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.
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:
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.