CN104004748A - Lysis solution used for plant parasitic nematode genome DNA micro-extraction and application thereof - Google Patents
Lysis solution used for plant parasitic nematode genome DNA micro-extraction and application thereof Download PDFInfo
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Abstract
The invention provides a lysis solution for plant parasitic nematode genome DNA micro-extraction and application of the lysis solution. The lysis solution is characterized by being allowed to be directly used for plant parasitic nematode genome DNA micro-extraction without cutting, extruding and breaking, grinding and freezing and thawing nematodes, operation steps are simpler and more convenient to implement, and sample pollution and loss are reduced to the maximum degree in the DNA extraction process, extraction efficiency is obviously improved, the success rate is obviously increased, and the problems that in the prior art, efficiency is low, stability is poor, operation is difficult and the steps are numerous in the plant parasitic nematode DNA extraction process are solved effectively. Nematode DNA obtained through extraction by the adoption of the method meets the requirements of plant parasitic nematode DNA barcode identification, RFLP enzyme digestion identification, specific primer PCR identification and other molecular detection technologies, and the lysis solution can be applied and popularized in quarantine identification work of China ports, agricultural departments and forestry departments.
Description
Technical field
The present invention relates to plant nematode quarantine evaluation field, a kind of lysate and application thereof of extracting for plant nematode genomic dna trace is provided, particularly, described method is by being used new DNA extraction lysate, greatly simplified plant nematode extracting genome DNA program, DNA extraction efficiency and success ratio have been significantly improved, effectively solved the efficiency existing in former plant nematode DNA trace leaching process low, poor stability, not easy to operate, the problems such as step is various, be applicable to the relevant quarantine of port and agroforestry identification experiment chamber application.
Background technology
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 that a large amount of extractions of nematode DNA and nematode DNA trace extract two kinds of methods.And wherein nematode DNA trace extracting method because of its need nematode amount few, without nematode purifying, there is the advantages such as simple and direct, cheap simultaneously, thereby enjoy favor in nematode Molecular Identification.
About 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, are also the nematode DNA setting up the earliest trace extracting method that can review at present.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.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. propose to be used 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 nematode that can be efficient, stable, 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.
Although the method that nematode DNA trace extracts is a lot, most methods because of complicated operation, step is various or efficiency is not high, stability is inadequate etc., and reason is eliminated, and only has enzymatic lysis method to be widely used.But put into practice, show, the efficiency of enzymatic lysis method and stability still need to optimize raising, and operation steps needs further simplification, to be applicable to the extraction of more various nematode DNA.This research be take relatively ripe a kind of enzymatic lysis method as basis, to its key reagents---component and the concentration of lysate have been carried out systematic screening and optimization, to propose a kind of more efficient, stable, cheap, simple and direct nematode DNA trace extracting method, for the quarantine of plant nematode, identify part technical support is provided.
Summary of the invention
The technical problem to be solved in the present invention is to overcome the defects such as the efficiency existing in existing plant nematode genomic dna trace extractive technique is low, success ratio is low, step is numerous and diverse, a kind of lysate and application thereof of extracting for plant nematode genomic dna trace is provided, and described lysate is as follows:
The formula of described lysate comprises:
10mM Tris-HCl (pH8.3), 1.5mM MgCl
2, 60mM KCl, 10mg/mL TritonX-100 (being triton x-100), 0.2mM DTT (being dithiothreitol dithio) and 300 μ g/mL Proteinase K (being Proteinase K).
Further, the application provides a kind of preparation method of above-mentioned lysate, it is characterized in that:
Described lysate is by 10 * PCR Buffer (Mg that contains 15mM
2+), the KCl solution of 100mM, the Proteinase K solution of the TritonX-100 solution of 100mg/mL, 3000 μ g/mL, the DTT solution of 1mM and distilled water by 1: 1: 1: the volume ratio mixed preparing of 1: 2: 4 forms.
Further, the present invention relates to a kind of application that above-mentioned lysate is extracted for plant nematode genomic dna trace, it is characterized in that:
First with distilled water, nematode is cleaned up, then picking wall scroll nematode is put into the PCR pipe containing lysate described in 5～20 μ L, last 56 ℃ of insulation 0.5～4h, 95 ℃ of heating 10min.Through above-mentioned steps, extract the supernatant liquor obtaining and can be used for molecule experiment.
Preferably, the usage quantity of lysate is 10～15 μ L, and the time of 56 ℃ of insulations is 1～3h.
The genomic dna trace that the application's lysate is applicable to various plants nematode extracts, especially, be applicable to Cobb root (Pratylenchus penetrans), Si Shi Pratylenchidae (Pratylenchus scribneri), pine wood nematode (Bursaphelenchus xylophilus), intend pine wood nematode (B.mucronatus), aphelenchoides besseyi (Aphelenchoides besseyi).
Described distilled water is the sterilized water through twice distillation, and it is generally used for molecular biology experiment.
Described lysate is prepared the 10 * PCR Buffer (Mg that contains 15mM in required reagent
2+) and Proteinase K (being Proteinase K) be TAKARA company and produce, product article No. is respectively R001B and 9033; KCl in described lysate (being Repone K) is Tianjin Fengchuan Chemical Reagent Science & Technology Co., Ltd.'s production, analytical pure, and lot number is 2013408; TritonX-100 in described lysate (being Triton-100) and DTT (being dithiothreitol dithio) are Yuan Ye bio tech ltd, Shanghai and produce, and product article No. is respectively 12023 and 11175.
Compared with prior art, progress of the present invention is: the trace that lysate of the present invention can be directly used in plant nematode complete genome DNA extracts, without cutting, rack, grinding, freeze thawing nematode, operation steps is simpler and more direct, farthest reduced the link of sample contamination and loss in DNA extraction process, extraction efficiency and success ratio significantly improve, and have effectively solved the problems such as the efficiency existing in former plant nematode DNA trace leaching process is low, poor stability, not easy to operate, step is various.Utilize lysate of the present invention to extract the nematode DNA obtaining and meet the demand that plant nematode DNA bar code is identified, RFLP enzyme is cut evaluation, specific primer PCR is identified equimolecular detection technique.
Accompanying drawing explanation
Fig. 1 is used the nematode DNA sample of lysate 1 extraction for rrna ITS district pcr amplification detected result;
Fig. 2 is used the nematode DNA sample of lysate 2 extractions for rrna ITS district pcr amplification detected result;
Fig. 3 is used the nematode DNA sample of lysate 3 extractions for rrna ITS district pcr amplification detected result;
Fig. 4 is used the nematode DNA sample of lysate 4 extractions for rrna ITS district pcr amplification detected result;
In figure, each alphabetical implication is as follows: swimming lane 1-4: Nanjing aphelenchoides besseyi; Swimming lane 5-8: intend pine wood nematode; Swimming lane 9-12: pine wood nematode; Swimming lane 13-16: Cobb root; Swimming lane 17-20: Si Shi Pratylenchidae.
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 5 nematode populations, comprising 1 Cobb root colony, 1 Si Shi Pratylenchidae colony, 1 pine wood nematode colony, 1 Ge Ni pine wood nematode colony, 1 aphelenchoides besseyi colony.Details see the following form:
Reagent source
10 * PCRBuffer is (containing the Mg of 15mM
2+) and Proteinase K (being Proteinase K) be TAKARA company and produce, product article No. is respectively R001B and 9033; KCl (being Repone K) is Tianjin Fengchuan Chemical Reagent Science & Technology Co., Ltd.'s production, analytical pure, and lot number is 2013408; TritonX-100 (being Triton-100) and DTT (being dithiothreitol dithio) are Yuan Ye bio tech ltd, Shanghai and produce, and product article No. is respectively 12023 and 11175; Polysorbas20 (being Tween20) is the production of Tianjin recovery fine chemistry industry institute, chemical pure, and lot number is 2012817; MgCl
2(being magnesium chloride) produces from Tianjin Fengchuan Chemical Reagent Science & Technology Co., Ltd., analytical pure, and lot number is 2012602; Gelatin (being gelatin) Tianjin Fengchuan Chemical Reagent Science & Technology Co., Ltd. produces, and lot number is 2013502; From Beijing Bo Run Lai Te Science and Technology Ltd., product article No. is 74385 to Nonidet P-40 (being the poly-di-alcohol of ethylphenyl).
The effect that embodiment 1, different lysate (lysate 1～4) extract 5 kind of plant parasitic nematode genomic dnas compares nematode DNA extraction:
With distilled water, nematode is cleaned up, picking wall scroll nematode is put into the 200 μ LPCR pipes containing 10 μ L lysates, then 56 ℃ of heating (56 ℃ of heating 1h of aphelenchoides besseyi, 56 ℃ of heating 2h of pine wood nematode and plan pine wood nematode, 56 ℃ of heating 3h of Pratylenchidae), 95 ℃ of heating 10min, the extraction supernatant liquor of instantaneous centrifugal rear acquisition can be used for molecule experiment.4 repetitions are set.
Different lysates extract the effect assessment of nematode DNA:
The nematode DNA sample of extraction is carried out to rrna ITS district's pcr amplification and by electrophoresis detection, to assess different lysates, extracts the effect of nematode gene group DNA.
1. the formula of different lysates:
Lysate 1 formula: 10mM Tris-HCl (pH8.3), 50mM KCl, 2.5mM MgCl
2, 4.5mg/mL Tween20 (being polysorbas20), 4.5mg/mL Gelatin (being gelatin), 60 μ g/mL Proteinase K (being Proteinase K).Described lysate is by 10 * PCR Buffer (Mg that contains 15mM
2+), the MgCl of 10mM
2the Proteinase K solution of the Tween20 solution of solution, 45mg/mL, the Gelatin solution of 45mg/mL, 600 μ g/mL and distilled water were by 1: 1: 1: the volume ratio mixed preparing of 1: 1: 5 forms.This lysate formula is derived from the paper (.Genetics, the 1992.131:609-624 such as Williams) that Williams in 1992 etc. deliver.
Lysate 2 formulas: 10mM Tris-HCl (pH8.3), 50mM KCl, 2.5mM MgCl
2, 10mg/mLTritonX-100 (being triton x-100), 4.5mg/mL Nonidet P-40 (being the poly-di-alcohol of ethylphenyl), 100 μ g/mL Proteinase K (being Proteinase K).Described lysate is by 10 * PCR Buffer (Mg that contains 15mM
2+), the MgCl of 10mM
2the Nonidet P-40 solution of the TritonX-100 solution of solution, 100mg/mL, 45mg/mL, the Proteinase K of 1000 μ g/mL and distilled water were by 1: 1: 1: the volume ratio mixed preparing of 1: 1: 5 forms.This lysate formula is derived from the paper that Zipperlen in 2005 etc. deliver, only by 4.5mg/mL Tween20 with 10mg/mL TritonX-100 replace (the .Genome biology such as Zipperlen, 2005,6:R19).
Lysate 3 formulas: 10mM Tris-HCl (pH8.3), 50mM KCl, 1.5mM MgCl
2, 100 μ g/mL Proteinase K (being Proteinase K).Described lysate is by 10 * PCR Buffer (Mg that contains 15mM
2+), the Proteinase K solution of 1000 μ g/mL and distilled water form by the volume ratio mixed preparing of 1: 1: 8.This lysate formula be derived from the paper that Wang Jincheng etc. delivers for 2012 (Wang Jincheng etc. animal classification journal, 2012,37 (4): 687-693).
Lysate 4 formulas: 10mM Tris-HCl (pH8.3), 1.5mM MgCl
2, 60mM KCl, 10mg/mL TritonX-100 (being triton x-100), 0.2mM DTT (being dithiothreitol dithio) and 300 μ g/mL Proteinase K (being Proteinase K).Described lysate is by 10 * PCR Buffer (Mg that contains 15mM
2+), the KCl solution of 100mM, the TritonX-100 solution of 100mg/mL, the Proteinase solution of 3000 μ g/mL, the DTT solution of 1mM and distilled water by 1: 1: 1: the volume ratio mixed preparing of 1: 2: 4 forms.This lysate formula is derived from the present invention.
2.PCR amplification:
Pcr amplification system (50 μ L): 10 * PCR Buffer (Mg
2+) 5 μ L, dNTP (2.5mmol/L) 2 μ L, upstream primer (10 μ mol/L) 1 μ L, downstream primer (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.
Pine wood nematode and plan pine wood nematode amplimer: F194 (5 '-CGTAACAAGGTAGCTGTAG-3 '), F195 (5 '-TCCTCCGCTAAATGATATG-3 ');
Aphelenchoides besseyi and Pratylenchidae amplimer: TW81 (5 '-GTTTCCGTAGGTGAACCTGC-3 '), AB28 (5 '-ATATGCTTAAGTTCAGCGGGT-3 ').
Pcr amplification system (25 μ L): 10 * PCR Buffer (Mg
2+) 2.5 μ L, dNTP (2.5mmol/L) 1 μ L, upstream primer (10 μ mol/L) 0.5 μ L, downstream primer (10 μ mol/L) 0.5 μ L, rTaq archaeal dna polymerase (5U/ μ L) 0.2 μ L, template DNA 5 μ L, distilled water 15.3 μ L.
PCR reaction conditions: 94 ℃ of denaturation 3min; 40 circulating reactions: 94 ℃ of sex change 40sec, 55 ℃ of annealing 40sec, 72 ℃ are extended 1.5min; Last 72 ℃ of insulation 5min, fully extend reactant.
3. 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.
4. amplification
From pcr amplification result (Fig. 1-4), except using lysate (Fig. 4 of the present invention, beyond the DNA sample of 5 kinds of nematodes of lysate 4) extracting is all positive, all there is certain problem in the lysates of scholar's preparation before other 3 kinds on efficiency and broad spectrum.Use lysate 1 (Fig. 1) can only extract preferably aphelenchoides besseyi and intend pine wood nematode, and poor to the extraction effect of pine wood nematode, to Cobb root and Si Shi Pratylenchidae, cannot extract completely, pcr amplification result is negative.Use lysate 2 (Fig. 2) can well extract aphelenchoides besseyi and extract preferably pine wood nematode and intend the DNA of pine wood nematode, but still cannot extract the DNA of Cobb root and Si Shi Pratylenchidae.The DNA that uses lysate 3 (Fig. 3) to can be good at extracting aphelenchoides besseyi and intend pine wood nematode, while extracting pine wood nematode, effect is slightly poor, and cannot extract equally the DNA of Cobb root and Si Shi Pratylenchidae.
5. conclusion
Experimental result shows, lysate (Fig. 4 of the present invention, lysate 4) can be good at extracting the genomic dna of the plant nematodes such as aphelenchoides besseyi, plan pine wood nematode, pine wood nematode, Cobb root, Si Shi Pratylenchidae, meet the demand of the Molecular Detection evaluation of follow-up PCR-based technology.Compare with nematode DNA extraction lysate (Fig. 1-3, lysate 1-3) in the past, while using lysate of the present invention (Fig. 4, lysate 4) to extract nematode gene group DNA efficiency and have higher success rate, applicable line insect types is more various.And, while using lysate of the present invention to extract nematode DNA, without cutting, rack, grinding, freeze thawing nematode, operation steps is simpler and more direct, farthest reduce the link of sample contamination and loss in DNA extraction process, effectively solved the problems such as the efficiency existing in former plant nematode DNA trace leaching process is low, poor stability, not easy to operate, step is various.
A kind of lysate extracting for plant nematode genomic dna trace of the present invention and application thereof are 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 (5)
1. the lysate extracting for plant nematode genomic dna trace, is characterized in that, the formula of described lysate comprises:
10mM Tris-HCl (pH8.3), 1.5mM MgCl
2, 60mM KCl, 10mg/mL TritonX-100,0.2mM DTT and 300 μ g/mL Proteinase K.
2. the preparation method of lysate according to claim 1, is characterized in that:
Described lysate by the KCl solution of 10 * PCR Buffer, 100mM, the Proteinase K solution of the TritonX-100 solution of 100mg/mL, 3000 μ g/mL, the DTT solution of 1mM and distilled water by 1: 1: 1: the volume ratio mixed preparing of 1: 2: 4 forms, and described 10 * PCR Buffer is containing the Mg of 15mM
2+.
3. the application that lysate according to claim 1 extracts for plant nematode genomic dna trace, is characterized in that:
First with distilled water, nematode is cleaned up, then picking wall scroll nematode is put into the PCR pipe containing lysate described in 5～20 μ L, last 56 ℃ of insulation 0.5～4h, 95 ℃ of heating 10min.Through above-mentioned steps, extract the supernatant liquor obtaining and can be used for molecule experiment.
4. according to the application of claim 3, it is characterized in that, the usage quantity of lysate is 10～15 μ L, and the time of 56 ℃ of insulations is 1～3h.
5. according to the application of claim 3, it is characterized in that, described plant nematode is Cobb root (Pratylenchus penetrans), Si Shi Pratylenchidae (Pratylenchus scribneri), pine wood nematode (Bursaphelenchus xylophilus), intends pine wood nematode (B.mucronatus), aphelenchoides besseyi (Aphelenchoides besseyi).
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| CN106048004A (en) * | 2016-06-01 | 2016-10-26 | 华南农业大学 | Real-time fluorescence quantification PCR primers and probes for identifying three kinds of pratylenchus coffeae on sugarcane and kit thereof |
| CN111826420A (en) * | 2019-04-15 | 2020-10-27 | 南京林业大学 | Pine wood nematode nucleic acid extraction reagent and application thereof |
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Application publication date: 20140827 |