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 nematodes by using a single nematode body or a single nematode egg, and belongs to the technical field of molecular biology detection and identification. In molecular biology experiments, due to the small size of nematodes, it is easy to be contaminated by other reagents in the process of extracting a single nematode DNA template or to cause serious material loss during the process of cutting a single nematode. The present invention includes a step of directly piercing the nematode body or egg in the PCR tube without transferring the nematode lysate from other carriers to the PCR tube, thus ensuring the existence of the nematode and its lysate, namely With the presence of DNA templates, the efficiency of template preparation is high. The present invention carries out PCR verification and partial further enzyme cutting experiment verification on molecular diagnostic markers currently used in multiple aspects of plant parasitic nematode identification research, etc., and the effect is remarkable. The invention also discloses for the first time that a single nematode egg is used as a template to identify plant parasitic nematodes.

Description

Method for identifying plant parasitic nematodes by using single nematode body or single nematode egg

technical field

The invention belongs to the technical field of molecular biology detection and identification, and in particular relates to a method for identifying plant parasitic nematodes by using a single nematode body or a single nematode egg.

Background technique

Plant parasitic nematodes are a class of plant pathogens that endanger agricultural and forestry production worldwide. About 4100 species of plant nematodes have been described so far, accounting for about 15% of all nematode species described (Decraemer & Hunt, 2006). Many plant-parasitic nematodes, such as Radopholus similis, Meloidogyne spp. Forestry trees cause serious damage, so many countries have listed these plant nematodes as quarantine pests (CABI & EPPO, 1998). The traditional classification and identification of plant parasitic nematodes relies on morphological characteristics. This method requires rich experience in classification and identification. At the species level, the accurate identification of some similar species is more complicated and difficult. Since the invention of PCR technology and the complete sequencing of the genome of Caenorhabditis elegans, molecular diagnostic and identification methods have been widely used in free-living nematodes, animal-parasitic nematodes and plant-parasitic nematodes, and for the phylogenetic evolution of these nematodes. and classification studies provide richer information (Floyd et al, 2002; Powers, 2004; Abebe et al, 2011).

In the taxonomy of nematodes, the arrangement of ribosomal RNA and its spacer and the sequence characteristics of some intervals in the mitochondrial genome are often used as the basis for identification. According to the conservation of the sequence interval, many general or specific primers have been designed for the identification or phylogenetic analysis of nematodes. For example, sequences amplified with universal primers for the internal transcribed spacer (ITS) as markers for identification, sequences amplified with universal primers for the D2-D3 extension region of the 28S rRNA gene were used to analyze nematode phylogenetic evolution. Specific primers (CI O or COII) for some conserved intervals of the mitochondrial cytochrome oxidase gene for evolutionary analysis (Powers & Harris, 1993; Al-Banna et al, 1997; Subbotin et al, 2000; Powers, 2004; Subbotin et al, 2005a ; De Ley et al, 2005).

Molecular identification of plant-parasitic nematodes requires DNA templates. Plant nematodes mixed with multiple species can usually be isolated from soil and plant tissues in nature at one time, so the DNA extraction of a single plant parasitic nematode is more meaningful in taxonomy, phylogenetic evolution, and nematode detection and identification. The acquisition of single plant nematode DNA usually includes two steps, one is to obtain the nematode lysate, and the other is to obtain the crude DNA extract. For the first step, the common method is to pick a single worm on a glass slide, cut it into 2 to 3 sections under a stereo microscope, and transfer it to the nematode lysate (WLB) with a pipette. The lysate was then repeatedly frozen and thawed to fully lyse the parasite 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 ); You can also put nematode body fragments or whole worms into a simple nematode lysis solution (such as 1 times polymerase buffer) (Subbotin et al, 2005, 2006) to obtain nematode lysates. The second step is to digest the obtained nematode lysate with proteinase K for about 1 hour, and then inactivate the protease to finally obtain the crude extract of DNA template for the next step of PCR reaction. In addition, other simple and rapid methods for obtaining nematode lysates have also been reported, such as direct lysis of whole worms with NaOH and Triton X-100 (Stanton et al, 1998; Floyd et al, 2002), or whole worms with SDS lysate (Sakai, 2010; Sakai et al, 2011), and then directly used in PCR reactions. In a word, these methods all need to add reagents before the PCR reaction, so they are relatively time-consuming and complicated to operate, and trace reagent residues may affect the PCR reaction or subsequent operations. Moreover, due to the small size of plant parasitic nematodes, mature male and female worms are generally 0.5-1.2mm, and the diameter of the worm body is 30-60μm, so that the above-mentioned single nematode fragmentation and transfer process will cause a large loss or loss of sample templates, which may lead to experimental results. Use the defect of insufficient amount of DNA template in PCR.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for identifying plant-parasitic nematodes with a single nematode body or a single nematode egg. This method can realize simple and fast PCR reaction identification, and can ensure the PCR target fragment Subsequent test operations such as enzyme digestion and sequencing will not be affected.

The present invention realizes above-mentioned object through following technical scheme:

A method for identifying plant-parasitic nematodes by using a single nematode body or a single nematode egg is a method for using a single nematode body or a single nematode egg as a PCR template, amplifying a conserved sequence, and determining the nematode species after identification. Specific steps are as follows:

(1) Washing of plant nematode samples: drop sterile deionized water on a sterile glass slide, and pick in a single nematode or a single egg with a nematode needle;

(2) PCR template preparation: drop three-distilled water onto the inner wall of the PCR tube, pick a single nematode or a single egg washed in step (1), and use a sterile insect needle to puncture the nematode body or worm under a stereomicroscope. Eggs, throw the nematode lysate into the bottom of the tube. The nematode lysate can be used directly as a PCR template or stored in a -80°C ultra-low temperature refrigerator for later use; After the worm lysate is thrown into the bottom of the tube, check under the microscope whether there are nematode worms or eggs at the bottom of the tube;

(3) PCR reaction: select the conserved region of nematodes to design primers, and use the PCR template prepared in step (2) as a template to perform PCR;

(4) Identification: The obtained PCR product is analyzed to determine the type of nematode.

The amount of triple-distilled water described in step (2) depends on the size of the nematode. For the nematode body or eggs of nematodes, the amount of triple-distilled water for nematodes and eggs shorter than 1.5mm is 6 μl. The amount of triple-distilled water for nematodes longer than 1.5 mm is 12 μl; for larger non-nematodes such as spherical and pear-shaped worms, the amount of triple-distilled water is 24 μl;

The position where the three-distilled water drops onto the inner wall of the PCR tube in step (2), preferably the position where the water drop is about 1 cm away from the mouth of the tube;

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

The sterile insect needle described in step (2) refers to soaking the insect needle in absolute alcohol, and then burning it in the flame of an alcohol lamp;

The nematode lysate described in step (2) is a mixture of nematode lysates obtained from punctured nematode bodies or eggs and three-distilled water;

The conserved region described in step (3) is preferably mitochondrial cytochrome oxidase gene II and LrRNA gene sequence, internal transcription spacer ITS sequence, 28S D2-D3 amplification region sequence and plant nematode cytochrome oxidase gene Co Ⅰ interval one or at least two of the sequence;

The primers of the mitochondrial cytochrome oxidase gene II and LrRNA gene sequences are as follows:

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

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

The primers of the internal transcription spacer ITS sequence are as follows:

Upstream primer: 5'-CGTAACAAGGTAGCTGTAG-3'

Downstream primer: 5'-TTTCACTCGCCGTTACTAAGG-3';

The primers of the 28S D2-D3 amplification region sequence are as follows:

Upstream primer: 5'-ACAAGTACCGTGAGGGAAAGTTG-3'

Downstream primer: 5'-TCGGAAGGAACCAGCTACTA-3';

The primers of the plant nematode cytochrome oxidase gene Co I interval sequence are as follows:

Upstream primer: 5'-CCTACTATGATTGGTGGTTTTGGTAATTG-3'

Downstream primer: 5'-GTAGCAGCAGTAAAATAAGCACG-3';

The reaction conditions of the PCR reaction described in step (3) are as follows: pre-denaturation at 94°C for 3 minutes; denaturation at 98°C for 10 sec, annealing at 52°C to 55°C for 30 sec, extension at 68°C for 1 min, 35 cycles; and extension at 68°C for 10 min;

The analysis described in step (4) includes electrophoresis analysis, enzyme digestion analysis and sequencing analysis.

The method for identifying plant-parasitic nematodes with a single nematode body or a single nematode egg can also be obtained by using a single nematode body or a single nematode egg to prepare a PCR template for amplification of a conserved sequence. Desired nematode DNA sequence.

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

(1) Compared with the traditional single plant parasitic nematode identification PCR, the present invention does not need to use nematode lysate (WLB) prepared by reagents or alkaline lysis or repeated freezing and thawing for 1 h of proteinase K cleavage, so the present invention avoids The pollution of other reagents saves the operation steps, saves the time of PCR preparation, and improves the work efficiency.

(2) Compared with other reported single plant parasitic nematodes that do not need to be prepared with lysate for PCR, the method of the present invention avoids first chopping up the nematodes on the glass slide and then transferring them to the PCR tube, because this process is easy to occur Nematode material loss and DNA degradation. The present invention includes a step of directly piercing the worm body or egg in the PCR tube, which not only ensures the existence of the nematode body or egg and its lysate, that is, the existence of the DNA template, but also does not need to carry out the nematode The lysate is transferred from other carriers to PCR tubes, so the identification success rate is higher.

(3) Most of the reported methods are only used for the amplification experiment of a certain genomic interval, and there are no reports of systematic and extensive application research, and the scope of application and practicability are not clear. The present invention verifies the current molecular diagnostic markers in various aspects such as plant parasitic nematode identification and phylogenetic research, and also verifies further enzyme cutting experiments, so the method has clear practicability and wide application range. In addition, the present invention reports for the first time that a single worm egg can be used to amplify the target fragment.

Description of drawings

Figure 1 is the electrophoresis results of PCR products of root-knot nematode amplified with Co Ⅱ-LrRNA gene universal primers, in which: lanes 1-2 in Figure (1) are eggs of M. incognita, and lanes 3-4 are M. incognita Nematode larvae, lanes 5-6 are females of M. incognita, lanes 7-10 are males of M. incognita; lane 1 in Figure (2) is the second instar of M. incognita, and lane 2 is M. incognita Second instars, lane 3 is second instars of root-knot nematode javanica, lane 4 is second instars of root-knot nematode elegans, and lane 5 is second instars of root-knot nematode peanut; lane M in the figure is Marker DL2000.

Figure 2 is the result of electrophoresis of the PCR products of Meloidogyne incognita amplified by using Co Ⅱ-LrRNA gene universal primers using different cleavage methods, wherein: Figure A is the second instar larvae of Meloidogyne incognita amplified by the patented method, lanes 1- 10 is the second instar larvae of different root-knot nematodes; Figure B uses the traditional method to amplify the second instar larvae of M. incognita, and 1-10 are the second instar larvae of different root-knot nematodes.

Figure 3 is the result of electrophoresis of PCR products of plant parasitic nematodes amplified using ITS universal primers, in which: the lane label M in the figure is Marker DL2000; Nematode worm body, 4. Egg of banana perforator nematode, 5. Short body nematode body, 6. Short trypanosoma worm body, 7. Dwarf nematode body, 8. Spiral worm body, 9. Meloidogyne incognita II Instars, 10. Meloidogyne incognita female, 11. Meloidogyne incognita male, 12. Microcircle nematode body, 13. Discocircle nematode body, 14. Pine wood nematode body, 15. Slippery blade Nematode worm body, 16. sword needle nematode worm body, 17. burr nematode worm body, 18. needle nematode worm body, 19. reniform nematode worm body, 20. small rod nematode worm body.

Figure 4 is the result of electrophoresis of PCR products amplified by 28SD2-D3 universal primers of plant parasitic nematodes, wherein: the lane numbers in the figure are 1. silkworm body, 2. D. destructor body, and 3. D. rotunda Insect body, 4. Short nematode body, 5. Banana perforator female, 6. Banana perforator egg, 7. Dwarf nematode body, 8. Spiral worm body, 9. Meloidogyne incognita second instar Worms, 10. Meloidogyne incognita worms, 11. Discocycline worm body, 12. Reniform worm body, 13. Acupunculus worm body, 14. Sword needle worm body, M is Marker DL2000.

Fig. 5 is the electrophoresis electrophoresis diagram of the PCR products of plant parasitic nematodes amplified using cytochrome Co Ⅰ universal primers, wherein: the number of swimming lanes in the figure is 1. D. velvetis worm body; 2. Short-bodied nematode worm body; 3. Dwarfing Nematode worm body; 4-5. Reniform nematode body; 6. Pine wood nematode body;

Figure 6 is an electrophoresis diagram of the results of enzyme digestion analysis of PCR products using restriction enzymes, wherein: in the figure, the ITS of the transcriptional spacer in the label of the swimming lane: 1. D. destructor worm body, 2. Brachynematode worm body, 3. Banana Perforator nematode body, 4. Trypanosoma brevis body; CO Ⅰ gene: 5. Brachyderm nematode body, 6. Reniform nematode body, 7. Rice stem tip nematode body, 8. Pine wood nematode body; D2-D3 extension area: 9. D. velvet worm body, 10. Brachyphyllum worm body, 11. Discocircle worm body, 12. Renal nematode body, 13. Meloidogyne incognita worm body; A: PCR products were digested with Hinf Ⅰ; B: PCR products were digested with Alu Ⅰ; M: Marker DL2000.

Detailed ways

The present invention will be further described in detail below with reference to the examples and drawings, but the implementation of the present invention is not limited thereto.

Kinds involved in the example material:

Banana punch nematode (Radopholus similis), Ditylenchus destructor (aka sweet potato stem nematode, Ditylenchus destructor), Bratylenchus spp., Helicotylenchus spp., Rotylenchulus spp., Southern root-knot nematode Meloidogyne incognita, M. javanica, Tylenchorhynchus spp., Filenchus spp., Ahelenchoides spp., Rhabditida, It has been published in the literature "Liu Yifan, Xu Chunling, Zhang Chao, Su Xiumin, Xie Hui. ITS-PCR method for direct detection of banana perforator nematode from mixed nematode samples and plant tissues. Chinese Agricultural Sciences, 2011, 44 (19): 3991- 3998" public.

Ditylenchus dipsaci (also known as Ditylenchus dipsaci), has been published in the literature "Zhao Lirong, Xu Chunling, Hu Xuenan, Zhong Guoqiang. Wu Mutao. Inspection and identification of intercepted Ditylenchus dipsaci. Plant Quarantine, 2011 , 25(5):45-47” Open.

Aphelenchoides besseyi has been published in the literature "Pei Yanyan, Cheng Xi, Xu Chunling, Yang Zaifu, Xie Hui. Determination of the pathogenicity of some populations of rice Aphelenchoides besseyi to rice. Chinese Rice Science, 2012, 26 (2 ): 218-226" Open.

Root-knot nematode (M.enterolobii) has been described in the literature "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" published.

Peanut root-knot nematode (M.arenaria), has been published in the literature "Wen Yanhua, Feng Zhixin, Chen Li, Xu Hanhong. The control of peanut root-knot nematode by the powder of three-pointed fir branches and leaves. Acta Plant Protection, 2004, 31 (2), 161- 165" open.

Pine wood nematode (Bursaphelenchus xylophilus), has been published in the literature "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” published.

Root-knot nematode graminearum (also known as root-knot nematode Gramineae, M.graminicola), has been published in the literature "Liu Guokun, Wang Yu, Xiao Shun, Zhang Shaosheng. Pathogen identification and infection source of rice root-knot nematode. China Rice Science, 2011, 25(4): 420-426" published.

Paratylenchus spp. has been published in the literature "Chen Lijie, Liu Weizhi, Qin Bo. Research on the host plant species and geographical distribution of Chinese needle nematodes. Liaoning Agricultural Sciences, 2002, 3:4-8".

Longidorus spp. and Tricodorus spp. have been published in the literature "Chi Yuanli, Zhang Weidong, Wu Changkun, Shao Xiaoyong, Liao Li, Chen Qiwen. Preliminary investigation report on the nematodes of Longidorus and Tricodorus spp. in garden plants in Guangdong Province . Phytosanitary, 2011, 25(4): 87-83" published.

Discocriconemella spp. has been published in the literature "Powers T.O., Harris T., Higgins R., Sutton L., Powers K.S.. Morphological and molecular characterization of Discocriconemella inarata, an endemic nematode from North American Nativetallgrassournprairies.Journal , 2010, 42(1):35–45” published.

Criconernella spp. has been published in the literature "Nyczepir A.P., Reilly C.C., Motsinger R.E., Okie W.R..Behavior, parasitism, morphology, and biochemistry of Criconernella xenoplax and C.ornata on peach. Journal of Nematology, 2( ):40-46" open.

Brachydorus spp., has been described in the literature "Koshy P.K., Raski D.j., and Sosamma V.K.. Brachydorus swarupi sp.n. (Nematoda: Dolichodorinae) from Soilabout Roots of Arecanut Palm in Kerala State, India. Journal of Nematology, 131, 198 (3):401-404" open.

Example 1 Amplification of the Conserved Sequences of the Meloidogynes pp. Mitochondrial Cytochrome Oxidase Gene II (CoII) and LrRNA Gene

The conserved sequences of mitochondrial cytochrome oxidase gene Ⅱ (Co Ⅱ) and LrRNA gene are commonly used to identify root-knot nematode, a plant parasitic nematode that is more harmful to agricultural production.

(1) Research materials

1) Nematode populations: Meloidogyne incognita, M.graminicola, M.javanica, M.enterolobii and peanut root The populations of knot nematodes (M.arenaria) were all preserved on the roots of water spinach by the Plant Nematode Research Laboratory of South China Agricultural University.

2) Primers: Co Ⅱ-LrRNA general primers are shown in the literature [Powers TO, Harris TS.1993. Apolymerase chain reaction method for identification of five major Meloidogyne species. Journal of Nematology 25(1):1-6], details are as follows :

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

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

(2) Test method

1) Isolation and collection of root-knot nematodes

Pull out the water spinach inoculated with root-knot nematodes, clean the root substrate soil, cut off the part with root knots, put it in a petri dish, dissect out the female insects under a stereo microscope, and obtain some male insects; Under the microscope, separate the oocysts at the end of the female, obtain the eggs, and put some eggs into the water to hatch, and collect the second instar larvae after 2 to 3 days;

2) Washing of nematode samples

Drop sterile deionized water on a sterile glass slide, pick the needle with nematodes, and pick the females, males, second-instar larvae and eggs obtained in the above step 1) into it for cleaning;

3) PCR template preparation

Use a micropipette to draw 6 μl triple-distilled water and drop it on the inner wall of the PCR tube about 1 cm away from the tube opening, and then pick the single eggs, larvae or males washed in step 2) respectively; draw 24 μl triple-distilled water and drop it into the PCR tube The wall is about 1cm away from the mouth of the nozzle, and then the single pear-shaped female worm that has been washed in step 2) is picked. Use a sterile stainless steel insect needle No. 1 to puncture the nematode body or nematode egg under a stereomicroscope, then throw the nematode lysate into the bottom of the tube, and check under the microscope whether there is a nematode body at the bottom of the tube, and if so, use it as a PCR template Use directly, or store in a -80°C ultra-low temperature freezer for later use;

4) PCR reaction

Prepare the PCR reaction system as follows: template 6 μl, 12.5 μl 2×buffer (2 times buffer), 5 μl 2.0mM dNTP (TOYOBO, Japan), 0.75 μl upstream primer (10 μM), 0.75 μl downstream primer (10 μM), 0.5 μl KOD Enzyme (TOYOBO, Japan), and the rest was supplemented with sterilized three-distilled water.

The PCR reaction program was: pre-denaturation at 94°C for 3 min, denaturation at 98°C for 10 sec, annealing at 52°C for 30 sec, extension at 68°C for 1 min, 35 cycles, and then extension at 68°C for 10 min.

Prepare the reaction product of the system and put it into the Bio-Rad C-1000 PCR instrument for PCR amplification.

5) Detection of PCR reaction results

Take 5 μl of PCR products and use agarose gel electrophoresis with a mass volume ratio of 1% to detect the PCR results, and observe the size and presence or absence of the amplified fragments of the PCR products on a gel imaging system (Figure 1). It can be seen from Figure 1 (1) that this method can amplify the conserved segments of females, males, eggs, and second-instar larvae of M. incognita. The amplification efficiency is high, and the target band is very clear. In addition, it can be seen from Fig. 1 (2) that different species of root-knot nematodes can be amplified using this method, and the target fragment can also be amplified very obviously, and the target fragment is consistent with that reported in the literature.

Example 2 Comparison of different lysis methods to amplify the conserved sequences of Co Ⅱ and LrRNA genes in root-knot nematode mitochondria

(1) Research materials

1) Nematode population: The root-knot nematode (Meloidogyne incognita) used in the test is a population preserved on the roots of water spinach by the Plant Nematode Research Laboratory of South China Agricultural University.

2) Primers: Same as Example 1.

(2) Test method

1) Isolation, collection and washing of root-knot nematodes

With embodiment 1.

2) PCR template preparation

Group A: the method provided by the present invention, the same as step (2) 3) of Example 1.

Group B: traditional method: pick a single second-instar larva on a sterile glass slide dripped with a small drop of sterile water, and then quickly cut the nematode body into 2~2~ 3 sections, and quickly use a pipette to suck the suspension into a pre-cooled PCR tube, add 1× polymerase buffer (Takara) to 10 μl, and put it into an ultra-low temperature incubator for freezing treatment for more than 30 minutes. Take out the frozen nematode lysate, add proteinase K to digest at 65°C for 1 hour, then treat at 95°C for 10 minutes to inactivate proteinase K, and obtain PCR template through these steps.

3) PCR reaction

With embodiment 1.

4) Detection of PCR reaction results

Take 5 μl of PCR products and use agarose gel electrophoresis with a mass volume ratio of 1% to detect the PCR results, and observe the size and presence or absence of amplified fragments of the PCR products on a gel imaging system (Figure 2A and B). It can be seen from Figure 2A that this method can bring 100% amplification efficiency, the target band is very clear, and the content of PCR products is high; Figure 2B shows that using the traditional method, some bands cannot be amplified repeatedly, and some bands are amplified. However, the content of PCR target fragments is small, so the brightness of the bands is weak.

The results show that the method for preparing the nematode DNA template used in the present invention has great progress and significant advantages in PCR amplification results compared with the traditional method of the above-mentioned group B.

Example 3 PCR Amplification of Transcribed Spacer (ITS) in Plant Parasitic Nematodes

The ITS sequence is an internal transcribed spacer, not expressed, including the internal transcribed spacer ITS1, ITS2 and the 5.8S gene in the middle, and is currently an important molecular marker for molecular identification of plant nematodes.

(1) Research materials

1) Nematode populations: Ditylenchus destructor, Radopholus similis, Pratylenchus sp., Brachydorus sp., Tylenchorhynchus sp., Spiral nematodes (Helicotylenchus sp.), Meloidogyne incognita, Criconemella sp., Discocrinemella sp., Bursaphelenchus xylophilus, Aphelenchoides besseyi , Xiphidorus sp., Trichodorus sp., Paratylenchus sp., Rotylenchulus sp. and Rhabditida, except for D. destructor, Banana borer nematode, pine wood nematode and root-knot nematode incognita were preserved populations in the Plant Nematode Laboratory of South China Agricultural University, and the rest were isolated from the rhizosphere soil of the Arboretum of South China Agricultural University.

2) Primers: General primers for ITS amplification reactions such as literature [Ferris VR, Ferris JM, and FaghihiJ.1993. VrainTC, Wakarchuk DA, Levesque AC, Hamilton RI.1992. Intraspecific rDNA restriction fragment length polymorphism in the Xiphinema americanum group. Fundamental and Applied Nematology 15:563-573.], as follows:

Upstream primer: 5'-CGTAACAAGGTAGCTGTAG-3'

Downstream primer: 5'-TTTCACTCGCCGTTACTAAGG-3';

(2) Test method

1) Isolation of plant nematodes

Using carrot callus to culture D. destructor and banana perforator nematodes, and using fungi to culture pine xylophilus, add sterile water directly to the petri dish, and then obtain a large number of worms in different stages. The acquisition of M. incognita was the same as in Example 1. Other soil nematodes were isolated using the Behmann funnel method [Xie Hui. 2005. Plant Nematode Taxonomy (Second Edition). Beijing: Higher Education Press, p 38-40].

2) Washing of nematode samples

Drop a few drops of sterile deionized water on a sterile glass slide, and pick the nematode worms and nematode eggs obtained in the above step 1) into sterile deionized water for cleaning;

3) PCR template preparation

The method for other nematodes is the same as in Example 1 except that Xiphidorus sp. worms longer than 1.5 mm are picked into a PCR tube filled with 12 μl of triple-distilled water for puncturing.

4) PCR reaction

The reaction system is the same as in Example 1. The reaction procedure is the same as in Example 1 except that the annealing temperature is 55°C.

5) Detection of PCR reaction results

Take 5 μl of PCR products and use agarose gel electrophoresis with a mass volume ratio of 1% to detect the PCR results, and observe the size and presence or absence of amplified fragments of the PCR products on a gel imaging system (Figure 3). It can be seen from Figure 3 that the target bands of all populations of nematodes can be amplified by this method, and the band concentration is high.

Example 4 PCR Amplification of Plant Parasitic Nematode 28S Gene D2-D3 Extension Region

The sequence of the 28S D2-D3 amplification region of plant nematodes is a region that mutates relatively quickly, but is conserved within the species, and is an important molecular marker for studying the molecular evolution of plant nematodes.

(1) Materials

1) Nematode population:

Silkworm (Filenchus sp.), Ditylenchus destructor, Ditylenchus destructor (D. dipsaci), Radopholus similis, Pratylenchus sp., Tylenchorhynchus sp. ), Helicotylenchus sp., Meloidogyne incognita, Discocrionemella sp., Xiphidorus sp., Paratylenchus sp., Reniform nematodes (Rotylenchulus sp.), the population source is obtained with embodiment 3.

2) Primers: Use general primers for amplifying D2D3 such as literature [Kaplan DT, Thomas WK, FrisseLM, SarahJL, Stanton JM, Speijer PR, Marin DH, Opperman CH.2000. motif.Journal of Nematology 32(2):134-142] as shown:

Upstream primer: 5'-ACAAGTACCGTGAGGGAAAGTTG-3'

Downstream primer: 5'-TCGGAAGGAACCAGCTACTA-3';

(2) Test method

1) Isolation of plant nematodes

With embodiment 3.

2) Washing of nematode samples

With embodiment 1.

3) PCR template preparation

With embodiment 1.

4) PCR reaction

Reaction system is with embodiment 3.

5) Detection of PCR reaction results

Take 5 μl of PCR products and use agarose gel electrophoresis with a mass volume ratio of 1% to detect the PCR results, and observe the size and presence or absence of the amplified fragments of the PCR products on a gel imaging system (Figure 4). It can be seen from Figure 4 that the target bands of all populations of nematodes can be amplified by this method, and the band concentration is high.

Example 5 Amplification of the Co Ⅰ interval of the plant parasitic nematode cytochrome oxidase gene

The Co Ⅰ interval sequence of plant nematode cytochrome oxidase gene is also conserved, which is an important molecular marker for studying the molecular evolution of plant nematodes.

(1) Materials

1) Nematode population:

Ditylenchus dipsaci, Pratylenchus sp., Tylenchorhynchus sp., Rotylenchulus sp., Bursaphelenchus xylophilus and Rice stem-point nematode ( Aphelenchoides besseyi), among which the short body nematodes, dwarf nematodes and reniform nematodes were isolated from the rhizosphere soil of the Arboretum of South China Agricultural University, and the others were preserved populations of the Plant Nematode Research Laboratory of South China Agricultural University.

2) Primers: Use universal primers that amplify the conserved region of the cytochrome oxidase CO Ⅰ gene (Kanzaki & Futai, 2002):

Upstream primer: 5'-CCTACTATGATTGGTGGTTTTGGTAATTG-3'

Downstream primer: 5'-GTAGCAGCAGTAAAATAAGCACG-3';

(2) Test method

1) Isolation of plant nematodes

Oryza sativa is also a population preserved in carrot tissue culture, and the isolation method is the same as in Example 3. The isolation of other nematode populations was the same as in Example 3.

2) Washing of nematode samples

With embodiment 1.

3) PCR template preparation

With embodiment 1.

4) PCR reaction

Reaction system is with embodiment 3.

5) Detection of PCR reaction results

Take 5 μl of PCR product and use agarose gel electrophoresis with a mass volume ratio of 1% to detect the PCR result, and observe the size and presence or absence of the amplified fragment of the PCR product on a gel imaging system (Figure 5). It can be seen from Figure 5 that the target bands of these populations of nematodes can be amplified by this method, and the band concentration is high.

Restriction enzyme analysis of embodiment 6 PCR product

Whether the PCR product is pure, whether it contains reagents that are not conducive to the operation of subsequent molecular biology experiments, restriction enzyme digestion analysis is a means, in addition, restriction enzyme digestion analysis is also a tool for detecting band diversity, and its use is relatively large. wide.

(1) Test material

PCR products from Examples 1-3.

Restriction enzymes: Hinf Ⅰ and Alu Ⅰ (Takara, Dalian Bao Biological Engineering Co., Ltd.).

(2) Test method

Restriction Enzyme Analysis System

Prepare 20 μl enzyme digestion system: Take 10 μl of PCR product, add 1 μl each of restriction enzymes Hinf Ⅰ and Alu Ⅰ, add 2 μl each of corresponding enzymes Buffer H and Buffer L, and make up the rest with ultrapure water.

The prepared enzyme digestion system was carried out in the C-1000 PCR instrument, and the program was set at 37°C for 1h, and then at 95°C for 5min.

(3) Analysis of enzyme digestion results

After the digested system, 5 μl of the reaction product was taken, electrophoresed on agarose gel electrophoresis with a mass volume ratio of 2%, and the size of the amplified fragment of the PCR product was observed on a gel imaging system (Figure 6). Since the two selected enzymes are commonly used enzymes in restriction enzyme analysis of fragments of the order Nematodes, many enzyme digestion results have been reported and analyzed. It can be seen from Figure 6 that all target PCR fragments can be digested well, and the positions of the restriction bands indicate that the restriction sites are accurately identified.

The results of the above examples show that the nematode gene sequence can be effectively amplified from a single nematode body or single nematode egg, and the amplified gene sequence can be digested with enzymes. Therefore, the invention provided by the present invention can be applied to the identification of nematode species.

The above embodiment is a preferred embodiment of the present invention, which is used to explain the technical solution of the present invention, but does not limit the present invention in any form, and any changes made under the premise of other not departing from the essence of the present invention, such as modification and substitution , combination, simplification and other ways should be included in the protection scope of the present invention.

Claims (10)

1. A method for identifying plant parasitic nematodes with a single nematode body or a single nematode ovum, characterized in that it comprises the following steps: (1) Washing of plant nematode samples: drop sterile deionized water on a sterile glass slide, and pick in a single nematode or a single egg with a nematode needle; (2) PCR template preparation: drop three-distilled water onto the inner wall of the PCR tube, pick a single nematode or a single egg washed in step (1), and use a sterile insect needle to puncture the nematode body or worm under a stereomicroscope. Eggs, throw the nematode lysate into the bottom of the tube, and use the nematode lysate directly as a PCR template or store it in a -80°C ultra-low temperature refrigerator for later use; (3) PCR reaction: select the conserved region of nematodes to design primers, and use the PCR template prepared in step (2) as a template to perform PCR; (4) Identification: The obtained PCR product is analyzed to determine the type of nematode. 2. The method for identifying plant-parasitic nematodes with a single nematode body or a single nematode egg according to claim 1, characterized in that: the amount of triple-distilled water described in step (2) depends on the size of the nematode, and for nematodes Nematode worms or eggs in the worm state, the amount of three-distilled water for nematodes and eggs shorter than 1.5mm is 6μl, and the amount of three-distilled water for nematodes longer than 1.5mm is 12μl; for swelling into spherical, pear-shaped, etc. For larger non-nematode worm bodies, the amount of three-distilled water is 24 μl. 3. The method for identifying plant-parasitic nematodes by using a single nematode body or single nematode egg according to claim 1, characterized in that: the triple distilled water described in step (2) is dropped onto the inner wall of the PCR tube at a distance of 1 cm from the tube. 4. The method for identifying plant-parasitic nematodes by using a single nematode body or a single nematode egg according to claim 1, characterized in that: the insect needle described in step (2) is stainless steel insect needle No. 1. 5. The method for identifying plant-parasitic nematodes with a single nematode body or a single nematode egg as claimed in claim 1, characterized in that: the sterile insect needle described in step (2) refers to the insect needle with absolute alcohol Soak and then burn in the flame of an alcohol burner. 6. The method for identifying plant-parasitic nematodes with a single nematode body or single nematode egg according to claim 1, characterized in that: the nematode lysate described in step (2) is a punctured nematode body or egg A mixture of the obtained nematode lysate and triple distilled water. 7. The method for identifying plant-parasitic nematodes with a single nematode body or a single nematode egg as claimed in claim 1, characterized in that: the conserved regions described in step (3) are mitochondrial cytochrome oxidase gene II and LrRNA gene One or at least two of the sequence, the internal transcription spacer ITS sequence, the 28S D2-D3 amplification region sequence and the plant nematode cytochrome oxidase gene Co Ⅰ interval sequence. 8. the method for identifying plant parasitic nematodes with a single nematode worm body or a single nematode ovum as claimed in claim 7, characterized in that: The primers of the mitochondrial cytochrome oxidase gene II and LrRNA gene sequences are as follows: Upstream primer #C2F3: 5'-GGTCAATGTTCAGAAATTTGTGG-3' Downstream primer #1108: 5'-TACCTTTGACCAATCACGCT-3'; The primers of the internal transcription spacer ITS sequence are as follows: Upstream primer: 5'-CGTAACAAGGTAGCTGTAG-3' Downstream primer: 5'-TTTCACTCGCCGTTACTAAGG-3'; The primers of the 28S D2-D3 amplification region sequence are as follows: Upstream primer: 5'-ACAAGTACCGTGAGGGAAAGTTG-3' Downstream primer: 5'-TCGGAAGGAACCAGCTACTA-3'; The primers of the plant nematode cytochrome oxidase gene Co I interval sequence are as follows: Upstream primer: 5'-CCTACTATGATTGGTGGTTTTGGTAATTG-3' Downstream primer: 5'-GTAGCAGCAGTAAAATAAGCACG-3'. 9. The method for identifying plant-parasitic nematodes by using a single nematode body or single nematode egg according to claim 1, characterized in that: the reaction conditions of the PCR reaction described in step (3) are as follows: pre-denaturation at 94°C for 3 minutes; Denaturation at 98°C for 10 sec, annealing at 52°C to 55°C for 30 sec, extension at 68°C for 1 min, 35 cycles; then extension at 68°C for 10 min. 10. The method for identifying plant-parasitic nematodes by using a single nematode body or single nematode egg according to claim 1, characterized in that the analysis in step (4) includes electrophoresis analysis, enzyme digestion analysis and sequencing analysis.