CN102732641A - Chip for screening Pelamoviroid and application thereof - Google Patents

Abstract

The invention discloses a chip for screening peach latent mosaic viroids and its application. The present invention provides probes for identifying or assisting in identifying peach latent mosaic viroids, which consist of 9 probes, and the nucleotide sequences of the 9 probes are sequence 1- 9. Also provided is a gene chip for screening peach latent mosaic viroids, which is a gene chip obtained by immobilizing the above-mentioned probes on the surface of a film substrate. Experiments of the present invention prove that the present invention adopts the method of bioinformatics to analyze the nucleotide sequence of peach latent mosaic viroid genus viroid, and designs the compatibility probe of this group, and the verification result of standard viroid sample proves that the probe The needle works well. The chip for screening peach latent mosaic viroids of the invention can be used for quarantine identification of peach latent mosaic viroids.

Description

Chip for Screening Peach Latent Mosaic Viroids and Its Application

technical field

The invention relates to the technical fields of bioinformatics and bioquarantine identification, in particular to a chip for screening peach latent mosaic viroids and its application.

Background technique

Peach latent mosaic virus (Pelamoviroid) belongs to the avocado sun spot virus family (Avsunviridae), according to the eighth classification report of the International Committee on Taxonomy of Viruses (ICTV), this genus includes peach latent Peach latent mosaic viroid (PLMVd) and Chrysanthemum chlorotic mottle viroid (CChMVd). Peach latent mosaic virus is distributed in North and South America, Australia, Austria, China, Croatia, France, Greece, Japan, Morocco, Romania, Serbia, Spain and Tunisia, and has been identified as the most dangerous system on fruit trees in many countries One of the invasive pathogens, it mainly infects stone fruit trees such as peaches, plums, and apricots. Infestation of peach trees generally manifests as mosaic leaves, delayed germination, premature aging and even death in serious cases, resulting in serious economic losses. This type of virus can be transmitted by grafting and peach aphid, and spreads along with the vegetative propagation material over a long distance. It is easy to cause the spread of the virus during the vegetative propagation of peach trees and the transfer of seedlings. Chrysanthemum chlorotic mottle-like virus is mainly distributed in Denmark, France, India, Japan, South Korea, and China. It infects chrysanthemums, etc., causing the leaves of the affected plants to appear mottled, and then completely chlorotic; growth retardation and dwarfing.

With the expansion of my country's foreign exchanges, international and domestic germplasm exchanges are becoming more and more frequent, and strengthening quarantine inspection will play an important role in blocking the spread of this genus of viruses (especially virulent strains). The establishment of effective screening technology is the key to improving the effect of viroid quarantine inspection, but the current international methods for the detection of viroids in this genus mainly include biological identification of woody indicator plants, RT-PCR, LAMP and nucleic acid molecular hybridization techniques. These technologies can only perform specific detection of known viroids of the genus, and are powerless for the monitoring of unknown and new viroids, so it is easy to cause dangerous viroids to be missed and spread, which will lead to huge economic losses and bad society. Influence.

Contents of the invention

An object of the present invention is to provide a probe set for identifying or assisting in identifying peach latent mosaic viroids.

The probe set provided by the present invention for identifying or assisting in identifying peach latent mosaic viroids is composed of a total of 9 probes from probe 1 to probe 9, and the nucleosides of probe 1 to probe 9 The acid sequences are respectively sequences 1-9 in the sequence listing.

In the above probes, the peach latent mosaic viroid is peach latent mosaic viroid or chrysanthemum chlorotic mottle viroid.

Another object of the present invention is to provide a gene chip for screening peach latent mosaic viroids.

The gene chip for screening peach latent mosaic viroids provided by the present invention is a gene chip obtained by fixing the above-mentioned probe group on the surface of a film base.

In the above-mentioned gene chip, the sheet substrate is an aldehydized glass sheet substrate, and in the embodiments of the present invention, Boao Biological Co., Ltd. is used Microarray substrate, catalog number: 420022.

In the above gene chip, the peach latent mosaic viroid is peach latent mosaic viroid or chrysanthemum chlorotic mottle viroid.

The third object of the present invention is to provide a kit for screening peach latent mosaic viroids.

The kit provided by the present invention includes the above-mentioned gene chip.

The above kit also includes a primer pair for amplifying the peach latent mosaic viroid genus cDNA, and the primer pair is specifically primer pair 1 or primer pair 2;

The primer pair 1 is composed of DNA molecules shown in sequence 10 in the sequence listing and sequence 11 in the sequence listing (for amplifying peach latent mosaic viroid);

The primer pair 2 consists of DNA molecules shown in sequence 12 in the sequence listing and sequence 13 in the sequence listing (for amplifying chrysanthemum chlorotic mottle viroid);

The peach latent mosaic viroid is peach latent mosaic viroid or chrysanthemum chlorotic mottle viroid.

The application of the above-mentioned probe set, the above-mentioned gene chip or the above-mentioned kit in the following 1)-4) is also within the protection scope of the present invention:

1) Identify or assist in the identification of peach latent mosaic viroids;

2) Prepare and identify or assist in the identification of peach latent mosaic viroid products;

3) To identify or assist in the identification of plants to be tested infected with peach latent mosaic viroids;

4) Preparation of virus-like products for identification or auxiliary identification of plants infected with peach latent mosaic virus.

In the above application, the peach latent mosaic viroid is specifically peach latent mosaic viroid or chrysanthemum chlorotic mottle viroid; and the plant to be tested is peach or chrysanthemum.

The fourth object of the present invention is to provide a method for screening or assisting in screening the plants to be tested infected with peach latent mosaic viroids.

The method provided by the invention comprises the steps of:

1) Labeling the cDNA of the tissue of the plant to be tested to obtain the labeled product;

2) Hybridize the labeled product obtained in step 1) with the above-mentioned gene chip to obtain a hybridized chip;

3) Scan the chip after hybridization obtained in step 2),

If the absolute signal value of at least one of the probes on the gene chip is not less than 600 and the signal-to-noise ratio is not less than 3.0, the plant to be tested is infected or candidate infected with Peach latent mosaic viroid.

In the above method, in step 1), the labeling is to use the cDNA as a template and perform PCR amplification with the primer pair in the above-mentioned kit to obtain a PCR product, and then carry out Klenow enzyme labeling on the PCR product , to obtain the labeled product;

In step 2), the hybridization temperature is 42°C, and the hybridization time is 12 hours;

After the step 2) and before the step 3), it also includes the step of washing the chip after hybridization; the at least one probe is any one of the above probe groups;

The tissue is a leaf;

The peach latent mosaic viroid, the primer pair and the plant to be tested are as follows 1) or 2):

1) The peach latent mosaic viroid is peach latent mosaic virus, and the primer pair is the primer pair 1; the plant to be tested is peach;

2) The peach latent mosaic viroid is a chrysanthemum chlorotic mottle viroid, and the primer pair is the primer pair 2; the plant to be tested is a chrysanthemum.

Experiments of the present invention prove that the probes provided by the present invention for screening the Peach Latent Mosaic Viroid chip have high compatibility at the level of Peach Latent Mosaic Viroids and specificity within the genus. Sex, the required sample amount is small, generally only 0.1g is required. In addition, data analysis is combined with computer image processing software to achieve intuitive and visualized analysis results. The invention adopts bioinformatics method to analyze the nucleotide sequence of peach latent mosaic viroid genus viroid, and designs the compatibility probe of the genus, and the verification result of standard viroid sample proves that the probe effect is good. The genus chip of the invention can be used for quarantine and identification of peach latent mosaic viroids.

Description of drawings

Figure 1 is a schematic diagram of the peach latent mosaic virus viroid screening chip probe array (10×12)

Fig. 2 is the result of using the chrysanthemum chlorotic mottle viroid sample to verify the peach latent mosaic viroid screening chip

Figure 3 shows the sensitivity test results of the screening chip

Fig. 4 is the detection result of PCR sensitivity

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1, Preparation of a Chip for Screening Peach Latent Mosaic Viroids

1. Design of genus-level highly compatible oligonucleotide probes

Download the whole genome and nucleic acid sequence of Viroids from the database of National Center for Biotechnology Information (NCBI) and the International Committee on Virology (ICTV); remove more than 90% of the nucleic acid sequences with 95% similarity to other sequences; use 5 Bases are used as intervals to continuously extract all 40mer nucleic acid sequences; 40%≤GC content≤60%, single base content≤50%, number of consecutive repeated bases≤4, and no hairpin structure of more than 6 bases is Nucleic acid sequences were screened for standards, and homology comparisons were performed in the NCBI database to ensure the specificity of the probes obtained.

Nine probes (probe 1-probe 9) of peach latent mosaic viroid genus viroids were designed according to the above principles, and their nucleotide sequences are respectively shown in sequence 1-9 in the sequence list.

The above nine probes can be artificially synthesized.

2. Chip preparation

芯片点样液,产品目录号:440010）溶解，浓度为50μM，每条探针横向重复3点点在醛基化玻璃片基芯片（博奥生物有限公司

微阵列基片,产品目录号:420022）上，每点约0.25nL，点直径约180μm，点间距为300μm，点样均匀度的标准方差为15％。将芯片点有探针的一面在65℃水浴锅上水合10s，芯片距水面距离为3cm，在空气中室温自然干燥，在进行一次水合。将点有探针的一面向上，放在紫外交联仪中250mJ交联。将芯片放在42℃预热，0.5％SDS清洗10min。将芯片转移到42℃预热蒸馏水中清洗2min。把芯片放在50mL锥形离心管中，2000rpm离心1min，以去除芯片表面的液体，获得筛查桃潜隐花叶类病毒属类病毒的芯片。The 9 probes obtained in the above 1 were respectively used in the spotting buffer (Boao Biological Co., Ltd.

Chip spotting solution, product catalog number: 440010) was dissolved at a concentration of 50 μM, and each probe was repeatedly spotted on an aldehyde-based glass chip chip (Boao Biotechnology Co., Ltd.

On a microarray substrate, catalog number: 420022), each spot is about 0.25nL, the spot diameter is about 180μm, the spot spacing is 300μm, and the standard deviation of spot uniformity is 15%. Hydrate the side of the chip with the probe on it in a 65°C water bath for 10 seconds. The distance between the chip and the water surface is 3 cm, and dry naturally at room temperature in the air, and then perform a hydration. Place the side with the probe pointing up, and place it in a UV cross-linker at 250mJ for cross-linking. Preheat the chip at 42°C and wash with 0.5% SDS for 10 minutes. Transfer the chips to preheated distilled water at 42°C for 2 min. Put the chip in a 50mL conical centrifuge tube and centrifuge at 2000rpm for 1min to remove the liquid on the surface of the chip to obtain a chip for screening peach latent mosaic viroids.

The chip for screening peach latent mosaic viroids is shown in Figure 1, and 1-9 in Figure 1 is the peach latent mosaic virus screening chip probes 1-9 (corresponding to sequence 1- 9), Hex is the positive quality control of chip immobilization, PC is the positive quality control of hybridization, and NC is the negative quality control of hybridization.

Example 2, application of the chip for screening peach latent mosaic viroids

1. Screening chip detection samples

1. Extraction of total RNA from samples used for detection

1) Take peach tree leaves infected with Peach latent mosaic viroid (Peach latent mosaic viroid, the Latin name for Peach latentmosaic viroid, is recorded in: Cloning and sequence analysis of Chinese isolate P3 of peach latent mosaic viroid. 2005 , Phytopathological Journal 35 (4): 300-304., the public can obtain from the Chinese Academy of Inspection and Quarantine.) and chrysanthemum leaves of chrysanthemum chlorotic mottle viroid (Chrysanthemum chlorotic mottle viroid. purchased from American type culture collection referred to as ATCC, PV-120) each 0.1g sample, add 1mL grinding buffer [Na ₂ HPO ₄ 12H ₂ O 3.58g, KH ₂ PO ₄ 0.27g, NaCl 8g, KCl 0.2g, Na ₂ SO ₃ 1.3g, polyvinylpyrrolidone (PVP) MW 24-40 000 20g, NaN ₃ 0.2g, chicken egg protein (pow dered egg albumin) 2.0g, Tween-20 20.0mL, dissolved in 1000mL deionized water, adjust the pH value 7.5], grind evenly, transfer to a 1.5mL centrifuge tube, centrifuge at 6000r/min for 5min, and take the supernatant;

2) Wash the nano magnetic beads twice with deionized water in the PCR tube, discard the deionized water, add 100 μL of supernatant, mix well, and place at room temperature for 10 min, during which the sample was mixed several times by inversion. Discard the supernatant after magnetically adsorbing the nano magnetic beads, add 200 μL of PBS [NaCl 8.0g, Na ₂ HPO ₄ , 1.15g, KH ₂ PO ₄ 0.2g, KCl 0.2g, add 900mL distilled water to dissolve, adjust the pH value to 7.4, add distilled water Dilute to 1L] to wash the nano magnetic beads;

3) After washing, add 50 μL ddH ₂ O to re-suspend the magnetic nano-beads, and treat at 95°C for 5 minutes; absorb the magnetic nano-beads with a magnet, absorb the supernatant as RNA, and store at -20°C for later use.

2. Sample labeling and hybridization

The RNA obtained above was reverse transcribed to obtain cDNA.

PCR amplification, that is, add 2 μL of cDNA product, 0.5 μL of upstream primer (final concentration of 0.5 mmol/L), 0.5 μL of downstream primer (final concentration of 0.5 mmol/L), dNTP Mix (10 mmol/L) to a 0.2 mL reaction tube L) 0.5 μL, Taq enzyme (5U/μL) 0.5 μL, PCR buffer (10×) 2 μL and DEPC-H ₂ O 14 μL, and then amplify according to the PCR reaction procedure.

The upstream and downstream primers consisted of primer pairs for amplifying peach latent mosaic viroid or chrysanthemum chlorotic mottle viroid respectively.

The above-mentioned primer pairs for amplifying peach latent mosaic viroid include

Upstream primer: 5'-CCAGGTAACGCCGTAGAAACTG-3' (SEQ ID NO: 10);

Downstream primer: 5'-ATCACACCCTCCTCGGAACCAA-3' (SEQ ID NO: 11);

The PCR reaction program is: 95°C for 3min; 94°C for 30s, 58°C for 30s, 72°C for 30s; 30 cycles; 72°C for 10min.

The above-mentioned primer pair for amplifying chrysanthemum chlorotic mottle viroid includes

Upstream primer: 5'-GGCACCTGATGTCGGTGT-3' (SEQ ID NO: 12);

Downstream primer: 5'-GACCTCTTGGGGGTTTCAAAC-3' (SEQ ID NO: 13);

The PCR reaction program is: 94°C 5min; 94°C 30s, 55°C 30s, 72°C 30s; 30 cycles; 72°C 8min.

The Klenow enzyme labeling system is 25 μL, that is, add 5 μL of PCR product, 2 μL of 9N random primer (invitrogen, Cat. No. 48190-011) (100 μmol/L) and 12 μL of H ₂ O into a 0.2 mL PCR reaction tube, denature at 95°C 3min, ice bath 5min. Then add 2.5 μL of 10× Klenow enzyme buffer, 2 μL of dNTP (2.5 mmol/L), 0.5 μL of cy3-dCTP (Amersham, Cat.No.PA 53021 final concentration is 5 nmol/L), klenow enzyme (5 U /μL) 1 μL. React at 37°C for 1.5h, denature at 70°C for 5min, and ice-bath for 5min to obtain labeled samples.

Hybridization: 16 μL system, including 2.4 μL SSC (final concentration 3×), 0.32 μL SDS (final concentration 0.2%), 4 μL formamide (final concentration 25%), 1.6 μL Denhardt’s (Ameresco, Cat.No.E717, final Concentration 5×) and 7.68 μL of labeled sample. Denature at 95°C for 3 minutes, ice-bath for 5 minutes, and centrifuge briefly. Add the hybridization solution to the chip, cover the cover glass, and hybridize overnight (12h) in a water bath at 42°C to obtain the hybridized chip (peach latent mosaic virus) and the hybridized chip (chrysanthemum chlorotic mottle viroid). ).

3. Washing and scanning

The cleaning system and procedures are as follows:

Preheat the lotions I and II in a microwave oven to 42°C, and transfer them to the cleaning box. After the hybridization, transfer the hybridized chip to the washing box containing the washing solution, with the hybridization side facing up, and slowly wash it on a horizontal shaker. After cleaning the chip, put it in a 50mL conical centrifuge tube, centrifuge at 2000rpm for 1min, remove the liquid on the surface of the chip, and obtain the chip to be detected (peach latent mosaic virus) and the chip to be detected (chrysanthemum chlorotic mottle virus).

Put the above chip to be detected (peach latent mosaic virus) and the chip to be detected (chrysanthemum chlorotic mottle virus) in the scanner for scanning analysis; PMT is set to 900, and the fluorescence intensity and background intensity of each point are obtained .

The data was extracted from the chip using the LuxScan 3.0 chip scanner of Boao Biotech, and the signal value of the probe was the median value of the foreground value of the probe minus the median value of the background value. The signal-to-noise ratio is the ratio of the median value of all signal values in the corresponding point of the image to the median value of the background value.

If the signal value of at least one of the probes is ≥600 and the signal-to-noise ratio is ≥3, it is judged to be positive (it is a peach latent mosaic virus-like infection); the signal value of the probe is <600 and the signal-to-noise ratio is <2 , judged negative (not peach latent mosaic viroid infection); the rest of the cases were judged suspicious and repeated verification was required.

The probe arrangement in Figure 2 is the same as that in Figure 1, and the positive probes are 1-9.

The verification results of chrysanthemum chlorotic mottle-like virus are shown in Figure 2. The signal values at the positions of probes 7, 8, and 9 are 1992, 20,970, and 18,483, respectively, all greater than 600; the signal-to-noise ratios are 6, 52, and 48, respectively. All greater than 3, illustrate that the present invention can detect the chrysanthemum chlorotic mottle viroid of peach latent mosaic viroid;

The fixed positive quality control, hybridization positive quality control and hybridization negative quality control of the above chip performed well, and the hybridization signal of the standard sample was strong, indicating that the designed probe and the established chip screening program had good working results.

2. Comparison of screening chip and PCR detection sensitivity

Accurately weigh 0.1 g of peach tree tissue infected with PMTV, extract total RNA, and use it for PMTV-like screening chip detection and PCR detection respectively. 10 ¹ and 10 ² -fold serial dilutions.

The chip detection method is the same as the above-mentioned one, and the result is shown in Figure 3. The probe arrangement in Figure 3 is the same as that in Figure 1, where A: 10 ¹ dilution; B: 10 ² dilution. The signal values at the positions of probes 1 and 2 in A are 3913 and 1004, both greater than 600; the signal-to-noise ratios are 8 and 3, both ≥3; therefore, probes 1 and 2 are judged as positive. The signal value at the position of probe 1 in B is 1531, greater than 600; the signal-to-noise ratio is 4; it can be determined that probe 1 is positive. Therefore, the peach latent mosaic viroid screening chip can detect the highest dilution factor of the object to be tested is 10 ² .

The primers for PCR detection are

Upstream primer: 5'-CCAGGTAACGCCGTAGAAACTG-3';

Downstream primer: 5'-ATCACACCCTCCTCGGAACCAA-3'.

The results of PCR detection are shown in Figure 4, 1: 10 ¹ dilution; 2: 10 ² dilution; 3: blank control; M: MarkerDL2000, it can be seen that both 10 ¹ and 10 ² dilution templates obtained a 337bp product (Genbank No. Nucleotides 1-337 of GU290549), can detect the dilution factor of 10 ² of the object to be tested.

Therefore, the sensitivity of the peach latent mosaic viroid screening microarray is equivalent to that of the PCR method.

Claims (10)

1. A probe group for identifying or assisting in identifying peach latent mosaic viroids, consisting of a total of 9 probes from probe 1 to probe 9, the nucleosides of said probe 1 to probe 9 The acid sequences are respectively sequences 1-9 in the sequence listing. 2 . The probe set according to claim 1 , wherein the peach latent mosaic viroid is peach latent mosaic viroid or chrysanthemum chlorotic mottle viroid. 3 . 3. A gene chip for screening peach latent mosaic viroids, which is the gene chip obtained by fixing the probe set according to claim 1 or 2 on the surface of the film base. 4. The gene chip according to claim 3, characterized in that: the sheet base is an aldehyde-based glass sheet base; the peach latent mosaic virus is a peach latent mosaic virus or a chrysanthemum Chlorotic mottle viroid. 5. A kit for screening peach latent mosaic viroids, comprising the gene chip according to claim 3 or 4. 6. kit according to claim 5, is characterized in that: described kit also comprises the primer pair that is used for amplifying described peach latent mosaic viroid genus viroid cDNA, and described primer pair is specifically primer Pair 1 or primer pair 2; said primer pair 1 consists of the DNA molecule shown in sequence 10 in the sequence listing and sequence 11 in the sequence listing; The primer pair 2 consists of DNA molecules shown in sequence 12 in the sequence listing and sequence 13 in the sequence listing; The peach latent mosaic viroid is peach latent mosaic viroid or chrysanthemum chlorotic mottle viroid. 7. the probe group described in claim 1 or 2, the gene chip described in claim 3 or 4, the test kit described in claim 5 or 6 The application in the following 1)-4) is also the protection scope of the present invention: 1) Identify or assist in the identification of peach latent mosaic viroids; 2) Prepare and identify or assist in the identification of peach latent mosaic viroid products; 3) To identify or assist in the identification of plants to be tested infected with peach latent mosaic viroids; 4) Preparation of virus-like products for identification or auxiliary identification of plants infected with peach latent mosaic virus. 8. application according to claim 7, is characterized in that: described peach latent mosaic viroid is specifically peach latent mosaic viroid or chrysanthemum chlorotic mottle viroid; described plant to be tested is peach or chrysanthemum. 9. A method for screening or assisting screening of plants to be tested infected with peach latent mosaic viroids, comprising the steps of: 1) Labeling the cDNA of the tissue of the plant to be tested to obtain the labeled product; 2) Hybridizing the labeled product obtained in step 1) with the gene chip described in claim 3 or 4 to obtain a hybridized chip; 3) Scan the chip after hybridization obtained in step 2), If the absolute signal value of at least one of the probes on the gene chip is not less than 600 and the signal-to-noise ratio is not less than 3.0, the plant to be tested is infected or candidate infected with Peach latent mosaic viroid. 10. The method of claim 9, wherein: In step 1), the labeling is to use the cDNA as a template and perform PCR amplification with the primer pair in the kit according to claim 6 to obtain a PCR product, and then carry out Klenow enzyme labeling on the PCR product , to obtain the labeled product; In step 2), the hybridization temperature is 42°C, and the hybridization time is 12 hours; After the step 2) and before the step 3), a step of washing the post-hybridization chip is also included; The at least one probe is any one of the above-mentioned probe groups; The tissue is a leaf; The peach latent mosaic viroid, the primer pair and the plant to be tested are as follows 1) or 2): 1) The peach latent mosaic viroid is peach latent mosaic virus, and the primer pair is the primer pair 1; the plant to be tested is peach; 2) The peach latent mosaic viroid is a chrysanthemum chlorotic mottle viroid, and the primer pair is the primer pair 2; the plant to be tested is a chrysanthemum.

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