CN103014157B - Method for identifying plant parasitic nematode by single nematode polypide or single nematode ovum - Google Patents

Abstract

The invention discloses a method for identifying plant parasitic nematode by single nematode polypide or single nematode ovum, and belongs to the technical field of molecular biological detection and identification. Other reagents are easily mixed in the process of extracting a DNA (deoxyribonucleic acid) template of a single nematode, resulting in pollution, or the material loss easily becomes serious in the process of cutting off and processing the single nematode because of small entity of the nematode in a molecular biology laboratory. The invention comprises a one-step operation for simply puncturing nematode polypide or ovum in a PCR (polymerase chain reaction) tube directly. Existence of the nematode and lysate thereof, namely existence of the DNA template is ensured without transferring the nematode lysate into the PCR tube from other carriers; and the preparation efficiency of the template is high. The PCR validation and partial further experimental verification of the enzyme are carried out on molecular diagnostic markers in a plurality of aspects such as identification and research of the plant parasitic nematode at present. The method is obvious in effect. The invention also discloses identification of the plant parasitic nematode by employing the single nematode ovum as the template for the first time.

Description

By the method for wall scroll nematode polypide or single nematode worm's ovum plant identification parasitic nematode

Technical field

The invention belongs to molecular Biological Detection authenticate technology field, be specifically related to a kind of method of wall scroll nematode polypide or single nematode worm's ovum plant identification parasitic nematode.

Background technology

Plant parasitic nematodes is the plant pathogeny organism that in world wide, class harm agroforestry are produced.Up to the present, nearly 4100 kind of plant nematodes are described, and account for the 15%(Decraemer & Hunt of all line insect types described, 2006).A lot of plant nematode, if radopholus similes thorne (Radopholussimilis), root knot nematode (Meloidogyne spp.) and pine wood nematode (Bursaphelenchus xylophilus) etc. are all destructive strong pathogens, on a lot of food crop, ornamental plant, vegetables and forestry trees, cause serious harm, so, these Plant nematodes are classified as quarantine harmful organisms (CABI & EPPO, 1998) by a lot of country.Traditional plant nematode taxonomic identification relies on morphological specificity, and the method needs abundant taxonomic identification experience, in kind level, and precise Identification more complicated and the difficulty especially of some allied specieses.Since invention round pcr, and after the genome of Caenorthaditis elegans ws123 (Caenorhabditis elegans) is completely sequenced, molecular diagnosis and authentication method have been widely used in free-living nematode, animal parasitic nematodes and plant nematode, and provide abundanter information (Floyd et al, 2002 for the phyletic evolution of these nematodes and sort research; Powers, 2004; Abebe et al, 2011).

On nematode taxonomy, the sequence signature in the arrangement of ribosome-RNA(rRNA) and its transcribed spacer and some intervals of Mitochondrial Genome Overview is often used as the foundation of qualification.According to the conservative property of sequence of interval, much general or special primer has been devised qualification for nematode or phylogenetic analysis at present.Such as, the sequence increased with the Internal Transcribed Spacer (ITS) universal primer is as the mark of qualification, with the sequential analysis nematode phyletic evolution that the universal primer of 28S rRNA gene D2-D3 expansion area increases, in addition, also have some for the mitochondrial cytochrome oxidase gene of phylogenetic analysis some conserved regions between special primer (CI O or COII) (Powers & Harris, 1993; Al-Banna et al, 1997; Subbotin et al, 2000; Powers, 2004; Subbotin et al, 2005a; De Ley et al, 2005).

The Molecular Identification of plant nematode, needs first to obtain DNA profiling.From the Plant nematode that usually once can be separated to the mixing of multiple kind natural soil, in plant tissue, so the DNA extraction of wall scroll plant nematode is more meaningful in the researchs such as taxonomy, phyletic evolution and nematode Testing and appraisal.The acquisition of wall scroll Plant nematode DNA generally includes two steps, and one is obtain nematode lysate, and two is obtain DNA coarse extract.For the first step, conventional method be exactly picking wall scroll worm on slide glass, under stereoscopic microscope, be cut into 2 ~ 3 sections, move on in nematode lysate (WLB) with pipettor, what have also can carry out multigelation lysate more again, makes the abundant cracking of body tissue (Subbotin et al, 2000; Wawyenberge et al, 2000; Elbadri et al, 2002; Bert et al, 2008; Troccoli et al, 2008; Huang et al, 2010); Also broken for nematode polypide section or whole piece worm can be put into the simple and easy lysate of nematode (1 times of damping fluid as polysaccharase) (Subbotin et al, 2005,2006), obtain nematode lysate.Second step is exactly the nematode lysate that will obtain, and carries out digesting about 1h with Proteinase K, and then inactivated proteases, finally obtains DNA profiling crude extract, carries out next step PCR reaction.In addition, some other method that is simple and acquisition nematode lysate fast also has report, such as, utilize NaOH and Triton X-100 By Direct Pyrolysis whole piece worm (Stantonet al, 1998; Floyd et al, 2002), or with SDS lysate whole piece worm (Sakai, 2010; Sakai etal, 2011), be then directly used in PCR reaction.In a word, these methods all needed to add reagent before PCR reaction, so relatively consuming time and complicated operation, and the reagent of trace remains the reaction or follow-up operation that may affect PCR.And due to plant nematode polypide tiny, the general 0.5-1.2mm of ripe female male worm, polypide diameter is 30-60 μm, makes the above-mentioned cataclasm transfer process of wall scroll nematode mentioned make sample form amount lose very large or lose, there is the defect causing DNA profiling quantity not sufficient in experiment PCR.

Summary of the invention

The object of the invention is to overcome the deficiencies in the prior art, a kind of method of wall scroll nematode polypide or single nematode worm's ovum plant identification parasitic nematode is provided, the method can realize simply, fast PCR reaction qualification, can ensure again the enzyme of PCR object fragment cut and the follow-up test such as order-checking operation unaffected.

The present invention is achieved through the following technical solutions above-mentioned purpose:

A method for wall scroll nematode polypide or single nematode worm's ovum plant identification parasitic nematode, for wall scroll nematode polypide or single nematode worm's ovum for pcr template, amplification obtains conserved sequence, determines the method for line insect types after qualification.Concrete steps are as follows:

(1) washing of Plant nematode sample: drip aseptic deionized water on aseptic slide glass, choose pin with nematode and choose into wall scroll nematode or single worm's ovum;

(2) pcr template preparation: tri-distilled water is dripped on PCR pipe inwall, choose into step (1) washed wall scroll nematode or single worm's ovum, under stereoscopic microscope, nematode polypide or worm's ovum is punctured with aseptic insect needle, nematode lysate is got rid of at the bottom of pipe, and this nematode lysate directly uses as pcr template or puts into-80 DEG C of Ultralow Temperature Freezers and saves backup; For preventing aseptic insect needle from nematode polypide or worm's ovum being taken away, preferably getting rid of after at the bottom of pipe at nematode lysate, checking whether have nematode polypide or worm's ovum at the bottom of pipe under the microscope;

(3) PCR reaction: the conservative region design primer choosing nematode, the pcr template prepared with step (2), for template, carries out PCR;

(4) identify: the PCR primer obtained is analyzed, thus determines the kind of nematode.

The amount of the tri-distilled water described in step (2) is determined according to nematode size, and for nematode polypide or the worm's ovum of linear worm state, polypide is shorter than the nematode of 1.5mm and worm's ovum tri-distilled water consumption is 6 μ l, and the nematode tri-distilled water consumption that polypide is longer than 1.5mm is 12 μ l; For the polypide enlarging into the larger non-linear worm state such as spherical, pyriform, tri-distilled water consumption is 24 μ l;

Tri-distilled water described in step (2) drips to the position of PCR pipe inwall, is preferably water droplet in the position apart from the mouth of pipe about 1cm;

Insect needle described in step (2) is preferably No. 1, stainless steel insect needle;

Aseptic insect needle described in step (2) refers to and is soaked by insect needle raw spirit, then in spirit lamp flame calcination process;

Nematode lysate described in step (2) is the mixed solution of the nematode lysate that obtains of the nematode polypide that punctures or worm's ovum and tri-distilled water;

Conservative region described in step (3) is preferably mitochondrial cytochrome oxidase gene II and the one in LrRNA gene order, the Internal Transcribed Spacer ITS sequence, 28S D2-D3 amplification region sequence and Plant nematode aibuhitensis cytochrome oxidase gene Co I sequence of intervals or at least two kinds;

Described mitochondrial cytochrome oxidase gene II is as follows with the primer of LrRNA gene order:

Upstream primer #C2F3:5 '-GGTCAATGTTCAGAAATTTGTGG-3 '

Downstream primer #1108:5 '-TACCTTTGACCAATCACGCT-3 ';

The primer of described the Internal Transcribed Spacer ITS sequence is as follows:

Upstream primer: 5 '-CGTAACAAGGTAGCTGTAG-3 '

Downstream primer: 5 '-TTTCACTCGCCGTTACTAAGG-3 ';

The primer of described 28S D2-D3 amplification region sequence is as follows:

Upstream primer: 5 '-ACAAGTACCGTGAGGGAAAGTTG-3 '

Downstream primer: 5 '-TCGGAAGGAACCAGCTACTA-3 ';

The primer of described Plant nematode aibuhitensis cytochrome oxidase gene Co I sequence of intervals is as follows:

Upstream primer: 5 '-CCTACTATGATTGGTGGTTTTGGTAATTG-3 '

Downstream primer: 5 '-GTAGCAGCAGTAAAATAAGCACG-3 ';

The reaction conditions of the PCR reaction described in step (3) is as follows: 94 DEG C of 3min denaturations; 98 DEG C of sex change 10sec, 52 DEG C ~ 55 DEG C annealing 30sec, 68 DEG C extend 1min, 35 circulations; Then 68 DEG C extend 10min again;

Analysis described in step (4) comprises electrophoretic analysis, restriction analysis and sequencing analysis.

The described method with wall scroll nematode polypide or single nematode worm's ovum plant identification parasitic nematode is owing to can prepare with wall scroll nematode polypide or single nematode worm's ovum the amplification that pcr template carries out conserved sequence, therefore, also required nematode DNA sequence dna is obtained by this method.

Compared with prior art, the present invention has following beneficial effect:

(1) compared with identifying PCR with traditional wall scroll plant nematode, the present invention does not need to use the nematode lysate (WLB) of preparation of reagents or alkaline lysis or multigelation method first to carry out the Proteinase K cracking of 1h, so present invention, avoiding the pollution of other reagent, save operation steps, save the time that PCR prepares, improve working efficiency.

(2) compared with the wall scroll plant nematode PCR that need not prepare lysate process reported with other, present method avoids and first on slide glass, nematode is shredded, then transfer in PCR pipe, lose and DNA degradation because nematode material easily occurs this process.The present invention contains the operation that a step directly simply punctures polypide or worm's ovum in PCR pipe, so both ensure that the existence of nematode polypide or worm's ovum and lysate thereof, the i.e. existence of DNA profiling, do not need to carry out nematode lysate to transfer to PCR pipe from other carrier, so qualification has higher success rate yet.

(3) method reported, most single experiment of the amplification for some genome intervals, does not have system, extensively carries out the report of applied research, range of application and practicality indefinite.The present invention verifies the current molecular diagnostic markers in plant nematode qualification and the many aspects such as phyletic evolution research, and conscientiously tests further enzyme and also verify, institute is clear and definite, the applied range of practicality in this way.In addition, reported first of the present invention can to increase object fragment with single worm's ovum.

Accompanying drawing explanation

Fig. 1 is the PCR primer electrophoresis result figure using Co II-LrRNA gene universal primer amplification root knot nematode, wherein: in figure (1), swimming lane 1 ~ 2 is Meloidogyne incognita ovum, swimming lane 3 ~ 4 is Meloidogyne incognita larvae, swimming lane 5 ~ 6 is the female worm of Meloidogyne incognita, and swimming lane 7 ~ 10 is Meloidogyne incognita male worm; In figure (2), swimming lane 1 is Meloidogyne incognita two instar, and swimming lane 2 is cereal root knot nematode two instar, and swimming lane 3 is javanese root knot nematode two instar, and swimming lane 4 is Meloidogyne enterolobii two instar, and swimming lane 5 is peanut root-knot nematode two instar; In figure, swimming lane M is Marker DL2000.

Fig. 2 is the PCR primer electrophoresis result figure utilizing different cleavage method to use Co II-LrRNA gene universal primer amplification Meloidogyne incognita, wherein: figure A is for utilizing this patent method amplification Meloidogyne incognita second instar larvae, and swimming lane 1 ~ 10 is different root knot second instar larvae polypides; Figure B utilizes traditional method amplification Meloidogyne incognita second instar larvae, and 1 ~ 10 is different root knot nematode second instar larvae polypides.

Fig. 3 is the PCR primer electrophoresis result figure using ITS universal primer amplification plant nematode, wherein: in figure, swimming lane label M is Marker DL2000, 1. rot stem nematodes polypide, 2. rot stem nematodes polypide, 3. radopholus similes thorne polypide, 4. radopholus similes thorne worm's ovum, 5. Pratylenchidae polypide, 6. short cone nematode polypide, 7. species of Tylenchorhynchus Nematodes polypide, 8. helicotylenchus polypide, 9. Meloidogyne incognita two instar, 10. the female worm of Meloidogyne incognita, 11. Meloidogyne incognita male worms, 12. little loop wire worm polypides, the little loop wire worm polypide of 13. dish, 14. pine wood nematode polypides, 15. aphelenchoides polypides, 16. sword needlework worm polypides, 17. burr nematode polypides, 18. Paratylenchus polypides, 19. reniform nematode polypides, 20. rhabditis axei polypides.

Fig. 4 is the PCR primer electrophoresis result figure using 28SD2-D3 universal primer amplification plant nematode, wherein: swimming lane label 1. filaria polypides in figure, 2. rot stem nematodes polypide, 3. fuller's teasel Ditylenchus dipsaci polypide, 4. Pratylenchidae polypide, 5. the female worm of radopholus similes thorne, 6. radopholus similes thorne worm's ovum, 7. species of Tylenchorhynchus Nematodes polypide, 8. helicotylenchus polypide, 9. Meloidogyne incognita two instar, 10. Meloidogyne incognita worm's ovum, the little loop wire worm polypide of 11. dish, 12. reniform nematode polypides, 13. Paratylenchus polypides, 14. sword needlework worm polypides, M is Marker DL2000.

Fig. 5 is the PCR primer electrophoresis electrophorogram using cytochrome C o I universal primer amplification plant nematode, wherein: swimming lane numbering 1. fuller's teasel Ditylenchus dipsaci polypide in figure; 2. Pratylenchidae polypide; 3. species of Tylenchorhynchus Nematodes polypide; 4 ~ 5. reniform nematode polypides; 6. pine wood nematode polypide; 7. aphelenchoides besseyi polypide; M:Marker DL2000.

Fig. 6 is the result electrophorogram using Restriction Enzyme PCR primer to be carried out to restriction analysis, wherein: swimming lane label the Internal Transcribed Spacer ITS:1. rot stem nematodes polypide in figure, and 2. Pratylenchidae polypide, 3. radopholus similes thorne polypide, 4. short cone nematode polypide; CO I gene: 5. Pratylenchidae polypide, 6. reniform nematode polypide, 7. aphelenchoides besseyi polypide, 8. pine wood nematode polypide; D2-D3 expansion area: 9. fuller's teasel Ditylenchus dipsaci polypide, 10. Pratylenchidae polypide, the little loop wire worm polypide of 11. dish, 12. reniform nematode polypides, 13. Meloidogyne incognita polypides; A is that PCR primer Hinf I carries out enzyme and cuts process; B is that PCR primer Alu I carries out enzyme and cuts process; M:Marker DL2000.

Embodiment

Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.

The kind related in embodiment material:

Radopholus similes thorne (Radopholus similis), rot stem nematodes (has another name called sweet potato stem nematode, Ditylenchusdestructor), Pratylenchidae (Pratylenchus spp.), helicotylenchus (Helicotylenchus spp.), reniform nematode (Rotylenchulus spp.), Meloidogyne incognita (Meloidogyne incognita), javanese root knot nematode (M.javanica), species of Tylenchorhynchus Nematodes (Tylenchorhynchus spp.), silk filaria (Filenchus spp.), aphelenchoides (Ahelenchoides spp.), rhabditis axei (Rhabditida), at document " Liu Yifan, Xu Chunling, Zhang Chao, Su Xiumin, Xie Hui. the ITS-PCR method of direct-detection radopholus similes thorne from mixing nematode sample and plant tissue. Scientia Agricultura Sinica, 2011, 44(19): 3991-3998 " open.

Fuller's teasel Ditylenchus dipsaci (having another name called Ditylenchus dip saci, Ditylenchus dipsaci), document " Zhao Lirong; Xu Chunling, Hu Xuenan, clock Guoqiang. military order great waves. the test sensitivity of the Ditylenchus dip saci of intercepting and capturing. Plant Quarantine; 2011,25(5): 45-47 " open.

Aphelenchoides besseyi (Aphelenchoides besseyi), document " Pei Yanyan, Cheng Xi; Xu Chunling, Yang Zaifu, Xie Hui. rice in China nematode aphelenchoide partial mass is to the Pathogenic Tests of paddy rice. rice in China science; 2012,26(2): 218-226 " open.

Meloidogyne enterolobii (M.enterolobii), open at document " Hu M.X.; Zhuo K.; and Liao J.L..Multiplex PCR for the simultaneous identification and detection of Meloidogyneincognita; M.enterolobii; and M.javanica using DNA extracted directly fromindividual galls.Pytopathology, 2011,101 (11): 1270-1277 ".

Peanut root-knot nematode (M.arenaria), document " Wen Yanhua, Feng Zhixin, Chen Li, Xu Hanhong. Cephalotaxus fortunei branches and leaves powder prevents and treats groundnut root knot nematode disease. plant protection journal, 2004,31(2), 161-165 " open.

Pine wood nematode (Bursaphelenchus xylophilus), open at document " Wang Xin-rong; ChengXi; Li Ya-dong; Zhang Jin-ai, Zhang Zhi-fen, Wu Han-rong.Cloning arginine kinasegene and its RNAi in Bursaphelenchus xylophilus causing pine wilt disease.Eur JPlant Pathol; 2012,134:521 – 532 ".

Cereal root knot nematode (having another name called Meloidogyne graminicola, M.graminicola), document " Liu Guokun; Wang Yu, Xiao Shun, Zhang Shaosheng. the Pathogen identification of rice root Root-knot and the research of source of infection thereof. rice in China science; 2011,25(4): 420-426 " open.

Paratylenchus (Paratylenchus spp.), document " Chen Lijie, Liu Weizhi, Qin Bo. the host plant kind of Chinese Paratylenchus and areal distribution research. Liaoning agricultural sciences, 2002,3:4-8 " open.

Sword needlework worm (Longidorus spp.) and burr nematode (Tricodorus spp.), document " Chi Yuanli; Zhang Weidong; Wu Changkun; Shao Xiaoyong, Liao Li, Chen Qiwen. ornamental plant minute hand section of Guangdong Province and Trichodoridae Preliminary Report of Investigation. Plant Quarantine; 2011,25(4): 87-83 " open.

Coil little loop wire worm (Discocriconemella spp.), open at document " Powers T.O.; Harris T.; Higgins R.; Sutton L., Powers K.S..Morphological and molecular characterizationof Discocriconemella inarata, an endemic nematode from North American Nativetallgrass prairies.Journal ofNematology; 2010,42 (1): 35 – 45 ".

Little loop wire worm (Criconernella spp.), open at document " Nyczepir A.P.; Reilly C.C.; Motsinger R.E., Okie W.R..Behavior, parasitism; morphology; and biochemistry ofCriconernella xenoplax and C.ornata on peach.Journal ofNematology, 1988,20 (1): 40-46 ".

Short cone nematode (Brachydorus spp.), open at document " Koshy P.K.; Raski D.j.; andSosamma V.K..Brachydorus swarupi sp.n. (Nematoda:Dolichodorinae) from Soilabout Roots ofArecanut Palm in Kerala State; India.Journal ofNematology; 1981,13 (3): 401-404 ".

The amplification of embodiment 1 root knot nematode (Meloidogynespp.) mitochondrial cytochrome oxidase gene II (Co II) and LrRNA gene conserved sequence

This mitochondrial cytochrome oxidase gene II (Co II) and LrRNA gene conserved sequence are commonly used at present identify and agriculture production endanger larger plant nematode---root knot nematode.

(1) research material

1) line insect population: be Plant nematode research department of Agricultural University Of South China for examination Meloidogyne incognita (Meloidogyne incognita), cereal root knot nematode (M.graminicola), javanese root knot nematode (M.javanica), Meloidogyne enterolobii (M.enterolobii) and peanut root-knot nematode (M.arenaria) population and be kept at population on mater convolvulus root system.

2) primer: Co II-LrRNA universal primer is shown in document [Powers TO, Harris TS.1993.Apolymerase chain reaction method for identification of five major Meloidogynespecies.Journal of Nematology 25 (1): 1-6] shown in, specific as follows:

Upstream primer #C2F3:5 '-GGTCAATGTTCAGAAATTTGTGG-3 '

Downstream primer #1108:5 '-TACCTTTGACCAATCACGCT-3 '.

(2) test method

1) separation of root knot nematode and collection

Choose and inoculate the mater convolvulus of root knot nematode, cleaning root substrate soil, there being the part of root knot to scale off, put into culture dish, under stereoscopic microscope, solution cuts female worm, and obtains part male worm; In addition, under stereoscopic microscope, be separated the egg capsule of female worm end, obtain ovum, and part ovum is put into water hatch, after 2 ~ 3 days, collect second instar larvae;

2) washing of nematode sample

Aseptic slide glass drips aseptic deionized water, chooses pin with nematode, by above-mentioned steps 1) in the female worm, male worm, second instar larvae and the ovum that obtain choose into wherein cleaning;

3) pcr template preparation

Draw 6 μ l tri-distilled waters with micropipet to drip on the position of the span mouth of pipe about 1cm in PCR pipe, then choose step 2 respectively) in washed single ovum, larva or male worm; Draw the position that 24 μ l tri-distilled waters drip to the span mouth of pipe about 1cm in PCR pipe, then choose step 2) in washed single head enlarge into the female worm of pyriform.Under Stereo microscope, nematode polypide or line eggs is punctured with No. 1, aseptic stainless steel insect needle, then nematode lysate is got rid of at the bottom of pipe, and check whether there is nematode polypide at the bottom of pipe, if having, directly use as pcr template under the microscope, or put into-80 DEG C of Ultralow Temperature Freezers and save backup;

4) PCR reaction

Preparation PCR reaction system is: template 6 μ l, 12.5 μ l 2 × buffer(2 times damping fluids), 5 μ l 2.0mM dNTP(TOYOBO, Japan), 0.75 μ l upstream primer (10 μMs), 0.75 μ l downstream primer (10 μMs), 0.5 μ l KOD enzyme (TOYOBO, Japan), all the other are supplied with sterilizing tri-distilled water.

PCR response procedures is: 94 DEG C of 3min denaturations, 98 DEG C of sex change 10sec, and 52 DEG C of annealing 30sec, 68 DEG C extend 1min, and 35 circulations, then 68 DEG C extend 10min again.

Prepare the reaction product of system, put into the enterprising performing PCR amplification of Bole C-1000PCR instrument.

5) PCR reaction result detects

The agarose gel electrophoresis getting 5 μ l PCR primer functional quality volume ratios 1% detects PCR result, gel imaging system is observed PCR primer amplified fragments size and with or without (Fig. 1).Can be increased to by Fig. 1 (1) known use the method the conservative section of the female worm of Meloidogyne incognita, male worm, ovum and second instar larvae, and amplification efficiency is high, and object band is very clear.In addition, from Fig. 1 (2), use the method to increase different types of root knot nematode, also can obviously increase object fragment, and this object fragment is consistent with bibliographical information.

Embodiment 2 more different cleavage method amplification root knot nematode plastosome Co II and LrRNA gene conserved sequence

(1) research material

1) line insect population: be kept at the population on mater convolvulus root system for examination Meloidogyne incognita (Meloidogyne incognita) for Plant nematode research department of Agricultural University Of South China.

2) primer: with embodiment 1.

(2) test method

1) separation of root knot nematode and collection, washing

With embodiment 1.

2) pcr template preparation

A group: be method provided by the invention, with embodiment 1 step (2) 3).

B group: traditional method: picking wall scroll second instar larvae has on the aseptic slide of a droplet sterilized water to dripping, then under stereoscope, fast nematode polypide is cut into 2 ~ 3 sections with the scalper that spirit lamp flame burnt, and rapid pipettor siphons away suspension in the PCR pipe of precooling, add 1 × polymerase buffer (Takara) to 10 μ l, put into very low temperature more than incubator freezing treatment 30min.Take out the nematode lysate that freezing treatment is crossed, then add Proteinase K in 65 DEG C of digestion 1h, then 95 DEG C of process 10min, make Proteinase K inactivation, obtain pcr template by these steps.

3) PCR reaction

With embodiment 1.

4) PCR reaction result detects

The agarose gel electrophoresis getting 5 μ l PCR primer functional quality volume ratios 1% detects PCR result, gel imaging system is observed PCR primer amplified fragments size and with or without (Fig. 2 A and B).Can be taken to the amplification efficiency of 100% by the known use the method for Fig. 2 A, object band is very clear, and PCR primer content is high; The indivedual repeat amplification protcol of Fig. 2 B known use traditional method does not go out band, and some amplifies band, but PCR object content of fragment is few, so band brightness is weak.

Result shows the traditional method that the used method preparing nematode DNA profiling of the present invention compares above-mentioned B group and has greatly improved in pcr amplification result and significant advantage.

The pcr amplification of embodiment 3 plant nematode the Internal Transcribed Spacer (ITS)

ITS sequence is the Internal Transcribed Spacer, does not express, and comprises the 5.8S gene of the Internal Transcribed Spacer ITS1, ITS2 and centre, is the important molecular markers of current Plant nematode Molecular Identification.

(1) research material

1) line insect population: rot stem nematodes (Ditylenchus destructor), radopholus similes thorne (Radopholus similis), Pratylenchidae (Pratylenchus sp.), short cone nematode (Brachydorus sp.), species of Tylenchorhynchus Nematodes (Tylenchorhynchus sp.), helicotylenchus (Helicotylenchus sp.), Meloidogyne incognita (Meloidogyne incognita), little loop wire worm (Criconemella sp.), coil little loop wire worm (Discocrionemella sp.), pine wood nematode (Bursaphelenchus xylophilus), aphelenchoides besseyi (Aphelenchoides besseyi), sword needlework worm (Xiphidorus sp.), burr nematode (Trichodorussp.), Paratylenchus (Paratylenchus sp.), reniform nematode (Rotylenchulus sp.) and Rhabditida nematode (Rhabditida), these populations are except rot stem nematodes, radopholus similes thorne, pine wood nematode and Meloidogyne incognita are that Plant nematode research department of Agricultural University Of South China preserves population, and all the other are separated in Agricultural University Of South China's arboretum rhizosphere soil.

2) primer: ITS amplified reaction universal primer is as document [Ferris VR, Ferris JM, and FaghihiJ.1993.Variation in spacer ribosomal DNA in some cyst-forming species of plantparasitic nematodes.Fundamental and Applied Nemato 14logy 16:177-184; VrainTC, Wakarchuk DA, Levesque AC, Hamilton RI.1992.Intraspecific rDNA restrictionfragment length polymorphism in the Xiphinema americanum group.FundamentalandAppliedNematology 15:563-573.] shown in, specific as follows:

Upstream primer: 5 '-CGTAACAAGGTAGCTGTAG-3 '

Downstream primer: 5 '-TTTCACTCGCCGTTACTAAGG-3 ';

(2) test method

1) separation of Plant nematode

The rot stem nematodes using carrot callus to cultivate and radopholus similes thorne, and use the pine wood nematode of fungus culture, directly in culture dish, add sterilized water, then can obtain the polypide of a large amount of different worm state.The acquisition of Meloidogyne incognita is with embodiment 1.In other soil nematode use shellfish graceful funnel method carry out being separated [Xie Hui .2005. Plant nematode taxonomy (second edition). Beijing: Higher Education Publishing House, p 38-40].

2) washing of nematode sample

Several aseptic deionized waters on aseptic slide glass drips, by above-mentioned steps 1) in the nematode polypide that obtains and line eggs, choose pin with nematode and choose in aseptic deionized water and clean;

3) pcr template preparation

Outside sword needlework worm (Xiphidorus sp.) polypide of being longer than 1.5mm is chosen in the PCR pipe that 12 μ l tri-distilled waters are housed and punctures, the method for nematodes is with embodiment 1.

4) PCR reaction

Reaction system is with embodiment 1.Response procedures except annealing temperature is 55 DEG C, the other the same as in Example 1.

5) PCR reaction result detects

The agarose gel electrophoresis getting 5 μ l PCR primer functional quality volume ratios 1% detects PCR result, gel imaging system is observed PCR primer amplified fragments size and with or without (Fig. 3).As shown in Figure 3, can amplify the object band of all population nematodes by this method, band concentration is high.

The pcr amplification of embodiment 4 plant nematode 28S gene D2-D3 expansion area

Plant nematode 28S D2-D3 amplification region sequence is that variance ratio is interval faster, but conservative in planting, and is used to the important molecular markers studying Plant nematode molecular evolution.

(1) material

1) line insect population:

Silk filaria (Filenchus sp.), rot stem nematodes (Ditylenchus destructor), fuller's teasel Ditylenchus dipsaci (D.dipsaci) radopholus similes thorne (Radopholus similis), Pratylenchidae (Pratylenchussp.), species of Tylenchorhynchus Nematodes (Tylenchorhynchus sp.), helicotylenchus (Helicotylenchus sp.), Meloidogyne incognita (Meloidogyne incognita), coil little loop wire worm (Discocrionemella sp.), sword needlework worm (Xiphidorus sp.), Paratylenchus (Paratylenchus sp.), reniform nematode (Rotylenchulus sp.), population source obtains with embodiment 3.

2) primer: use the universal primer of amplification D2D3 as document [Kaplan DT, Thomas WK, FrisseLM, SarahJL, Stanton JM, Speijer PR, Marin DH, Opperman CH.2000.Phylogenetic analysis of geographically diverse Radopholus similis via DNAsequence reveals a monomorphic motif.Journal ofNematology 32 (2): 134-142] shown in:

Upstream primer: 5 '-ACAAGTACCGTGAGGGAAAGTTG-3 '

Downstream primer: 5 '-TCGGAAGGAACCAGCTACTA-3 ';

(2) test method

1) separation of Plant nematode

With embodiment 3.

2) washing of nematode sample

With embodiment 1.

3) pcr template preparation

With embodiment 1.

4) PCR reaction

Reaction system is with embodiment 3.

5) PCR reaction result detects

The agarose gel electrophoresis getting 5 μ l PCR primer functional quality volume ratios 1% detects PCR result, gel imaging system is observed PCR primer amplified fragments size and with or without (Fig. 4).As shown in Figure 4, can amplify the object band of all population nematodes by this method, band concentration is high.

The amplification in embodiment 5 plant nematode aibuhitensis cytochrome oxidase gene Co I interval

Plant nematode aibuhitensis cytochrome oxidase gene Co I sequence of intervals is also conservative, is used to the important molecular markers studying Plant nematode molecular evolution.

(1) material

1) line insect population:

Fuller's teasel Ditylenchus dipsaci (Ditylenchus dipsaci), Pratylenchidae (Pratylenchus sp.), species of Tylenchorhynchus Nematodes (Tylenchorhynchus sp.), reniform nematode (Rotylenchulus sp.), pine wood nematode (Bursaphelenchus xylophilus) and aphelenchoides besseyi (Aphelenchoides besseyi), wherein Pratylenchidae, species of Tylenchorhynchus Nematodes and reniform nematode are separated from Agricultural University Of South China's arboretum rhizosphere soil, and other is that Plant nematode research department of Agricultural University Of South China preserves population.

2) primer: use the universal primer (Kanzaki & Futai, 2002) between amplifying cells chromo-oxidase CO I gene conserved regions:

Upstream primer: 5 '-CCTACTATGATTGGTGGTTTTGGTAATTG-3 '

Downstream primer: 5 '-GTAGCAGCAGTAAAATAAGCACG-3 ';

(2) test method

1) separation of Plant nematode

Aphelenchoides besseyi also cultivates for carrot tissue the population preserved, and separation method is with embodiment 3.The separation of nematodes population is with embodiment 3.

2) washing of nematode sample

With embodiment 1.

3) pcr template preparation

With embodiment 1.

4) PCR reaction

Reaction system is with embodiment 3.

5) PCR reaction result detects

The agarose gel electrophoresis getting 5 μ l PCR primer functional quality volume ratios 1% detects PCR result, gel imaging system is observed PCR primer amplified fragments size and with or without (Fig. 5).As shown in Figure 5, can amplify the object band of these population nematodes by this method, band concentration is high.

The restricted enzyme cutting analysis of embodiment 6PCR product

Whether PCR primer is pure, and whether containing being unfavorable for the reagent that follow-up molecular biology test operates, using Restriction Enzyme to carry out restriction analysis is means, and in addition, restricted enzyme cutting analysis is also the multifarious a kind of instrument of test strip, and purposes is wider.

(1) test materials

From the PCR primer in embodiment 1 ~ 3.

Restriction Enzyme: Hinf I and Alu I (Takara, the precious biotechnology company limited in Dalian).

(2) test method

Restricted enzyme cutting analysis system

The enzyme preparing 20 μ l cuts system: get PCR primer 10 μ l, adds each 1 μ l of Restriction Enzyme Hinf I and Alu I respectively, and add each 2 μ l of corresponding enzyme Buffer H and Buffer L, all the other are supplied with ultrapure water.

The enzyme prepared is cut system and is carried out in C-1000PCR instrument, and setting program is 37 DEG C of 1h, then uses 95 DEG C of 5min.

(3) enzyme cuts interpretation of result

The system that enzyme cuts, gets 5 μ l reaction product, the agarose gel electrophoresis of mass volume ratio 2% carries out electrophoresis, and gel imaging system is observed the size (Fig. 6) of PCR primer amplified fragments.Due to the enzyme that the two kinds of enzymes selected are all conventional in Plant nematode object fragment restricted enzyme cutting analysis, a lot of enzyme cuts result analysis of report mistake.As shown in Figure 6, all object PCR fragment can well be carried out enzyme and be cut, and enzyme slitting band position description restriction enzyme site is accurately identified.

The result of above embodiment illustrates, effectively can increase obtain the gene order of nematode with wall scroll nematode polypide or single nematode worm's ovum, and the gene order that obtains of increasing can be carried out enzyme and cut.Therefore, invention provided by the present invention can be applied to the qualification of the kind of nematode.

Above embodiment is the present invention's preferably embodiment; for explaining technical scheme of the present invention; but any pro forma restriction is not done to the present invention, any Change Example done under other do not deviate from the prerequisite of essence of the present invention as modified, substituting, combination, the mode such as simplification all should be included within protection scope of the present invention.

Claims (10)

1., by a method for wall scroll nematode polypide or single nematode worm's ovum plant identification parasitic nematode, it is characterized in that comprising following steps:

(1) washing of Plant nematode sample: drip aseptic deionized water on aseptic slide glass, choose pin with nematode and choose into wall scroll nematode or single worm's ovum;

(2) pcr template preparation: tri-distilled water is dripped on PCR pipe inwall, choose into step (1) washed wall scroll nematode or single worm's ovum, under stereoscopic microscope, nematode polypide or worm's ovum is punctured with aseptic insect needle, nematode lysate is got rid of at the bottom of pipe, and this nematode lysate directly uses as pcr template or puts into-80 DEG C of Ultralow Temperature Freezers and saves backup;

(3) PCR reaction: the conservative region design primer choosing nematode, the pcr template prepared with step (2), for template, carries out PCR;

(4) identify: the PCR primer obtained is analyzed, thus determines the kind of nematode.

2. the method for wall scroll nematode polypide according to claim 1 or single nematode worm's ovum plant identification parasitic nematode, it is characterized in that: the amount of the tri-distilled water described in step (2) is determined according to nematode size, for nematode polypide or the worm's ovum of linear worm state, polypide is shorter than the nematode of 1.5mm and worm's ovum tri-distilled water consumption is 6 μ l, and the nematode tri-distilled water consumption that polypide is longer than 1.5mm is 12 μ l; For the polypide enlarging into the larger non-linear worm state such as spherical, pyriform, tri-distilled water consumption is 24 μ l.

3. the method for wall scroll nematode polypide according to claim 1 or single nematode worm's ovum plant identification parasitic nematode, is characterized in that: the tri-distilled water described in step (2) drips to the position of span pipe 1cm in PCR pipe.

4. the method for wall scroll nematode polypide according to claim 1 or single nematode worm's ovum plant identification parasitic nematode, is characterized in that: the insect needle described in step (2) is No. 1, stainless steel insect needle.

5. the method for wall scroll nematode polypide according to claim 1 or single nematode worm's ovum plant identification parasitic nematode, it is characterized in that: the aseptic insect needle described in step (2) refers to and soaked by insect needle raw spirit, then in spirit lamp flame calcination process.

6. the method for wall scroll nematode polypide according to claim 1 or single nematode worm's ovum plant identification parasitic nematode, is characterized in that: the nematode lysate described in step (2) is the mixed solution of the nematode lysate that obtains of the nematode polypide that punctures or worm's ovum and tri-distilled water.

7. the method for wall scroll nematode polypide according to claim 1 or single nematode worm's ovum plant identification parasitic nematode, is characterized in that: the conservative region described in step (3) is mitochondrial cytochrome oxidase gene II and the one in LrRNA gene order, the Internal Transcribed Spacer ITS sequence, 28S D2-D3 amplification region sequence and Plant nematode aibuhitensis cytochrome oxidase gene Co I sequence of intervals or at least two kinds.

8. the method for wall scroll nematode polypide according to claim 7 or single nematode worm's ovum plant identification parasitic nematode, is characterized in that:

Described mitochondrial cytochrome oxidase gene II is as follows with the primer of LrRNA gene order:

Upstream primer #C2F3:5 '-GGTCAATGTTCAGAAATTTGTGG-3 '

Downstream primer #1108:5 '-TACCTTTGACCAATCACGCT-3 ';

The primer of described the Internal Transcribed Spacer ITS sequence is as follows:

Upstream primer: 5 '-CGTAACAAGGTAGCTGTAG-3 '

Downstream primer: 5 '-TTTCACTCGCCGTTACTAAGG-3 ';

The primer of described 28S D2-D3 amplification region sequence is as follows:

Upstream primer: 5 '-ACAAGTACCGTGAGGGAAAGTTG-3 '

Downstream primer: 5 '-TCGGAAGGAACCAGCTACTA-3 ';

The primer of described Plant nematode aibuhitensis cytochrome oxidase gene Co I sequence of intervals is as follows:

Upstream primer: 5 '-CCTACTATGATTGGTGGTTTTGGTAATTG-3 '

Downstream primer: 5 '-GTAGCAGCAGTAAAATAAGCACG-3 '.

9. the method for wall scroll nematode polypide according to claim 1 or single nematode worm's ovum plant identification parasitic nematode, is characterized in that: the reaction conditions of the PCR reaction described in step (3) is as follows: 94 DEG C of 3min denaturations; 98 DEG C of sex change 10sec, 52 DEG C ~ 55 DEG C annealing 30sec, 68 DEG C extend 1min, 35 circulations; Then 68 DEG C extend 10min again.

10. the method for wall scroll nematode polypide according to claim 1 or single nematode worm's ovum plant identification parasitic nematode, is characterized in that: the analysis described in step (4) comprises electrophoretic analysis, restriction analysis and sequencing analysis.