阅读说明：本技术 一种快速检测甜樱桃李属坏死环斑病毒的方法 (Method for rapidly detecting prunus necrotic ringspot virus ) 是由 王磊 宿红艳 吴凡林 朱冬姿 王雪辉 王先钰 于 2020-04-17 设计创作，主要内容包括：本发明涉及植物病毒检测技术领域,特别涉及一种甜樱桃李属坏死环斑病毒的检测方法。包括以下步骤：1)、DNA片段与探针的设计合成；2)纳米金的制备；3)、纳米金-探针1复合物的制备；4)、试纸条的组装；5)、样品的制备；6)、核酸外切酶Ⅲ信号放大；7)、试纸条检测。本发明采用核酸检测探针制备试纸条,不需要制备抗体,制备工艺简单,成本低廉；针对甜樱桃李属坏死环斑病毒的高度特异保守区设计探针,检测效果好,特异性强,不易产生假阳性；利用核酸外切酶Ⅲ循环酶切进行信号放大,提高了检测灵敏度,且无需专业人员和专门的仪器,携带、检测方便,结果凭肉眼可测；显色时间短,仅需10min。(The invention relates to the technical field of plant virus detection, in particular to a method for detecting prunus necrotic ringspot virus. The method comprises the following steps: 1) designing and synthesizing a DNA fragment and a probe; 2) preparing nano gold; 3) preparing a nano gold-probe 1 compound; 4) assembling the test strip; 5) preparing a sample; 6) amplifying exonuclease III signals; 7) and detecting by using a test strip. The test strip is prepared by adopting the nucleic acid detection probe, an antibody does not need to be prepared, the preparation process is simple, and the cost is low; the probe is designed aiming at the highly specific conserved region of the prunus necrotic ringspot virus, the detection effect is good, the specificity is strong, and false positive is not easy to generate; signal amplification is carried out by using exonuclease III circulating enzyme digestion, so that the detection sensitivity is improved, professional personnel and special instruments are not needed, the carrying and the detection are convenient, and the result can be detected by naked eyes; the color development time is short, and only 10min is needed.)

1. The test strip for rapidly detecting the prunus necrotic ringspot virus is characterized by consisting of a sample pad, a gold-labeled binding pad coated with a nanogold-probe compound, a nitrocellulose membrane provided with a detection line probe and a quality control line probe, a water absorption pad and an adhesive base plate, wherein the sample pad, the gold-labeled binding pad, the nitrocellulose membrane and the water absorption pad are sequentially adhered to the adhesive base plate, the parts are overlapped by 2-3 mm, and are compacted after being assembled and dried and stored at 4 ℃, and the preparation process of the test strip is as follows:

1) DNA fragment and Probe design

Finding out highly specific conserved regions through 95 PNRSV nucleic acid sequences found in NCBI database, determining detection sequences, and designing hairpin DNA, nanogold Probe1, Probe2 and Probe3 based on the detection sequences. Wherein, the 5' end of Probe1 has a sulfydryl label, which can be combined with the nano-gold;

2) preparation of nano gold

The preparation of the nano-gold adopts a chloroauric acid reduction method, and the prepared nano-gold solution is stored at 4 ℃ in a dark place for later use;

3) synthesis of Nanogold-Probe 1 Complex

Concentrating the 1m L nanogold solution, covalently bonding the sulfhydryl modified Probe1 on the surface of the nanogold through an Au-S bond to assemble a nanogold-Probe 1 compound, and storing the nanogold-Probe 1 compound at 4 ℃ in a dark place;

4) and assembly of the test strip

The test strip is prepared by assembling a sample pad, a gold-labeled combination pad, a nitrocellulose membrane, an absorption pad and an adhesive base plate;

(1) the sample pad is composed of glass fiber, and is soaked in buffer solution and dried at 37 ℃;

(2) the gold-labeled bonding pad is composed of glass fiber, the nano gold-probe compound prepared in the step is dripped on the glass fiber pad, and the nano gold-probe compound is dried at 37 ℃;

(3) the nitrocellulose membrane was soaked in buffer and dried at 37 ℃. Scribing 10 MuM of Probe2 solution as a detection line at one end of the nitrocellulose membrane, scribing 10 MuM of Probe3 solution as a quality control line at the other end, and fixing and crosslinking the two lines at a distance of 5 mm;

(4) the absorption pad is composed of absorbent paper, and is soaked in PBS buffer solution and dried at 37 ℃;

(5) and (3) sequentially adhering the sample pad, the gold-labeled binding pad, the nitrocellulose membrane and the absorption pad on an adhesive base plate, overlapping each part by 2-3 mm, compacting by a heavy object overnight after assembly, and drying and storing at 4 ℃.

2. The detection method of the test strip for rapidly detecting the prunus necrotic ringspot virus according to claim 1, which is characterized by comprising the following steps:

1) preparation of samples

Taking PNRSV positive cherry plants and virus-free tissue culture seedling leaves, washing the leaves with tap water, then washing the leaves with distilled water, extracting total RNA of the leaves by adopting a CTAB method, taking 1 mu g of the total RNA to carry out DNase I treatment to remove residual DNA, heating the leaves at 95 ℃ for 5min to inactivate enzyme, cooling the leaves on ice, and carrying out reverse transcription by adopting a cDNA 1 st chain synthesis kit to obtain cDNA;

2) exonuclease III enzyme digestion amplification

Taking a proper amount of cDNA prepared in the step, adding a hybridization Buffer, heating at 95 ℃ for 5min, then quickly placing in an ice-water bath to form a free straight-chain state, adding 10 mu M Hairpin DNA solution 5 mu L into 10 mu L double distilled water, carrying out denaturation at 95 ℃ for 5min, then slowly cooling to room temperature to anneal the DNA solution to form a Hairpin-shaped secondary structure, then mixing the two DNA solutions, adding Exo III exonuclease 4 mu L, supplementing the double distilled water to 50 mu L, carrying out enzyme digestion at 37 ℃ for 60min, heating the reacted solution at 80 ℃ for 10min to inactivate the Exo III exonuclease, terminating the enzyme digestion reaction, and finally cooling the enzyme digestion reaction solution to room temperature for detection of a test strip;

3) and the test strip detection

And (3) dropwise adding the enzyme digestion reaction solution on a sample pad of the nucleic acid detection test strip, adding 100 mu L PBS buffer solution as developing solution to wash the test strip, and finally carrying out result judgment according to the color development conditions of the detection line and the quality control line, wherein the result is positive if the detection line and the quality control line simultaneously display red strips, the result is judged to be negative if the detection line does not display color and the quality control line displays red strips, and the detection is invalid if the quality control line does not display color.

Technical Field

The invention relates to the technical field of plant virus detection, in particular to a method for detecting prunus necrotic ringspot virus.

Background

The sweet cherry (Cerasus avium (L.) Moench), also named as a large cherry, is a rosaceous Prunus plant and is the main cultivated variety of the cherry in China at present, the sweet cherry originates from Europe and Western Asia, the cultivation history is long, the cultivated variety is many, the distribution range is wide, the sweet cherry is introduced into China before and after 1871 year, and the sweet cherry is mainly cultivated in the area around Bohai gulf at present, and has extremely high nutritional value and commercial value due to the fact that the fruit is bright in color, clear, fragrant, sweet, delicious and rich in nutrition, enjoys the reputation of 'treasure in fruit', and is deeply favored by consumers.

With the continuous development of the sweet cherry industry in China, the virus diseases are getting more and more serious, and become the main bottleneck restricting the healthy development of the industry. Wherein, Prunus Necrotic Ring Spot Virus (PNRSV) is one of the most common and serious diseases in China, and the sweet cherry disease caused by the Prunus necrotic ring spot virus can reduce the yield of orchards by 25 to 50 percent. At present, no effective medicament for preventing and treating prunus necrotic ring spot disease exists, and once the prunus necrotic ring spot disease occurs, the prunus necrotic ring spot disease is difficult to radically treat. The effective prevention and control measures carried out at the present stage are to strengthen the early quarantine of the nursery stock, popularize and plant disease-free nursery stock and stop the spread of the disease-free nursery stock. Therefore, the development of a simple, sensitive and efficient sweet cherry virus detection technology and product with the independent intellectual property rights of China is an urgent need for guaranteeing the healthy development of the sweet cherry industry of China.

At present, the PNRSV virus in China mainly adopts a serological E L ISA method and a molecular biology-based RT-PCR detection technology, but the methods have the defects of difficult preparation, tedious detection, long time consumption, need of special equipment, higher technical requirements on operators, easy occurrence of false positive and the like.

Disclosure of Invention

Aiming at the sweet cherry high-disease virus PNRSV, the invention establishes a cherry virus visual detection technology and a product research and development platform, combines an exonuclease III auxiliary amplification strategy with a nano-gold labeled nucleic acid probe, and establishes a brand-new rapid and sensitive cherry virus test strip detection method. The immune test strip broken by the test strip established by the invention has the dependence on the antibody, the artificially synthesized nucleic acid probe replaces the antibody, the bottleneck of preparing the monoclonal antibody by the hybridoma technology is avoided, and the test probe can be directly synthesized in a biological company, so that the production cost and the preparation period are greatly reduced. The color development process only needs 10min, no special instrument is needed, the result is directly read by naked eyes, and the method is convenient and quick.

The test strip for rapidly detecting the prunus necrotic ringspot virus is characterized by consisting of a sample pad, a gold-labeled binding pad coated with a nanogold-Probe (Probe1) compound, a nitrocellulose membrane provided with a detection line Probe (Probe2) and a quality control line Probe (Probe3) and a water absorption pad, wherein the sample pad, the gold-labeled binding pad, the nitrocellulose membrane and the water absorption pad are sequentially adhered to an adhesion bottom plate, the parts are overlapped by 2-3 mm, and are compacted after being assembled and dried and stored at 4 ℃, and the preparation process of the test strip is as follows:

1. design of DNA fragments and probes

Through the 95 PNRSV nucleic acid sequences found in NCBI database, highly specific conserved regions are found, detection sequences are determined, and hairpin DNA (hairpin DNA), nanogold Probe1, Probe2 and Probe3 are designed based on the detection sequences. Wherein, the 5' end of Probe1 has a sulfydryl label, which can be combined with the nano-gold;

2. preparation of nano gold

The preparation of the nano-gold adopts a chloroauric acid reduction method, and the prepared nano-gold solution is stored at 4 ℃ in a dark place for later use;

3. synthesis of Nanogold-Probe 1 Compound

Concentrating the 1m L nanogold solution, covalently bonding the sulfhydryl modified Probe1 on the surface of the nanogold through an Au-S bond to assemble a nanogold-Probe 1 compound, and storing the nanogold-Probe 1 compound at 4 ℃ in a dark place;

4. assembly of test strips

The test strip is prepared by assembling a sample pad, a gold-labeled combination pad, a nitrocellulose membrane, an absorption pad and an adhesive base plate;

(1) a sample pad (17mm × 5mm) consisting of glass fibers, which was soaked in buffer and dried at 37 ℃;

(2) the gold-labeled conjugate pad (8mm × 5mm) was composed of glass fiber, and the nanogold-probe complex prepared in step 2 of 40 μ L was dropped on the glass fiber pad and dried at 37 ℃.

(3) A nitrocellulose membrane (25mm × 5mm) was soaked in a buffer solution, dried at 37 ℃ and then cross-linked by fixing, with a Probe2 solution of 10. mu.M drawn at one end of the nitrocellulose membrane as a detection line and a Probe3 solution of 10. mu.M drawn at the other end as a quality control line, with a 5mm interval between the two lines.

(4) An absorbent pad (30mm × 5mm) consisting of absorbent paper was soaked in PBS buffer and dried at 37 ℃.

(5) And (3) sequentially adhering the sample pad, the gold-labeled binding pad, the nitrocellulose membrane and the absorption pad on an adhesive bottom plate (77mm × 5mm), overlapping each part by 2-3 mm, compacting by a heavy object overnight after assembly, and drying and storing at 4 ℃.

The invention relates to a detection method of a test strip for rapidly detecting prunus necrotic ringspot virus, which is characterized by comprising the following steps:

1. preparation of samples

Taking leaves of PNRSV positive cherry plants and virus-free tissue culture seedlings (negative control), washing the leaves with tap water, cleaning the leaves with distilled water, extracting total RNA of the leaves by a CTAB (cetyl trimethyl ammonium bromide) method, taking 1 mu g of the total RNA, carrying out DNase I treatment to remove residual DNA, heating at 95 ℃ for 5min to inactivate enzyme, cooling on ice, and carrying out reverse transcription by a cDNA 1 st chain synthesis kit to obtain cDNA;

2. exosucrase III (Exonuclease III, Exo III) enzyme digestion amplification

In order to improve the detection sensitivity, signal amplification is carried out by means of exonuclease III circulating enzyme digestion, firstly, a 68nt Hairpin DNA is designed, the 5 'end 20nt part of the Hairpin DNA is a PNRSV virus sequence, the 3' end 28nt part of the Hairpin DNA is a PNRSV virus complementary sequence, and the middle 20nt part of the Hairpin DNA is a sequence which can be combined with a quality control line probe;

the specific principle is as follows: when the target PNRSV exists, the stem-loop structure of the Hairpin DNA is opened and can be complementarily hybridized with the target PNRSV to form double-stranded DNA, ExoIII only acts on the complementary single strand in the double-stranded DNA, the double-stranded DNA is cut along the 3'→ 5' direction, the free DNA single strand cannot be cut, because the two ends of the cherry virus sequence and the 5 'end of the Hairpin DNA are free, the ExoIII only can cut 28nt of complementary part from the 3' end of the Hairpin DNA, then the cherry virus sequence and the residual part of the Hairpin DNA are released, the released cherry virus sequence can be hybridized with the Hairpin DNA again, the 5 'end part of the Hairpin DNA is released again through the ExoIII cutting, the circulation is repeated, and finally, the 5' end and the middle part of the Hairpin DNA are accumulated, and the cherry virus can be used as an enrichment signal for detection of a test strip. The specific operation steps are as follows:

adding hybridization Buffer into the cDNA prepared in the step, heating for 5min at 95 ℃, then rapidly placing in an ice-water bath to form a free straight chain state, adding 10 mu M hairpin DNA solution 5 mu L into 10 mu L double distilled water, denaturing for 5min at 95 ℃, then slowly cooling to room temperature to anneal the DNA solution to form a hairpin secondary structure, then mixing the two DNA solutions, adding Exo III exonuclease 4 mu L, supplementing the double distilled water to 50 mu L, carrying out enzyme digestion for 60min at 37 ℃, heating the reacted solution at 80 ℃ for 10min to inactivate the Exo III exonuclease, terminating the enzyme digestion reaction, and finally cooling the enzyme digestion reaction solution to room temperature for detection of a test strip;

3. detection of test strips

And (3) dropwise adding the enzyme digestion reaction solution on a sample pad of the nucleic acid detection test strip, adding 100 mu L PBS buffer solution as developing solution to wash the test strip, and finally carrying out result judgment according to the color development conditions of the detection line and the quality control line, wherein the result is positive if the detection line and the quality control line simultaneously display red strips, the result is judged to be negative if the detection line does not display color and the quality control line displays red strips, and the detection is invalid if the quality control line does not display color.

After the solution is unfolded for 5-10 min, a test strip detection line and a quality control line for detecting a positive plant sample simultaneously display red strips, which indicates that the test strip successfully detects the existence of PNRSV (human immunodeficiency virus) in a positive plant; the test strip for detecting the nontoxic tissue culture seedling sample only displays red strips on a quality control line, which shows that the test strip prepared by the invention is effective. The enzyme digestion product of the PNRSV nucleic acid is diluted by 10000 times and then is dripped onto the PNRSV test strip, and the detection line still develops color, which shows that the PNRSV test strip has extremely high sensitivity.

In order to research the specificity of the test strip, positive plant leaves of prunus necrotic ringspot virus (PNRSV), cherry-small-fruited virus-2 (L ChV-2), Plum Dwarf Virus (PDV), Cherry Virus A (CVA), cherry green ringspot virus (CGRMV) and cherry latent mosaic virus (C L MV) are respectively collected to extract RNA, after cDNA is reversely transcribed, the RNA is subjected to Exo III enzyme digestion, and enzyme digestion liquid is respectively dripped on the PNRSV virus detection test strip to detect.

Compared with the prior art, the nano gold nucleic acid test strip method established by the invention has the following advantages:

1. the test strip is prepared by adopting the nucleic acid detection probe, an antibody does not need to be prepared, the preparation process is simple, and the cost is low;

2. the probe is designed by taking the highly specific conserved region of the PNRSV as a target spot, the detection effect is good, the specificity is strong, and false positive is not easy to generate;

3. exo III circular enzyme digestion is used for signal amplification, so that the detection sensitivity is improved, no professional and special instrument is needed, the carrying and the detection are convenient, and the result can be detected by naked eyes;

4. the color development time is short, and only 10min is needed;

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.