1 μ L, rTaq archaeal dna polymerase (5U/ μ L) 0.4 μ L, template DNA 10 μ L, ddH 2o30.6 μ L.Agents useful for same is all purchased from precious biological (TAKARA) company.
Pcr amplification program: 95 ℃ of denaturation 3min; 40 circulating reactions: 95 ℃ of sex change 40sec, 55 ℃ of annealing 40sec, 72 ℃ are extended 1min; Last 72 ℃ of insulation 5min, fully extend reactant.
2. electrophoresis detection
Each sample is got 3 μ L amplified productions, uses 2% sepharose to carry out electrophoresis, and voltage 160V is set, and after electrophoresis 30min, gel is put into the EB dye liquor 15min that dyes, and with being placed on, observing and take pictures in gel imaging system.
3.PCR amplification
From pcr amplification result (Fig. 1), wall scroll nematode DNA success ratio in pcr amplification subsequently of 6 kinds of nematodes of extracting by the method that comprises cutting, cleavage step of the present invention is 100%, and electrophoretic band is bright, illustrate that the efficiency that adopts cutting of the present invention, cleavage step to extract wall scroll nematode DNA is very high, meet the demand of the Molecular Detection based on nucleic acid such as follow-up pcr amplification.
Embodiment 2, " adopting cutting, freeze thawing and cleavage step " are extracted the effect of single line molitor genomic dna
Wall scroll nematode DNA extraction method: with distilled water, nematode is cleaned up, picking wall scroll nematode is put into containing 8 μ L ddH 2in the PCR pipe of O, instantaneous centrifugal, in PCR pipe, with small-sized scalper, nematode is cut, 2 times (liquid nitrogen is processed 1min to multigelation, 65 ℃ of water-bath 2min) after, (described lysate is the mixed solution (volume ratio 10:9:1) of 10 * PCR Buffer, distilled water and 20mg/mL Proteinase K to add 2 μ L lysates, 10 * PCR Buffer and 20mg/mL Proteinase K are all purchased from TAKARA company), 56 ℃ of insulation 1h, 95 ℃ of heating 10min, instantaneous centrifugal rear supernatant liquor can be used for follow-up Molecular Detection.
Wall scroll nematode DNA extraction effect assessment: wall scroll nematode DNA extraction effect is evaluated by the efficiency of D2D3 gene fragment on pcr amplification nematode rrna 28S rRNA.
1.PCR amplification:
Pcr amplification primer: upstream primer is D2A (5 '-ACAAGTACCGTGAGGGAAAGTTG-3 '), downstream primer is D3B (5 '-TCGGAAGGAACCAGCTACTA-3 ');
Pcr amplification system (50 μ L): 10 * PCR Buffer (Mg 2+) 5 μ L, dNTP (2.5mmol/L) 2 μ L, D2A (10 μ mol/L) 1 μ L, D3B (10 μ mol/L) 1 μ L, rTaq archaeal dna polymerase (5U/ μ L) 0.4 μ L, template DNA 10 μ L, ddH 2o30.6 μ L.Agents useful for same is all purchased from precious biological (TAKARA) company.
Pcr amplification program: 95 ℃ of denaturation 3min; 40 circulating reactions: 95 ℃ of sex change 40sec, 55 ℃ of annealing 40sec, 72 ℃ are extended 1min; Last 72 ℃ of insulation 5min, fully extend reactant.
2. electrophoresis detection
Each sample is got 3 μ L amplified productions, uses 2% sepharose to carry out electrophoresis, and voltage 160V is set, and after electrophoresis 30min, gel is put into the EB dye liquor 15min that dyes, and with being placed on, observing and take pictures in gel imaging system.
3.PCR amplification
From pcr amplification result (Fig. 2), wall scroll nematode DNA success ratio in pcr amplification subsequently of 6 kinds of nematodes of extracting by the method that comprises cutting, freeze thawing and cleavage step of the present invention is 100%, and electrophoretic band is bright, illustrate that method of the present invention is extracted the efficiency of wall scroll nematode DNA very high, meet the demand of the Molecular Detection based on nucleic acid such as follow-up pcr amplification.Meanwhile, in the extraction of root knot nematode DNA, non-specific amplification relatively only adopts cutting, cleavage step slightly to weaken, and extraction effect is slightly improved but not obvious (Fig. 1-B, Fig. 2-B).
Embodiment 3, " adopting freeze thawing, cleavage step " are extracted the effect of single line molitor genomic dna
Wall scroll nematode DNA extraction method: with distilled water, nematode is cleaned up, picking wall scroll nematode is put into containing 8 μ L ddH 2in the PCR pipe of O, instantaneous centrifugal, 2 times (liquid nitrogen is processed 1min to multigelation, 65 ℃ of water-bath 2min) after, add 2 μ L lysates (described lysate is the mixed solution (volume ratio 10:9:1) of 10 * PCR Buffer, distilled water and 20mg/mL Proteinase K, and 10 * PCR Buffer and 20mg/mL Proteinase K are all purchased from TAKARA company), 56 ℃ of insulation 1h, 95 ℃ of heating 10min, instantaneous centrifugal rear supernatant liquor can be used for follow-up Molecular Detection.
Wall scroll nematode DNA extraction effect assessment: wall scroll nematode DNA extraction effect is evaluated by the efficiency of D2D3 gene fragment on pcr amplification nematode rrna 28S rRNA.
1.PCR amplification:
Pcr amplification primer: upstream primer is D2A (5 '-ACAAGTACCGTGAGGGAAAGTTG-3 '), downstream primer is D3B (5 '-TCGGAAGGAACCAGCTACTA-3 ');
Pcr amplification system (50 μ L): 10 * PCR Buffer (Mg 2+) 5 μ L, dNTP (2.5mmol/L) 2 μ L, D2A (10 μ mol/L) 1 μ L, D3B (10 μ mol/L) 1 μ L, rTaq archaeal dna polymerase (5U/ μ L) 0.4 μ L, template DNA 10 μ L, ddH 2o30.6 μ L.Agents useful for same is all purchased from precious biological (TAKARA) company.
Pcr amplification program: 95 ℃ of denaturation 3min; 40 circulating reactions: 95 ℃ of sex change 40sec, 55 ℃ of annealing 40sec, 72 ℃ are extended 1min; Last 72 ℃ of insulation 5min, fully extend reactant.
2. electrophoresis detection
Each sample is got 3 μ L amplified productions, uses 2% sepharose to carry out electrophoresis, and voltage 160V is set, and after electrophoresis 30min, gel is put into the EB dye liquor 15min that dyes, and with being placed on, observing and take pictures in gel imaging system.
3.PCR amplification
From pcr amplification result (Fig. 3), there is notable difference in wall scroll nematode DNA success ratio in pcr amplification subsequently of 6 kinds of nematodes of extracting by the method that comprises freeze thawing, cleavage step of the present invention, extraction effect with pine wood nematode is best, six the end of a thread Tylenchidas take second place, though the extraction effect of Cobb root is better, but there is non-specific amplification problem, root knot nematode and plan pine wood nematode effect are unstable, and the extraction effect of little loop wire worm is the poorest, and non-specific amplification is obvious, visible nematode cutting is remarkable on the impact of different nematode DNA extraction.
Embodiment 4, " employing cleavage step " are extracted the effect of single line molitor genomic dna
Wall scroll nematode DNA extraction method: nematode is cleaned up with distilled water, picking wall scroll nematode is put into the PCR pipe containing 8 μ L distilled waters, instantaneous centrifugal after, (described lysate is the mixed solution (volume ratio 10:9:1) of 10 * PCR Buffer, distilled water and 20mg/mL Proteinase K to add 2 μ L lysates, 10 * PCR Buffer and 20mg/mL Proteinase K are all purchased from TAKARA company), 56 ℃ of insulation 1h, 95 ℃ of heating 10min, instantaneous centrifugal rear supernatant liquor can be used for follow-up Molecular Detection.
Wall scroll nematode DNA extraction effect assessment: wall scroll nematode DNA extraction effect is evaluated by the efficiency of D2D3 gene fragment on pcr amplification nematode rrna 28S rRNA.
1.PCR amplification:
Pcr amplification primer: upstream primer is D2A (5 '-ACAAGTACCGTGAGGGAAAGTTG-3 '), downstream primer is D3B (5 '-TCGGAAGGAACCAGCTACTA-3 ');
Pcr amplification system (50 μ L): 10 * PCR Buffer (Mg 2+) 5 μ L, dNTP (2.5mmol/L) 2 μ L, D2A (10 μ mol/L) 1 μ L, D3B (10 μ mol/L) 1 μ L, rTaq archaeal dna polymerase (5U/ μ L) 0.4 μ L, template DNA 10 μ L, ddH 2o30.6 μ L.Agents useful for same is all purchased from precious biological (TAKARA) company.
Pcr amplification program: 95 ℃ of denaturation 3min; 40 circulating reactions: 95 ℃ of sex change 40sec, 55 ℃ of annealing 40sec, 72 ℃ are extended 1min; Last 72 ℃ of insulation 5min, fully extend reactant.
2. electrophoresis detection
Each sample is got 3 μ L amplified productions, uses 2% sepharose to carry out electrophoresis, and voltage 160V is set, and after electrophoresis 30min, gel is put into the EB dye liquor 15min that dyes, and with being placed on, observing and take pictures in gel imaging system.
3.PCR amplification
From pcr amplification result (Fig. 4), the wall scroll nematode DNA of 6 kinds of nematodes that method by cleavage step of the present invention is extracted is poorer compared with freezing-thawing and cracking method (example 3) in the effect of pcr amplification subsequently, illustrate that the efficiency that does not have the By Direct Pyrolysis of freeze thawing link method to extract DNA is lower and stable not, especially true concerning root knot nematode and little loop wire worm.For Cobb root, intend pine wood nematode and six the end of a thread Tylenchidas need 56 ℃ of insulations just PCR product can be detected above in 3 hours, and the pine wood nematode Diapause of cultivating through BC even needs to be incubated 4 hours and just can reach the object of extraction above.
A kind of method of utilizing pipe inscribe worm to extract wall scroll Plant nematode genomic dna of the present invention is described by concrete example, those skilled in the art can use for reference content of the present invention, the links such as appropriate change raw material, processing condition realize corresponding other object, its relevant change does not all depart from content of the present invention, within all similar replacements and change will become apparent to those skilled in the art that and be all deemed to be included in scope of the present invention.

Claims (4)

1. utilize pipe inscribe worm to extract a method for wall scroll Plant nematode genomic dna, described method comprises the steps:
Step 1, pre-treatment: nematode to be extracted is cleaned up with distilled water, and picking wall scroll nematode is put into the PCR pipe containing 8 μ L distilled waters, and palm whizzer is instantaneous centrifugal;
Step 2, cutting cracking: in above-mentioned PCR pipe, with small-sized scalper, nematode is cut, added subsequently 2 μ L lysates, described lysate is that volume ratio is the mixed solution of 10 * PCR Buffer, distilled water and the 20mg/mL Proteinase K of 10:9:1;
Step 3, aftertreatment: the nematode by step 2 through cutting cracking is after 56 ℃ of insulation 1h, and 95 ℃ are heated 10min, and instantaneous centrifugal rear supernatant liquor can be used for follow-up Molecular Detection.
2. according to the extracting method of claim 1, it is characterized in that: the 10 * PCR Buffer in described lysate and the Proteinase K of 20mg/mL are TAKARA company and produce, and goods number is respectively R001B and 9033.
3. according to the extracting method of claim 1, it is characterized in that: after the cutting step of step 2, before cleavage step, increase freeze thawing step applicable equally, be specially: in above-mentioned PCR pipe, with small-sized scalper, nematode is cut, multigelation 2 times, described freeze thawing adopts frozen-thawed 1min, 65 ℃ of water-bath 2min, add subsequently 2 μ L lysates, then carry out the post-processing step of step 3.
4. according to the extracting method of claim 1, it is characterized in that: described Plant nematode is Cobb root (Pratylenchus penetrans), Meloidogyne incognita (Meloidogyne incognita), pine wood nematode (Bursaphelenchus xylophilus), intends pine wood nematode (B.mucronatus), six the end of a thread Tylenchidas (Cephalenchus hexalineatus) or little loop wire worm (Criconemella sp.).
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Cited By (4)

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Publication number Priority date Publication date Assignee Title
CN104004748A (en) * 2014-06-11 2014-08-27 天津出入境检验检疫局动植物与食品检测中心 Lysis solution used for plant parasitic nematode genome DNA micro-extraction and application thereof
CN104032002A (en) * 2014-06-11 2014-09-10 天津出入境检验检疫局动植物与食品检测中心 New combination of universal amplification primers for plant parasitic nematode ribosome ITS (internal transcribed spacer) and application method thereof
CN105483119A (en) * 2015-12-25 2016-04-13 华南农业大学 DNA crude extraction liquid for fixed single nematode as well as preparation method and application of DNA crude extraction liquid
CN109750034A (en) * 2019-03-25 2019-05-14 河北省农林科学院植物保护研究所 Utilize the method for pipe lid inscribe worm rapidly extracting single nematode DNA

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* Cited by examiner, † Cited by third party
Title
WANG JIN CHENG: "Description of a new species (Nematoda, Aphelenchoididae) isolated from wood packaging material", 《ACTA ZOOTAXONOMICA SINICA》, vol. 30, no. 2, 30 April 2005 (2005-04-30), pages 314 *
王金成等: "基于核糖体ITS区和28S rRNA D2~D3区的短体线虫系统发育研究", 《动物分类学报》, vol. 37, no. 4, 31 October 2012 (2012-10-31) *
魏庄等: "4种禾谷孢囊线虫基因组DNA提取方法的比较和改进", 《甘肃农业大学学报》, vol. 47, 31 December 2012 (2012-12-31), pages 64 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104004748A (en) * 2014-06-11 2014-08-27 天津出入境检验检疫局动植物与食品检测中心 Lysis solution used for plant parasitic nematode genome DNA micro-extraction and application thereof
CN104032002A (en) * 2014-06-11 2014-09-10 天津出入境检验检疫局动植物与食品检测中心 New combination of universal amplification primers for plant parasitic nematode ribosome ITS (internal transcribed spacer) and application method thereof
CN104032002B (en) * 2014-06-11 2016-04-13 天津出入境检验检疫局动植物与食品检测中心 Plant nematode rrna ITS district's universal amplification primer Combination nova and using method thereof
CN105483119A (en) * 2015-12-25 2016-04-13 华南农业大学 DNA crude extraction liquid for fixed single nematode as well as preparation method and application of DNA crude extraction liquid
CN109750034A (en) * 2019-03-25 2019-05-14 河北省农林科学院植物保护研究所 Utilize the method for pipe lid inscribe worm rapidly extracting single nematode DNA

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