CN104946774A - Method for gathering and detecting DON toxin production genes in corn - Google Patents

Abstract

The invention belongs to the field of biotechnology and relates to a method for enriching and detecting DON toxin-producing genes in corn. The detection steps include: extracting total DNA by CTAB method; using the extracted DNA as a template to enrich DON toxin-producing genes with carboxyl magnetic nanoparticles, and then performing PCR amplification; agarose gel electrophoresis for PCR products to detect whether there are target strips Band to determine whether the detected maize contains DON toxin-producing genes. The invention enriches the DON toxin-producing gene in the grain by a fast and simple method. It can reduce the interference of other environmental factors on the extraction of DON toxin-producing genes, and effectively enrich them for subsequent PCR detection.

Description

A method for enrichment and detection of DON toxin-producing gene in maize

technical field

The invention relates to a method for enriching DON toxin-producing genes in maize with magnetic nanoparticles and subsequent detection, and belongs to the field of biotechnology.

Background technique

Fusarium graminearum is a fungus widely distributed in warm and humid areas, which can cause root rot, seedling rot and ear rot of wheat crops, as well as ear rot and stem rot of corn. Fusarium graminearum infects wheat and causes scab of wheat. It is the most important disease in Northeast my country and the Yangtze River Basin, and it has a tendency to spread in recent years. In the process of grain storage, especially in the high temperature and humid environment in summer, Fusarium graminearum grows fast, and the toxin will accumulate continuously with the growth of the bacteria. When the accumulation of toxins exceeds the national standard, the food cannot be eaten by humans and animals, and loses its commodity value, so it needs to be processed separately. Therefore, huge economic losses will be caused. Seed borne fungus is an important way for Fusarium graminearum to infect cereal crops. Sowing contaminated seeds increases the probability of cereal crops being infected with head blight, which will have a direct impact on grain yield.

The types and abilities of toxins produced by different strains of Fusarium graminearum (different collection places, hosts, infection sites) are very different. But there are two main categories: Trichothecenes and Zearalenone (ZEN).

The basic structure of trichothecenes is a tetracyclic sesquiterpene, and the epoxy group on it is the main source of toxicity. There are four types of A, B, C, and D according to the specific functional groups. Types A and B are naturally contaminating trichothecene compounds. Among the type B compounds, deoxynivalenol (DON) and nivalenol (NIV) are the main two toxins. Symptoms of acute poisoning of trichothecene compounds are vomiting, nausea, dizziness, blood in the stool, and numbness of hands and feet. Chronic poisoning includes genotoxicity, cytotoxicity and immunotoxicity.

At present, the methods for detecting DON toxin in grain mainly include ELISA and high performance liquid chromatography. ELISA has strong specificity and high sensitivity, but enzyme-linked immunosorbent (ELISA) kits and detection equipment are more expensive. High-performance liquid chromatography has high sensitivity and can quantitatively detect the toxin content in samples, but the operation process is cumbersome and requires professionals to operate. In addition, the detection equipment is expensive and cannot be used for the detection of grain in grass-roots grain depots. The method of using magnetic beads to enrich DON toxin-producing genes and then using polymerase chain reaction (PCR) to detect toxin genes has not been reported at home and abroad.

Contents of the invention

The purpose of the present invention is to provide an effective and simple method to quickly and easily enrich and detect DON toxin-producing genes in maize. It can reduce the interference of other environmental factors on the extraction of DON toxin-producing genes, and effectively enrich them for subsequent PCR detection.

A method for enriching and detecting DON toxin-producing genes in corn, characterized in that the detection steps include: extracting total DNA by the cetyltrimethylammonium bromide (CTAB) method; using the extracted DNA as a template using carboxyl Magnetic nanoparticles enrich DON toxin-producing genes, and then perform PCR amplification; PCR products are subjected to agarose gel electrophoresis to detect whether there is a target band to determine whether the tested corn contains DON toxin-producing genes.

Described detection method, concrete steps are as follows:

1. Extraction of total DNA:

a. Corn samples were crushed at 15000-25000rpm for 1-5min.

b. Take 1-5g sample powder in a centrifuge tube, add appropriate amount of CTAB extraction buffer, and bathe in 60-70°C water bath for 15-25min;

c. Cool to room temperature, add phenol:chloroform:isoamyl alcohol (25:24:1), centrifuge at 10,000-20,000rpm for 10-20min, and obtain supernatant 1;

Step d.c to obtain the supernatant 1, add 1/10 volume of 10% CTAB-0.7M NaCl, then add an equal volume of chloroform:isoamyl alcohol (24:1), centrifuge at 10,000-20,000rpm for 10-20min to obtain the supernatant 2;

Step e.d to obtain the supernatant 2, add an equal volume of frozen isopropanol to the upper aqueous phase, centrifuge at 3,000-5,000 rpm for 10-20 minutes, remove the supernatant, add 70% ethanol, and change and wash the 70% ethanol 2-3 times;

f. Pour off 70% ethanol, air-dry and dissolve DNA with an appropriate amount of Tris-ethylenediaminetetraacetic acid (TE) buffer, and store at -20°C.

2. DON toxin gene enrichment

a. Carboxylated magnetic nanoparticles 100-500 μL, separated by magnetic finger cot, discarded supernatant;

b. Add 100-500 μL of activation buffer to wash the magnetic nanoparticles, separate them with magnetic finger cots, repeat 2-3 times, and discard the supernatant;

c. Add 100-200 μL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) solution and 100-200 μL N-hydroxysuccinimide (NHS) solution to centrifuge In the tube, vortex to mix, activate at 20-30°C for 30-60min, separate with magnetic finger cot, and discard the supernatant;

d. Add 10-50 μL of amino primers and 100-180 μL of phosphate buffer saline (PBS) solution (pH 8.0), mix gently, couple at 20-30°C for 1.5-2.5 hours, separate with magnetic finger cots, and discard the supernatant;

e. Add 100-500 μL of blocking buffer, resuspend the magnetic nanoparticles, react at 20-30°C for 1-2 hours to block the unreacted activated carboxyl groups on the surface of the magnetic nanoparticles, separate with magnetic finger cots, and discard the supernatant;

f. Add 100-500 μL PBS solution (pH 7.2) to wash 2-3 times, separate with magnetic finger cot, and discard the supernatant;

g. Add 150-200 μL saline sodium citrate (SSC) solution, 30-50 μL formamide solution and 30-50 μL single-stranded corn genomic DNA, hybridize at room temperature for 20-40 minutes, separate with magnetic finger cots, and discard the supernatant;

h. Add 100-500 μL PBS solution (pH 7.2) to wash 2-3 times, separate with magnetic finger cots, and discard the supernatant;

i. Add 20-50 μL of double-distilled water (ddH ₂ O), heat at 94°C for 5 minutes, and transfer the supernatant to another centrifuge tube for storage at -20°C.

3. DON toxin production gene detection

a.PCR amplification system: 2.5 μL of 10×enzyme-specific reaction amplification buffer (PCR Buffer); 2.5 μL of Mg ²⁺ (10 μM); 2.5 μL of deoxyribonucleoside triphosphates (dNTPs) (2 mM); DON toxin 0.5 μL upstream and downstream primers for toxin-producing genes; 1.5 μL DNA template; 0.3 μL thermostable DNA polymerase (Taq enzyme) (5 U/μL); 14.7 μL double distilled water (ddH ₂ O). The primer sequences are: upstream primer: TACGTGAAACATTGTTGGC; downstream primer: GGTGTCCCAGGATCTGCG. Note: A is adenine; T is thymine; G is guanine; C is cytosine.

b. Reaction conditions:

c. Agarose gel electrophoresis

PCR products and nucleic acid molecular weight reference (DL 2000), on a 1% agarose gel with a constant voltage of 120-130V, electrophoresis for 20-30min. If there is a DON toxin-producing gene in the sample, there will be a 237bp amplification product.

In the described total DNA extraction method, the composition of the b step CTAB damping fluid is: 100mM Tris-hydrochloric acid (Tris-HCl) (pH 8.0), 20mM ethylenediaminetetraacetic acid disodium (EDTA-Na ), 1.4mM NaCl, 2% CTAB, 2% β-mercaptoethanol;

In the described total DNA extraction method, the composition of step d 10%CTAB-0.7M NaCl is: 4.1%NaCl, 10%CTAB;

In the described total DNA extraction method, the fth step TE buffer composition is: 10mM Tris-HCl (pH8.0), 1 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0).

In the described DON toxin-producing gene enrichment method, the components of the b-step activation buffer are: 100mM 2-(N-morpholine)ethanesulfonic acid monohydrate (MES) (pH 5.0), 0.05% Tween- 20 (Tween20);

In the DON toxin-producing gene enrichment method, the EDC solution and the NHS solution in step c use an activation buffer as a dispersant;

In the method for enriching the DON toxin-producing gene, the amino primer in step d is the amino group connected to the 5' end of the upstream and downstream primers of the DON toxin-producing gene in the PCR system

In the DON toxin-producing gene enrichment method, step d adds 0.05% Tween 20 to the PBS solution (pH 8.0) to improve dispersibility;

In the described DON toxin-producing gene enrichment method, the components of the blocking buffer in step e are: PBS solution (pH 7.2), 1% bovine serum albumin (BSA);

In the method for enriching DON toxin-producing genes, the components of the SSC solution in step g are: 3M NaCl, 0.3M sodium citrate.

The invention uses a fast and simple method to enrich and detect DON toxin-producing genes in maize. It can reduce the interference of other environmental factors on the extraction of DON toxin-producing genes, and effectively enrich them for subsequent PCR detection.

The implementation of the invention will be described in detail below through specific examples, the purpose of which is to help readers better understand the essence of the present invention, but not as a limitation to the implementation scope of the present invention.

detailed description

Example 1

1. Extraction of total DNA from corn sample 1:

a. Corn sample 1 was crushed at 15,000rpm for 5min.

b. Take 1.5g sample powder in a 1.5mL centrifuge tube, add an appropriate amount of CTAB extraction buffer, and bathe in 60°C water for 15 minutes;

c. After cooling to room temperature, add phenol:chloroform:isoamyl alcohol (25:24:1), and centrifuge at 10,000rpm for 20min;

d. Add 1/10 volume of 10% CTAB-0.7M NaCl to the upper aqueous phase, then add an equal volume of chloroform:isoamyl alcohol (24:1), mix gently, and centrifuge at 10,000rpm for 20min;

e. Add an equal volume of frozen isopropanol to the upper aqueous phase, centrifuge at 3,000 rpm for 20 minutes, remove the supernatant, add 70% ethanol to soak the DNA, and change and wash the 70% ethanol 2-3 times;

f. Pour off 70% ethanol, dissolve DNA with appropriate amount of TE buffer after air drying, and store at -20°C.

2. DON toxin gene enrichment

a. 100 μL of carboxyl magnetic nanoparticles, separated by magnetic finger cot, and discarded the supernatant;

b. Add 100 μL of activation buffer to wash the magnetic beads, separate them with magnetic finger cots, repeat twice, and discard the supernatant;

c. Add 100 μL EDC solution and 100 μL NHS solution to the centrifuge tube, vortex to mix, activate at 20°C for 40 minutes, separate with magnetic finger cot, and discard the supernatant;

d. Add 10 μL of amino primers and 180 μL of PBS solution (pH 8.0), couple at 20°C for 2.5 hours, separate with magnetic finger cots, and discard the supernatant;

e. Add 100 μL of blocking buffer, resuspend the magnetic nanoparticles, react at 20°C for 2 hours to block the unreacted activated carboxyl groups on the surface of the magnetic nanoparticles, separate with magnetic finger cots, and discard the supernatant;

f. Add 100 μL of PBS solution (pH 7.2) to wash 3 times, separate with magnetic finger cots, and discard the supernatant;

g. Add 160 μL of SSC solution, 30 μL of formamide solution and 30 μL of single-stranded maize genomic DNA, hybridize at room temperature for 20 minutes, separate with magnetic finger cots, and discard the supernatant;

h. Add 100 μL PBS solution (pH 7.2) to wash 3 times, separate with magnetic finger cots, and discard the supernatant;

i. Add 30 μL of ddH ₂ O, heat at 94°C for 5 minutes, and transfer the supernatant to another centrifuge tube for storage at -20°C.

3. DON toxin production gene detection

a.PCR amplification system: 10×PCR Buffer 2.5μL; Mg ²⁺ (10μM) 2.5μL; dNTPs (2mM) 2.5μL; DON toxin-producing gene primers upstream and downstream each 0.5μL; DNA template 1.5μL; Taq enzyme ( 5U/μL) 0.3 μL; ddH ₂ O 14.7 μL. .

b. PCR reaction conditions:

c. Agarose gel electrophoresis

The PCR product and nucleic acid molecular weight reference (DL 2000) were placed on a 1% agarose gel at a constant voltage of 120V and electrophoresed for 30 minutes.

Example 2

1. Extraction of total DNA from corn sample 2:

a. Corn sample 2 was crushed at 20,000rpm for 3min.

b. Take 3g of sample powder in a 1.5mL centrifuge tube, add an appropriate amount of CTAB extraction buffer, and bathe in water at 65°C for 20 minutes;

c. After cooling to room temperature, add phenol:chloroform:isoamyl alcohol (25:24:1), and centrifuge at 15,000rpm for 15min;

d. Add 1/10 volume of 10% CTAB-0.7M NaCl to the upper aqueous phase, mix gently, then add an equal volume of chloroform:isoamyl alcohol (24:1), and centrifuge at 15,000rpm for 15min;

e. Add an equal volume of frozen isopropanol to the upper aqueous phase, centrifuge at 4,000 rpm for 15 minutes, remove the supernatant, add 70% ethanol to soak the DNA, and change and wash the 70% ethanol 2-3 times;

f. Pour off 70% ethanol, dissolve DNA with appropriate amount of TE buffer after air drying, and store at -20°C.

2. DON toxin gene enrichment

a. 300 μL of carboxyl magnetic nanoparticles, separated by magnetic finger cot, and discarded the supernatant;

b. Add 300 μL of activation buffer to wash the magnetic beads, separate them with magnetic finger cots, repeat twice, and discard the supernatant;

c. Add 150 μL EDC solution and 150 μL NHS solution to the centrifuge tube, vortex to mix, activate at 25°C for 40 minutes, separate with magnetic finger cot, and discard the supernatant;

d. Add 30 μL of amino primers and 140 μL of PBS solution (pH 8.0), couple at 25°C for 2 hours, separate with magnetic finger cots, and discard the supernatant;

e. Add 300 μL of blocking buffer, resuspend the magnetic nanoparticles, react at 25°C for 1.5 hours to block the unreacted activated carboxyl groups on the surface of the magnetic nanoparticles, separate with magnetic finger cots, and discard the supernatant;

f. Add 300 μL PBS solution (pH 7.2) to wash 3 times, separate with magnetic finger cots, and discard the supernatant;

g. Add 180 μL SSC solution, 40 μL formamide solution and 40 μL single-stranded maize genomic DNA, hybridize at room temperature for 35 minutes, separate with magnetic finger cots, and discard the supernatant;

h. Add 300 μL PBS solution (pH 7.2) to wash 3 times, separate with magnetic finger cot, and discard the supernatant;

i. Add 40 μL of ddH ₂ O, heat at 94°C for 5 minutes, and transfer the supernatant to another centrifuge tube for storage at -20°C.

3. DON toxin production gene detection

a.PCR amplification system: 10×PCR Buffer 2.5μL; Mg ²⁺ (10μM) 2.5μL; dNTPs (2mM) 2.5μL; DON toxin-producing gene primers upstream and downstream each 0.5μL; DNA template 1.5μL; Taq enzyme ( 5U/μL) 0.3 μL; ddH ₂ O 14.7 μL.

b. PCR reaction conditions:

c. Agarose gel electrophoresis

The PCR product and nucleic acid molecular weight reference (DL 2000) were stabilized at 125V on 1% agarose gel, and electrophoresed for 25 minutes.

Example 3

1. Extraction of total DNA of corn sample 3:

a. Corn sample 3 25,000rpm crushed for 1min.

b. Take 5g of sample powder in a 1.5mL centrifuge tube, add an appropriate amount of CTAB extraction buffer, and bathe in 70°C water for 15 minutes;

c. After cooling to room temperature, add phenol:chloroform:isoamyl alcohol (25:24:1), and centrifuge at 20,000rpm for 10min;

d. Add 1/10 volume of 10% CTAB-0.7M NaCl to the upper aqueous phase, then add an equal volume of chloroform:isoamyl alcohol (24:1), and centrifuge at 20,000rpm for 10min;

e. Add an equal volume of frozen isopropanol to the upper aqueous phase, centrifuge at 5,000 rpm for 10 minutes, remove the supernatant, add 70% ethanol to soak the DNA, and change and wash the 70% ethanol 2-3 times;

f. Pour off 70% ethanol, dissolve DNA with appropriate amount of TE buffer after air drying, and store at -20°C.

2. DON toxin gene enrichment

a. 500 μL of carboxyl magnetic nanoparticles, separated by magnetic finger cot, and discarded the supernatant;

b. Add 500 μL of activation buffer to wash the magnetic beads, separate them with magnetic finger cots, repeat twice, and discard the supernatant;

c. Add 200 μL EDC solution and 200 μL NHS solution to the centrifuge tube, vortex to mix, activate at 30°C for 30 minutes, separate with magnetic finger cot, and discard the supernatant;

d. Add 50 μL of amino primers and 100 μL of PBS solution (pH 8.0), couple at 30°C for 1.5 h, separate with magnetic finger cots, and discard the supernatant;

e. Add 500 μL of blocking buffer, resuspend the magnetic nanoparticles, react at 30°C for 1 hour to block the unreacted activated carboxyl groups on the surface of the magnetic nanoparticles, separate with magnetic finger cots, and discard the supernatant;

f. Add 500 μL PBS solution (pH 7.2) to wash 3 times, separate with magnetic finger cot, and discard the supernatant;

g. Add 200 μL SSC solution, 50 μL formamide solution and 50 μL single-stranded genomic DNA, hybridize at constant temperature for 40 minutes, separate with magnetic finger cots, and discard the supernatant;

h. Add 500 μL PBS solution (pH 7.2) to wash 3 times, separate with magnetic finger cots, and discard the supernatant;

i. Add 50ddH ₂ O, heat at 94°C for 5min, transfer the supernatant to another centrifuge tube and store at -20°C.

3. DON toxin production gene detection

a.PCR amplification system: 10×PCR Buffer 2.5μL; Mg ²⁺ (10μM) 2.5μL; dNTPs (2mM) 2.5μL; DON toxin-producing gene primers upstream and downstream each 0.5μL; DNA template 1.5μL; Taq enzyme ( 5U/μL) 0.3 μL; ddH ₂ O 14.7 μL.

b. PCR reaction conditions:

c. Agarose gel electrophoresis

The PCR product and nucleic acid molecular weight reference (DL 2000) were stabilized at 130V on 1% agarose gel and electrophoresed for 20 minutes.

Genomic DNA was extracted by CTAB method from three kinds of corn samples, then enriched by carboxyl magnetic nanoparticles, and then PCR was used to detect DON toxin-producing genes. According to the results of agarose gel electrophoresis of its genomic DNA, all three kinds of maize have bands of about 20,000 bp, indicating that the genomic DNA was successfully extracted. According to the results of agarose gel electrophoresis of the products obtained by PCR, the corn samples were directly subjected to PCR. Although the DON toxin-producing gene can be amplified, there are many miscellaneous bands, the bands of the gene are blurred, and there will be dragging. , which is not conducive to subsequent analysis; and after enrichment by carboxyl magnetic nanoparticles, the amplified DON toxin gene is relatively clear and does not contain miscellaneous bands, indicating that this method can effectively remove some miscellaneous bands, and can be used for DON The enrichment and purification of toxin-producing genes is beneficial to subsequent detection work.

Claims (10)

1. in corn, DON toxin produces a method for virus gene enrichment and detection, and it is characterized in that, detecting step comprises: CTAB method extracts STb gene; Utilize using carried DNA as template carboxyl magnetic nanoparticle to produce virus gene to DON toxin and carry out enrichment, then carry out pcr amplification; Whether PCR primer carries out agarose gel electrophoresis detection has target stripe thus determines whether produce virus gene containing DON toxin in detected corn.

2. in corn as claimed in claim 1, DON toxin produces the method for virus gene enrichment and detection, and it is characterized in that, described detection method concrete steps are as follows:

1), the extraction of STb gene:

A. corn sample 15000-25000rpm pulverizes 1-5min;

B. get 1-5g sample powder in centrifuge tube, add CTAB Extraction buffer, 60-70 DEG C of water-bath 15-25min;

C. be cooled to room temperature, add phenol: chloroform: primary isoamyl alcohol, ratio is 25:24:1, and the centrifugal 10-20min of 10,000-20,000rpm, obtains supernatant liquor 1;

The supernatant liquor 1 that d.c step obtains adds the 10%CTAB-0.7M NaCl of 1/10 volume, then adds isopyknic chloroform and primary isoamyl alcohol, and the ratio of chloroform and primary isoamyl alcohol is 24:1, and the centrifugal 10-20min of 10,000-20,000rpm, obtains supernatant liquor 2;

The supernatant liquor 2 of e.d step adds isopyknic freezing Virahol, and the centrifugal 10-20min of 3,000-5,000rpm, removes supernatant, adds 70% ethanol, changes clothes 70% ethanol therebetween 2-3 time;

F. 70% ethanol is outwelled, air-dry rear use appropriate TE buffer solution DNA ,-20 DEG C of preservations;

2), DON toxin produces virus gene enrichment

A. carboxyl magnetic nanoparticle 100-500 μ L, magnetic fingerstall is separated, and abandons supernatant;

B. add 100-500 μ L activation buffer washing magnetic nanoparticle, magnetic fingerstall is separated, and repeats 2-3 time, abandons supernatant;

C. add 100-200 μ L EDC solution and 100-200 μ L NHS solution in centrifuge tube, whirlpool mixes, 20-30 DEG C of activation 30-60min, and magnetic fingerstall is separated, and abandons supernatant;

D. add the amino primer of 10-50 μ L and 100-180 μ L PBS solution (pH8.0), softly mix, 20-30 DEG C of coupling 1.5-2.5h, magnetic fingerstall is separated, and abandons supernatant;

E. add 100-500 μ L Block buffer, resuspended magnetic nanoparticle, 20-30 DEG C of reaction 1-2h closes the unreacted activated carboxyl group in magnetic nanoparticle surface, and magnetic fingerstall is separated, and abandons supernatant;

F. add 100-500 μ L PBS solution (pH7.2) to wash 2-3 time, magnetic fingerstall is separated, and abandons supernatant;

G. add 150-200 μ L SSC solution, 30-50 μ L formamide soln and 30-50 μ L strand corn gene group DNA, room temperature hybridization 20-40min, magnetic fingerstall is separated, and abandons supernatant;

H. add 100-500 μ L PBS solution (pH7.2) to wash 2-3 time, magnetic fingerstall is separated, and abandons supernatant;

I. 20-50 μ L distilled water (ddH is added ₂o), 94 DEG C of heating 5min, supernatant liquor is transferred to-20 DEG C of preservations in another centrifuge tube;

3), DON toxin produces virus gene detection

A.PCR amplification system: 10 × PCR Buffer2.5 μ L; Mg ²⁺(10 μMs) 2.5 μ L; DNTPs (2mM) 2.5 μ L; DON toxin produces each 0.5 μ L of virus gene primer upstream and downstream; DNA profiling 1.5 μ L; Taq enzyme 5U/ μ L 0.3 μ L; ddH ₂o 14.7 μ L.Primer sequence is: upstream primer:

TACGTGAAACATTGTTGGC; Downstream primer: GGTGTCCCAGGATCTGCG; Note: A is VITAMIN B4; T is thymus pyrimidine; G is guanine; C is cytosine(Cyt);

B. reaction conditions:

C. agarose gel electrophoresis:

PCR primer and nucleic acid molecular weight object of reference (DL 2000), voltage stabilizing 120-130V on 1% sepharose, electrophoresis 20-30min; If there is deoxynivalenol enol (DON) in sample, have 237bp amplified production.

3. in a kind of corn as claimed in claim 2, DON toxin produces the method for virus gene enrichment and detection, it is characterized in that, in described Total DNA extraction method, the composition that b walks CTAB damping fluid is: 100mM Tris-HCl (pH8.0), 20mM EDTA-Na, 1.4mM NaCl, 2%CTAB, 2% beta-mercaptoethanol.

4. in a kind of corn as claimed in claim 2, DON toxin produces the method for virus gene enrichment and detection, it is characterized in that, in described Total DNA extraction method, the composition that d walks 10%CTAB-0.7M NaCl is: 4.1%NaCl, 10%CTAB.

5. in a kind of corn as claimed in claim 2, DON toxin produces the method for virus gene enrichment and detection, it is characterized in that, in described Total DNA extraction method, f walks TE buffer components and is: 10mMTris-HCl (pH8.0), lmM EDTA (pH8.0).

6. in a kind of corn as claimed in claim 2, DON toxin produces the method for virus gene enrichment and detection, it is characterized in that, described DON toxin produces in virus gene enriching method, and b walks activation buffer composition and is: 100mM MES (pH5.0), 0.05%Tween 20.

7. in a kind of corn as claimed in claim 2, DON toxin produces the method for virus gene enrichment and detection, it is characterized in that, described DON toxin produces in virus gene enriching method, and c walks EDC solution and NHS solution uses activation buffer as dispersion agent.

8. in a kind of corn as claimed in claim 2, DON toxin produces the method for virus gene enrichment and detection, it is characterized in that, described DON toxin produces in virus gene enriching method, and it is that in PCR system, DON toxin produces the upper amino group of virus gene upstream and downstream primer 5 ' end connection that d walks amino primer.

9. in a kind of corn as claimed in claim 2, DON toxin produces the method for virus gene enrichment and detection, it is characterized in that, described DON toxin produces in virus gene enriching method, and d walks PBS solution (pH8.0) and adds 0.05%Tween 20 to improve dispersiveness.

10. in a kind of corn as claimed in claim 2, DON toxin produces the method for virus gene enrichment and detection, it is characterized in that, described DON toxin produces in virus gene enriching method, and e walks Block buffer composition and is: PBS solution (pH7.2), 1%BSA; G walks SSC solution composition: 3M NaCl, 0.3M Trisodium Citrate.