阅读说明：本技术 一种棘沟赖利绦虫鉴定的pcr方法 (PCR method for identifying echinocandis labyrinthi tapeworm ) 是由 张鼎 刘晶英 杨述远 田文霞 于 2020-05-14 设计创作，主要内容包括：本发明具体涉及一种棘沟赖利绦虫鉴定的PCR方法,属于感染家禽的赖利属绦虫种属的鉴别领域。棘沟赖利绦虫是威胁鸡健康和养殖最大的赖利属绦虫种类,目前对于棘沟赖利绦虫的鉴定仍然依赖于传统的虫体形态学特征差异分析,存在用时长、操作复杂和判断不准确等缺点。本发明参考已公布的赖利属绦虫核糖体基因序列,自主设计18s rDNA和ITS-2 rDNA引物,通过对PCR方法进行优化,准确扩增棘沟赖利绦虫18s rDNA和ITS-2 rDNA基因产物,建立一种基于18srDNA和ITS-2 rDNA双基因确认的快速、准确鉴定棘沟赖利绦虫的PCR方法。(The invention particularly relates to a PCR (polymerase chain reaction) method for identifying echinochloa lisnerica tapeworm, belonging to the field of identification of Lysidium tapeworm species infecting poultry. The echinochloa lisnerica tapeworm is the largest Lyricia tapeworm species threatening the health and cultivation of the chicken, the identification of the echinochloa lisnerica tapeworm still depends on the traditional morphological characteristic difference analysis of the polypide, and the defects of long time, complex operation, inaccurate judgment and the like exist. The invention refers to a published ribosomal gene sequence of the Lysidium tapeworm, autonomously designs 18s rDNA and ITS-2rDNA primers, and establishes a PCR method for quickly and accurately identifying the Lysidium tapeworm based on the confirmation of double genes of 18srDNA and ITS-2rDNA by optimizing a PCR method and accurately amplifying gene products of the Lysidium tapeworm 18s rDNA and ITS-2 rDNA.)

1. A PCR method for identifying the echinocandi tapeworm is characterized in that two gene detection primers, namely 18srDNA and ITS-2rDNA, identified by the echinocandi tapeworm species are provided, and the method comprises the following steps:

step 1, collection and morphological identification of echinochloa lisneri tapeworm: collecting a chicken case infected by tapeworm, killing a sick chicken by a bleeding method, taking out a complete digestive tract, carefully cutting off the intestinal tract, placing the found tapeworm and the intestinal tract into clear water together, washing the tapeworm in normal saline after the head section of the tapeworm is separated from the intestinal mucosa and is fully stretched, washing the tapeworm in the normal saline, independently preserving each worm after the tapeworm is washed, freezing a part of the worm in a refrigerator at-80 ℃ to extract DNA of the worm, fixing a part of the worm in glutaraldehyde aqueous solution, observing the worm by using a scanning electron microscope, fixing a part of the worm in paraformaldehyde aqueous solution, and dyeing hematoxylin-eosin;

step 2, DNA extraction: 30mg of tapeworm body, and extracting the tapeworm body DNA by using a tissue DNA extraction kit;

step 3, PCR amplification:

(1) designing a primer:

the nucleotide sequence of the 18s rDNA gene upstream primer for identifying the echinocandis lissora is shown as SEQ ID NO. 1: 5'-CTGAGAAACGGCTACCACT-3' for 18 s-F;

the nucleotide sequence of the 18s rDNA gene downstream primer for identifying the echinocandis lissora is shown as SEQ ID NO. 2: 18s-R: 5'-ACGCCAGACGAACTACAC-3';

the nucleotide sequence of the ITS-2rDNA gene upstream primer for identifying the echinocandis lissora is shown as SEQ ID NO. 3: ITS-2-F: 5'-TATTGCGGCCATAGGTTT-3';

the nucleotide sequence of the ITS-2rDNA gene upstream primer for identifying the echinocandis lissora is shown as SEQ ID NO. 4: ITS-2-R: 5'-CGTGAGTACCCGCTGAAC-3';

(2) and (3) PCR reaction:

preparing a first PCR reaction tube and a second PCR reaction tube, adding 2 XMasterMix into the first PCR reaction tube, taking the tapeworm DNA extracted in the step 2 as a template, using 18s rDNA upstream and downstream primers and deionized water, and carrying out PCR reaction; 2 XMasterMix is added into a second PCR reaction tube, the tapeworm DNA extracted in the step 2 is taken as a template, ITS-2rDNA upstream and downstream primers and deionized water are used for carrying out PCR reaction; preparing agarose gel solution, using TAE electrophoresis buffer solution, adding GelRed fluorescent dye before gel solidification to trace PCR amplification products in gel electrophoresis; adding a PCR amplification product solution into the sample application hole after the gel is solidified, carrying out electrophoresis under the voltage condition, observing the molecular size and the electrophoretic band definition of the PCR amplification product under a gel imager, and taking a picture;

and 4, sequencing the PCR amplification product: the electrophoresis strips of PCR amplification products of the first PCR reaction tube and the second PCR reaction tube are recovered by an agarose gel DNA recovery kit, the recovered 18s rDNA and ITS-2rDNAPCR amplification products are respectively connected with a pMD19-T vector, the obtained product is transformed into DH5 alpha competent cells after being connected with the pMD19-T vector, the DH5 alpha competent cells transformed by the pMD19-T vector are positively screened by an LB agar plate culture medium containing ampicillin, and PCR sequencing is carried out on colonies after the positive colonies are amplified and cultured to obtain a gene sequence of the 18s rDNA amplification products and a gene sequence of the ITS-2rDNAPCR amplification products;

and 5, sequence analysis: respectively comparing the gene sequences of the amplification products of 18s rDNA and ITS-2rDNA NAPCR with the sequences of 18s rDNA and ITS-2rDNA of the Lysidium tapeworm disclosed in a GenBank database, and respectively carrying out genetic evolution relation analysis on the gene sequences of the PCR amplification products of 18s rDNA and ITS-2rDNA and the published sequences by using MEGA software.

2. The PCR method for identification of Taenia echinocandis of claim 1, wherein the volume fraction of the glutaraldehyde aqueous solution in step 1 is 2.5%, and the formulation is: adding 50mL of 0.2M phosphate buffer solution and 40mL of distilled water into 10mL of glutaraldehyde water solution with the volume fraction of 25%, adjusting the pH value to be 7.4, and adding distilled water to make up to 100mL, wherein the formula of the 0.2M phosphate buffer solution is as follows: adding distilled water into 2.6g of sodium dihydrogen phosphate and 29g of disodium hydrogen phosphate to make up to 500mL, and adjusting the pH value to 7.4;

the scanning mirror observation comprises the following specific steps: shearing 1-2 cm and fixing for 4h by using 2.5% glutaraldehyde aqueous solutionIncubating polypide fragments with 0.2M aqueous solution of sodium arsenate A containing 1% g/mL osmium tetroxide by mass/volume for 1 hr, dehydrating with gradient ethanol, and adding CO₂Drying the critical point, spraying gold, and observing under a scanning electron microscope.

3. The PCR method for identification of Taenia echinocandis of claim 1, wherein the mass/volume fraction of the aqueous paraformaldehyde solution in step 1 is 4% g/mL, and the formulation is: weighing 40g of paraformaldehyde, placing the paraformaldehyde into a triangular flask, adding 500-800 mL of 0.1mol/L phosphate buffer, heating to 55-60 ℃, continuously stirring to completely dissolve the powder, dropwise adding 1mol/L NaOH to clear the solution, and finally supplementing 0.1mol/L phosphate buffer to 1000 mL;

the hematoxylin-eosin staining method comprises the following specific steps: shearing 1-2 cm of polypide fragments which are fixed for 24 hours by using a paraformaldehyde aqueous solution with the mass/volume fraction of 4% g/mL, fully cleaning the polypide fragments by using tap water, placing the polypide fragments into a staining jar, staining the polypide fragments for 0.5-1 hour by using a hematoxylin-eosin staining solution, adjusting the color of the polypide fragments by using 1% hydrochloric acid alcohol for 30s, dehydrating by using gradient ethanol, sealing the fragments by using neutral gum after xylene is transparent, and observing and taking pictures under an optical microscope.

4. The PCR method for identification of Taenia echinocandis according to claim 2, wherein the gradient of ethanol is: volume fraction 70%, volume fraction 80%, volume fraction 95% and volume fraction 100%.

5. The PCR method for identification of Taenia echinocandis according to claim 3, wherein the gradient of ethanol is: volume fraction 70%, volume fraction 80%, volume fraction 95% and volume fraction 100%.

6. The PCR method for identification of Taenia echinocandis according to claim 1, wherein the volume of the first PCR reaction tube in step 3(2) is 200 μ L; the reaction system for the first PCR reaction tube to carry out PCR reaction is 50 mu L;

adding 2 XMasterMix into the first PCR reaction tube to 25 μ L, extracting tapeworm DNA from step 2 to 1 μ L, extracting 18s rDNA upstream and downstream primers to 1 μ L respectively, the concentration is 0.5 μ M, and deionized water is 22 μ L;

the reaction conditions of the first PCR reaction tube for PCR reaction are pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30 seconds, annealing at 58 ℃ for 60 seconds, extension at 72 ℃ for 30 seconds and amplification for 40 cycles.

7. The PCR method for identification of Taenia echinocandis according to claim 1, wherein the volume of the second PCR reaction tube in step 3(2) is 200 μ L; the reaction system for carrying out PCR reaction in the second PCR reaction tube is 50 mu L;

adding 2 XMasterMix into a second PCR reaction tube to obtain 25 μ L, extracting tapeworm DNA from step 2 to obtain 1 μ L, and adding ITS-2rDNA upstream and downstream primers to obtain 1 μ L, wherein the concentration is 0.5 μ M and the deionized water is 22 μ L;

the reaction conditions of the second PCR reaction tube for PCR reaction are pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30 seconds, annealing at 58 ℃ for 60 seconds, extension at 72 ℃ for 30 seconds and amplification for 40 cycles.

8. The PCR method for identification of Taenia echinocandis of claim 1, wherein in step 3(2), the mass/volume fraction of the agarose gel solution is 1.5% g/mL, the volume ratio of GelRed fluorescent dye to the agarose gel solution is 1: 5000-1: 10000, and the volume of the PCR amplification product solution is 5-8 μ L; the voltage under the voltage condition is 120V-150V, and the electrophoresis time is 30 min-40 min.

9. The PCR method for identification of Taenia echinocandis of claim 1, wherein the concentration of ampicillin in step 4 is 50 μ g/mL.

10. The PCR method for identification of Taenia echinocandis according to claim 1, wherein the gene sequence of the 18s rDNA amplification product in step 4 is shown in SEQ ID NO.5, and the gene sequence of the ITS-2rDNA PCR amplification product is shown in SEQ ID NO. 6.

Technical Field

The invention particularly relates to a PCR (polymerase chain reaction) method for identifying echinochloa lisnerica tapeworm, belonging to the field of identification of Lysidium tapeworm species infecting poultry.

Background

Taeniasis is a parasitic disease that seriously harms chicken health and the breeding industry. The cestodes parasitizing on chicken belong to the genus Lyricia of family Vitaceae, order Zoleyozoa, class Tastodiae. The taenia solitarica parasitizes in small intestines of various poultry such as domestic chicken, turkey, pheasant and the like, and causes the symptoms of stunted development, slow growth, emaciation, anemia, poisoning and the like of the chickens through nutrient absorption and toxin release. A large amount of tapeworm parasitism also causes intestinal obstruction of chickens, and induces intestinal obstruction and acute inflammation. The chicken can be infected with one kind of tapeworm alone or with a mixture of tapeworms. In recent years, the number of cases of chicken infected with the Lyricia tapeworm is greatly increased, and more than 200 Lyricia tapeworms are found. At present, the identification of the type of the tapeworm of the genus Lysidium mainly depends on morphological feature observation, and is distinguished by slight differences of the body size, the head segment shape, the apical process, the hook and the segment structure of the pregnant egg. The conventional morphological identification method has the defects of long time, complex operation, inaccurate judgment and the like, and along with the increase of the discovered quantity of the Lysidium tapeworms, the morphological description of the Lysidium tapeworms generates great divergence, and the difficulty of identifying the Lysidium tapeworms is greatly increased.

Ribosomes are organelles found widely in cells and are the major site of protein synthesis in cells. Eukaryotic ribosomal dna (rDNA) is a nuclear gene encoding ribosomal rna (rrna), and eukaryotic rDNA consists of repeated single-copy gene tandems, each single-copy gene consisting of 18s, 5.8s and 28s forming a transcription unit, separated by the rDNA internal transcribed spacers ITS1 and ITS2, respectively. Ribosome 18S is a gene encoding ribosomal small subunit RNA on the chromosome of eukaryotes, and has the slowest evolution rate in ribosomes and high conservation. The ITS sequence is an internal transcribed spacer in ribosomal DNA between 18S and 28S, the ITS1 in the first internal transcribed spacer of ribosomal DNA is between 18S rDNA and 5.8S rDNA, and the ITS second internal transcribed spacer of ribosomal DNA is between 5.8S rDNA and 28S rDNA. With the improvement of parasite gene sequencing data in recent years, 18s and ITS2 have been widely used for endoparasitic and intraspecific identification and genetic evolutionary relationship analysis. The invention provides a PCR method for quickly and accurately identifying echinochloa lissorosis tapeworm, which aims to solve the defect of species identification of the traditional echinochloa lissorosis tapeworm.

Disclosure of Invention

The echinochloa lisnerica tapeworm is the largest kind of the lisneria tapeworm threatening the health and cultivation of the chicken, the identification of the echinochloa lisnerica tapeworm still depends on the traditional morphological characteristics of the worm body, and the defects of long time, complex operation, inaccurate judgment and the like exist. The invention refers to a published ribworm ribosome gene sequence of Lysidium tenuisticis, autonomously designs 18s rDNA and ITS-2rDNA primers, optimizes a PCR method, accurately amplifies gene products of the 18s rDNA and ITS-2rDNA of the echinococcus ribworm, and establishes a PCR method for quickly and accurately identifying the echinococcus ribworm based on double gene confirmation of the 18s rDNA and the ITS-2 rDNA.

In order to achieve the purpose, the invention adopts the following technical scheme:

a PCR method for identifying the echinocandi tapeworm provides two gene detection primers of 18s rDNA and ITS-2rDNA identified by the species of the echinocandi tapeworm, and the method comprises the following steps:

step 1, collection and morphological identification of echinochloa lisneri tapeworm: collecting a chicken case infected by tapeworm, killing a sick chicken by a bleeding method, taking out a complete digestive tract, carefully cutting off the intestinal tract, placing the found tapeworm and the intestinal tract into clear water together, washing the tapeworm in normal saline after the head section of the tapeworm is separated from the intestinal mucosa and is fully stretched, washing the tapeworm in the normal saline, independently preserving each worm after the tapeworm is washed, freezing a part of the worm in a refrigerator at-80 ℃ to extract DNA of the worm, fixing a part of the worm in glutaraldehyde aqueous solution, observing the worm by using a scanning electron microscope, fixing a part of the worm in paraformaldehyde aqueous solution, and dyeing hematoxylin-eosin;

step 2, DNA extraction: 30mg of tapeworm body, and extracting the tapeworm body DNA by using a tissue DNA extraction kit;

step 3, PCR amplification:

(1) designing a primer:

the nucleotide sequence of the 18s rDNA gene upstream primer for identifying the echinocandis lissora is shown as SEQ ID NO. 1: 5'-CTGAGAAACGGCTACCACT-3' for 18 s-F;

the nucleotide sequence of the 18s rDNA gene downstream primer for identifying the echinocandis lissora is shown as SEQ ID NO. 2: 18s-R: 5'-ACGCCAGACGAACTACAC-3';

the nucleotide sequence of the ITS-2rDNA gene upstream primer for identifying the echinocandis lissora is shown as SEQ ID NO. 3: ITS-2-F: 5'-TATTGCGGCCATAGGTTT-3';

the nucleotide sequence of the ITS-2rDNA gene upstream primer for identifying the echinocandis lissora is shown as SEQ ID NO. 4: ITS-2-R: 5'-CGTGAGTACCCGCTGAAC-3';

(2) and (3) PCR reaction:

preparing a first PCR reaction tube and a second PCR reaction tube, adding 2 XMasterMix into the first PCR reaction tube, taking the tapeworm DNA extracted in the step 2 as a template, using 18s rDNA upstream and downstream primers and deionized water, and carrying out PCR reaction; 2 XMasterMix is added into a second PCR reaction tube, the tapeworm DNA extracted in the step 2 is taken as a template, ITS-2rDNA upstream and downstream primers and deionized water are used for carrying out PCR reaction; preparing agarose gel solution, using TAE electrophoresis buffer solution, GelRed fluorescent dye before gel solidification to trace PCR amplification products in gel electrophoresis; adding a PCR amplification product solution into the sample application hole after the gel is solidified, carrying out electrophoresis under the voltage condition, observing the molecular size and the electrophoretic band definition of the PCR amplification product under a gel imager, and taking a picture;

and 4, sequencing the PCR amplification product: the electrophoresis strips of PCR amplification products of the first PCR reaction tube and the second PCR reaction tube are recovered by an agarose gel DNA recovery kit, the recovered 18s rDNA and ITS-2rDNAPCR amplification products are respectively connected with a pMD19-T vector, the obtained product is transformed into DH5 alpha competent cells after being connected with the pMD19-T vector, the DH5 alpha competent cells transformed by the pMD19-T vector are positively screened by an LB agar plate culture medium containing ampicillin, and PCR sequencing is carried out on colonies after the positive colonies are enlarged and cultured to obtain gene sequences of 18s rDNA and ITS-2rDNA PCR amplification products;

and 5, sequence analysis: respectively comparing the gene sequences of the PCR amplification products of 18s rDNA and ITS-2rDNA with the sequences of 18s rDNA and ITS-2rDNA of the Lysidium tapeworm disclosed in a GenBank database, and respectively carrying out genetic evolution relation analysis on the gene sequences of the PCR amplification products of 18s rDNA and ITS-2rDNA and the published sequences by using MEGA software.

Further, the volume fraction of the glutaraldehyde aqueous solution in step 1 is 2.5%, and the formula is as follows: adding 50mL of 0.2M phosphate buffer solution and 40mL of distilled water into 10mL of glutaraldehyde aqueous solution with the volume fraction of 25%, adjusting the pH value to be 7.4, and adding distilled water to make up to 100mL, wherein the formula of the 0.2M phosphate buffer solution is as follows: adding distilled water into 2.6g of sodium dihydrogen phosphate and 29g of disodium hydrogen phosphate to make up to 500mL, and adjusting the pH value to 7.4;

the scanning mirror observation comprises the following specific steps: shearing 1-2 cm of polypide fragments fixed with 2.5% glutaraldehyde aqueous solution by volume fraction for 4h, incubating for 1h with 0.2M sodium arsenate aqueous solution containing 1% g/mL osmium tetroxide by mass/volume fraction, dehydrating with gradient ethanol, and adding CO₂Drying the critical point, spraying gold, and observing under a scanning electron microscope.

Further, the gradient of the gradient ethanol is as follows: volume fraction 70%, volume fraction 80%, volume fraction 95% and volume fraction 100%.

Further, the mass/volume fraction of the paraformaldehyde aqueous solution in the step 1 is 4% g/mL, and the formula is as follows: weighing 40g of paraformaldehyde, placing the paraformaldehyde into a triangular flask, adding 500-800 mL of 0.1mol/L phosphate buffer, heating to 55-60 ℃, continuously stirring to completely dissolve the powder, dropwise adding 1mol/L NaOH to clear the solution, and finally supplementing 0.1mol/L phosphate buffer to 1000 mL;

the hematoxylin-eosin staining method comprises the following specific steps: shearing 1-2 cm of polypide fragments which are fixed for 24 hours by using a paraformaldehyde aqueous solution with the mass/volume fraction of 4% g/mL, fully cleaning the polypide fragments by using tap water, placing the polypide fragments into a staining jar, staining the polypide fragments for 0.5-1 hour by using a hematoxylin-eosin staining solution, adjusting the color of the polypide fragments by using 1% hydrochloric acid alcohol for 30s, dehydrating by using gradient ethanol, sealing the fragments by using neutral gum after xylene is transparent, and observing and taking pictures under an optical microscope.

The gradient of the gradient ethanol is as follows: volume fraction 70%, volume fraction 80%, volume fraction 95% and volume fraction 100%.

The main points of judgment of acanthopanax senticosus and taenia are as follows: the body is 5-10 cm long, milky white and opaque. The head section is spherical, 4 circular suckers are arranged on the periphery of the head section, 1 telescopic top protrusion is arranged in the center of the head section, two circles of alternately arranged small hooks are distributed on the top protrusion, and the number of the small hooks in each circle is about 22-25. The neck segment is shorter, the starting part is as wide as the head segment, and the neck segment gradually widens backwards. The width of the cervical segment is larger than the length, the appearance is trapezoidal, the internal reproductive system is not developed and matured, and the dyeing is not obvious. The segments are rectangular, the length is slightly larger than the width, a set of reproductive system is arranged in the segment, and the reproductive holes are alternately arranged at the position of 1/3 on the outer edge of the segment. The male stem sacs of the male reproductive system are positioned outside the excretion tubes, and the number of the testicles is 30-40, and the testicles are distributed on two sides of the segment front section and above the ovary. The fertilized sac in the female reproductive system is developed and peaky and is located inside the excretory duct and below the testis. The ovary is in the shape of a leaf and is positioned in the center of the segment, and the yolk gland is positioned behind the ovary. The pregnant joint is fat, the joint is bigger than wide, the interior has no uterus, and the joint is replaced by the oocysts. The oocysts are circular or oval and are scattered at all parts of the pregnancies, the number of the oocysts is about 200-400, and each oocyst contains 1 embryonated oncosphere.

Further, the volume of the first PCR reaction tube in the step 3(2) is 200 μ L; the reaction system for the first PCR reaction tube to carry out PCR reaction is 50 mu L;

adding 2 XMasterMix into the first PCR reaction tube to 25 μ L, extracting tapeworm DNA from step 2 to 1 μ L, extracting 18srDNA upstream and downstream primers to 1 μ L respectively, the concentration is 0.5 μ M, and deionized water is 22 μ L;

the reaction conditions of the first PCR reaction tube for PCR reaction are pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30 seconds, annealing at 58 ℃ for 60 seconds, extension at 72 ℃ for 30 seconds and amplification for 40 cycles.

Further, the volume of the second PCR reaction tube in the step 3(2) is 200 μ L; the reaction system for carrying out PCR reaction in the second PCR reaction tube is 50 mu L;

adding 2 XMasterMix into a second PCR reaction tube to obtain 25 μ L, extracting tapeworm DNA from step 2 to obtain 1 μ L, and adding ITS-2rDNA upstream and downstream primers to obtain 1 μ L, wherein the concentration is 0.5 μ M and the deionized water is 22 μ L;

the reaction conditions of the second PCR reaction tube for PCR reaction are pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30 seconds, annealing at 58 ℃ for 60 seconds, extension at 72 ℃ for 30 seconds and amplification for 40 cycles.

Further, in the step 3(2), the mass/volume fraction of the agarose gel solution is 1.5% g/mL, the volume ratio of the GelRed fluorescent dye to the agarose gel solution is 1: 5000-1: 10000, and the volume of the PCR amplification product solution is 5-8 muL; the voltage under the voltage condition is 120V-150V, and the electrophoresis time is 30 min-40 min.

Further, the concentration of ampicillin in step 4 was 50. mu.g/mL.

Further, the gene sequence of the 18s rDNA amplification product in the step 4 is shown in SEQ ID NO.5, and the gene sequence of the ITS-2rDNAPCR amplification product is shown in SEQ ID NO. 6.

Compared with the prior art, the invention has the following advantages:

1) in the earlier stage, the PCR primers and the method are used for establishing the echinochloa lisica tapeworm through traditional morphological identification of worms. The traditional morphological identification of the polypide needs to dye and identify the segments of different parts of the tapeworm, and has the defects of large sample demand, large workload, long time consumption, complex dyeing process, difficulty in obtaining ideal dyeing results and the like (figure 1). The PCR identification method established by the invention needs less samples (about 30mg), has small workload (no need of distinguishing segment parts), takes short time (less than 24h) and has simple result judgment.

2) The primers designed by the invention can respectively and specifically amplify 18s rDNA and ITS-2rDNA sequences in a worm sample, and the band amplified by the PCR method of the invention is single and clear (figure 2). 18s rDNA (FIGS. 3 and 4) and ITS-2rDNA (FIGS. 5 and 6) can be aligned to the corresponding sequence of Lyricia tapeworm published in GeneBank, and the analysis of genetic evolution relationship shows that the DNA has high homology with the Oncorhynchus Lyricia tapeworm.

Drawings

FIG. 1 is a morphological identification of Echinochloa lissorica;

FIG. 2 shows PCR gel electrophoresis of 18s rDNA and ITS-2 rDNA;

FIG. 3 shows the result of the alignment of the 18s rDNA sequence GeneBank sequence amplified according to the present invention;

FIG. 4 is an analysis of the evolutionary relationship of the 18s rDNA sequence gene amplified according to the present invention;

FIG. 5 shows the result of alignment of ITS-2rDNA sequence GeneBank sequence amplified in the present invention;

FIG. 6 shows the analysis of the genetic evolution of the ITS-2rDNA gene sequence amplified according to the present invention.

Detailed Description