CN114196789A - Method for non-invasively and rapidly determining whether plants are infected with tomato yellow leaf curl virus - Google Patents

Abstract

The invention relates to the field of plant disease control, and discloses a method for determining whether a plant is infected by a virus tomato yellow leaf curl virus or not by honeydew secreted by vector insects, which can effectively reduce the workload of a traditional detection method and simultaneously avoid the problem that the plant is not easy to find due to early infection of the tomato yellow leaf curl virus; the method can find the diseases at the early stage of the infection of the plants with the tomato yellow leaf curl virus, and further prevent and treat the diseases as early as possible, thereby effectively inhibiting the development and the spread of the diseases.

Description

Method for non-invasively and rapidly determining whether plants are infected with tomato yellow leaf curl virus

Technical Field

The invention belongs to the field of plant disease control, and particularly relates to a method for rapidly determining whether a plant is infected with tomato yellow leaf curl virus in a non-invasive manner.

Background

The infection symptoms are obvious only after Tomato Yellow Leaf Curl Virus (TYLCV) infects tomato seedlings for 3-4 weeks, the plants grow badly, the tomato tender leaves curl upwards more or less, and the TYLCV infection of the tomato seedlings can cause the complete loss of the growth capacity of the tomato plants, thereby causing the reduction of the yield and the quality of crops.

The field detection mainly identifies the plant infected with TYLCV through the disease symptoms in the field, however, the identification of the field symptoms is only a preliminary empirical judgment, and further determination requires indoor molecular biological detection of the sample. The conventional PCR detection method is complex, the fluorescent quantitative PCR is easy to generate errors according to standard curve judgment, whether the plant is infected by the TYLCV can not be judged at the early stage of plant infection, and the existing detection method can not meet the field detection requirement. In addition, the existing molecular biological detection methods need to collect plant samples, so that damage to plants is irreversible.

Bemisia tabaci is an important plant virus transmission medium, serves as the most main transmitter of geminivirus, namely Tomato Yellow Leaf Curl Virus (TYLCV), and largely causes the sweep situation of the virus in China after 2006. The bemisia tabaci spreads tomato yellow leaf curl virus in a durable manner, and the bemisia tabaci eats plant juice with adults and nymphs, and the damaged leaves are faded, yellow and wilted, and the virus is also spread in the process of smoking.

Disclosure of Invention

Aiming at the technical problem that the infection condition can not be known at the early stage of Tomato Yellow Leaf Curl Virus (TYLCV) infection in the prior art, the invention provides a method for rapidly determining whether a plant is infected with tomato yellow leaf curl virus in a non-invasive way, which comprises the following steps:

(1) collecting whitefly honeydew on plant leaves by using a cotton swab stained with buffer ASL and extracting DNA from the honeydew;

(2) taking the DNA extracted in the step (1) as a template, adding primers with sequences of SEQ ID NO.1 and SEQ ID NO.2 and a probe with sequence of SEQ ID NO.3, and 2 XddPCR Super Mix For Probes and RNase free water to prepare a reaction system;

(3) adding the system prepared in the step (2) and the droplet generating oil into a droplet generating card, and placing the droplet generating card into a droplet generator to generate droplets;

(4) transferring the microdroplets generated in the step (3) to a ddPCR 96 pore plate for amplification reaction, and analyzing microdroplet fluorescence data after the reaction is finished;

(5) analyzing the data result, calculating the copy number contained in the sample by copies/mu L to obtain whether the collected sample is infected by TYLCV, and judging that the sample is infected by TYLCV when the number of the positive drops is more than 5.

On the basis of the scheme, the primer sequence in the step (2) is as follows: 5'-CCGAAAGCCCAGAATATACAGA-3' (SEQ ID NO.1), 5'-CCAGATCCACGAGTAACATCAC-3' (SEQ ID NO. 2). The probe sequence in the step (2) is as follows: 5'-CCCATGTAAAGTCCAGTCTTATGAGCAACG-3' (SEQ ID NO. 3). The 5 'end of the probe is marked with a fluorescent group, and the 3' end of the probe is marked with a quenching group; the fluorescent group is one of FAM, TET, HEX, CY3 or JOE; the quenching group is one of BHQ1, BHQ2, TAMRA, MGB or DABCYL.

Plants to which the above method is applicable include, but are not limited to, tomato, pepper, eggplant.

The primer and the probe can be simultaneously used as a detection means after Tomato Yellow Leaf Curl Virus (TYLCV) is infected, and the detection means uses a droplet type digital PCR technology.

The method for determining whether the plant is infected by the virus tomato yellow leaf curl virus or not by using honeydew secreted by vector insects can effectively reduce the workload of the traditional detection method and simultaneously avoid the problem that the plant is not easy to find due to early infection of the tomato yellow leaf curl virus; the method can find the diseases at the early stage of the infection of the plants with the tomato yellow leaf curl virus, and further prevent and treat the diseases as early as possible, thereby effectively inhibiting the development and the spread of the diseases.

Aiming at the problems that the existing TYLCV molecular biology detection methods need to sample plants, damage the integrity of the plants and cause irreversible damage to the plants, the method for detecting the virus infection condition through the bemisia tabaci honeydew belongs to non-invasive and can effectively avoid damage to the plants in the sampling process; and the mobility of the bemisia tabaci is large, and like a collector, a plurality of plants can be collected, compared with the traditional sampling means (one plant is used), the method disclosed by the patent uses the bemisia tabaci, and can simultaneously collect a plurality of plants by utilizing the characteristic of large mobility of the bemisia tabaci, so that the effect of enriching the sampling is achieved, the sampling workload is saved, and the sample collection range is expanded. Healthy plants cannot immediately get ill after being infected by TYLCV, and the detection error of the collected plant samples is large. And the bemisia tabaci can be transferred to other healthy plants after infecting the healthy plants, so that the bemisia tabaci sample cannot be obtained, and therefore, the problem that the vector insects cannot be obtained due to the transfer of the bemisia tabaci can be reasonably solved by collecting honeydew generated on leaves of the bemisia tabaci.

The primer and the probe sequence designed aiming at the specific capsid protein of the TYLCV have high detection sensitivity and accuracy; meanwhile, the invention uses the droplet type digital PCR technology to carry out fluorescence labeling on the probe sequence, detects the expression of TYLCV by the ddPCR technology, establishes a detection method based on the droplet type digital PCR technology, is applied to the judgment of the virus infection condition of plants in the field, has the characteristics of high efficiency and accuracy, and can quickly diagnose the TYLCV at the initial stage of the plant infection so as to effectively inhibit the prevalence of the TYLCV in the field.

Drawings

The advantages and benefits of the present invention will become apparent to those of ordinary skill in the art upon reading the following detailed description of the specific embodiments. The drawings are illustrative and not to be considered as limiting the invention. In the drawings:

FIG. 1 is a linear range fit standard curve of TYLCV detected in example 4;

FIG. 2 shows the results of quantitative determination of the amount of TYLCV in the Bemisia tabaci honeydew in example 6.

Detailed Description

The following examples are presented to illustrate the technical solutions of the present invention. It should be noted that the following examples do not limit the scope of the claimed invention.

The starting materials, reagents and apparatuses used in the following examples are conventionally commercially available or can be obtained by conventionally known methods, unless otherwise specified.

Experimental materials: tomato Zhongza No. 9 for breeding TYLCV bemisia tabaci populations was purchased from technical and scientific industries of Medium vegetable culture (Beijing) Co., Ltd; the bemisia tabaci population was provided by the biological intrusion laboratory of the university of Qingdao agriculture.

Main reagents and instruments: SPARKeasy fecal genome DNA rapid extraction kit (AA0701-B) is purchased from Qingdao Haoise science and technology Limited; high speed refrigerated centrifuge (Eppendorf, germany); a NanoDrop1000 micro uv spectrophotometer (seimer feishel, usa); Bio-Rad QX200TM Droplet Digital PCR System (including a Droplet generation card, a Droplet generator, a membrane sealer, a gradient PCR machine, and a Droplet fluorescence detector) (Burley, USA); pipette gun (edward, germany); 2 XDddPCR supermix for probes, droplet generation oil for probes, and droplet analysis oil (ddPCR droplet readoil) (Burley, USA).

Example 1

This example provides a microdroplet digital PCR primer and probe for detecting TYLCV, which is based on capsid protein sequence, and designed by analyzing specific conserved sequence of TYLCV and using molecular biology software.

Wherein the designed primer nucleotide sequence is as follows:

5’-CCGAAAGCCCAGAATATACAGA-3’(SEQ ID NO.1)，

5’-CCAGATCCACGAGTAACATCAC-3’(SEQ ID NO.2)。

wherein the designed probe nucleotide sequence is as follows:

5’-CCCATGTAAAGTCCAGTCTTATGAGCAACG-3’(SEQ ID NO.3)

the 5 'end of the probe is marked with a fluorescent group FAM, and the 3' end of the probe is marked with a quenching group BHQ 1.

The length of the fragment amplified by using the above primers is 150 bp.

Example 2

After two cotyledons of the tomato seedlings grow out, cultivating the tomato seedlings on a single seedling tray, infecting the tomato seedlings by TYLCV virus in a true leaf period of 4-5 days, putting bemisia tabaci into the tomato seedlings after infecting the tomato seedlings for 12 days, collecting honeydew generated by the bemisia tabaci on the tomato leaves after putting the bemisia tabaci into the tomato seedlings for 5 days, and extracting DNA, wherein the specific extraction method comprises the following steps:

the method for extracting the genomic DNA of the bemisia tabaci honeydew by utilizing the SPARKeasy fecal genomic DNA rapid extraction kit (AA0701-B) comprises the following specific steps:

(1) collecting Bemisia tabaci honey by using a cotton swab (provided with an AA0701-B kit) stained with buffer solution ASL, and exposing the Bemisia tabaci honey in a centrifuge tube filled with 1.4ml of buffer solution ASL;

(2) continuously carrying out vortex oscillation on the centrifuge tube in the step (1) for 1min or until the centrifuge tube is completely and uniformly mixed;

(3) incubating the resuspension at 70 ℃ for 5 min;

(4) vortex for 15s and let stand at room temperature for 1 min. Centrifuging at 12000rpm for 1min to precipitate fecal particles;

(5) transfer 900. mu.L of supernatant to a 1.5mL centrifuge tube, add 100. mu.L of impurity scavenger AB, immediately vortex and shake for 1min or until uniform, and stand at room temperature for 1 min. Centrifuging at 12000rpm for 3min to remove impurities;

(6) transferring all the supernatants to a 1.5mL centrifuge tube, and centrifuging at 12000rpm for 3 min;

(7) transfer 210. mu.L of the supernatant to a 1.5mL centrifuge tube, add 20. mu.L of proteinase K (20mg/mL) solution, mix well, add 200. mu.L of binding solution CB, vortex for 15s, mix well. Incubate at 70 ℃ for 10 min.

(8) Cooling, adding 100 μ L isopropanol, and mixing by vortex;

(9) a new silica gel membrane adsorption column was placed in the collection tube and 100. mu.L of the equilibrium solution was pipetted into the column. Centrifuging at 12000rpm for 1min, pouring out waste liquid in the collecting tube, and replacing the adsorption column into the collecting tube again. Finishing the pretreatment of the equilibrium solution;

(10) adding the solution obtained in the previous step and the precipitate which may appear into an adsorption column AC pretreated by equilibrium liquid, centrifuging at 12,000rpm for 30s, and pouring out the waste liquid in the collecting pipe;

(11) adding 500 μ L inhibitor removing solution IR, centrifuging at 12000rpm for 30s, and discarding the waste solution;

(12) adding 600 mu L of rinsing liquid WB, centrifuging at 12000rpm for 30s, discarding the waste liquid, and repeatedly rinsing once;

(13) putting the adsorption column AC back into an empty collection pipe, centrifuging at 12000rpm for 2min, and removing the rinsing liquid as much as possible to prevent residual ethanol in the rinsing liquid from inhibiting downstream reaction;

(14) the adsorption column AC was taken out, placed in a clean centrifuge tube, 50. mu.L of elution buffer EB (pre-warmed in a water bath at 65-70 ℃ C.) was added to the middle of the adsorption membrane, left at room temperature for 2min, and centrifuged at 12000rpm for 1 min. Adding the obtained solution into a centrifugal adsorption column again, standing at room temperature for 2min, centrifuging at 12000rpm for 1min, and collecting the solution to obtain a genome DNA solution;

(15) taking 1 μ L genome DNA solution obtained by the above method, measuring concentration with DNA concentration meter, making concentration mark on the tube wall, and storing at-20 deg.C for a long time.

Example 3

This example provides a method for non-invasive rapid determination of whether a plant is infected with tomato yellow leaf curl virus, comprising the steps of:

(1) the whitefly honeydew was smeared with cotton swabs wetted with buffer ASL and its DNA was extracted.

(2) The above sample DNA was used as a template, and the primers and Probes described in example 1, 2 XDdPCR Super Mix For Probes, and RNase free water were added to prepare a reaction system.

(3) Adding the system prepared in the step (2) and the droplet-generating oil into a droplet-generating card, and placing the card in a droplet generator to generate droplets.

(4) And (4) transferring the microdroplets generated in the step (3) to a ddPCR 96 pore plate for amplification reaction, and analyzing microdroplet fluorescence data after the reaction is finished.

Wherein the reaction system of the step (2) comprises the following components: 2 XDddPCR Super Mix for Probes 10. mu.L, 10. mu. mol/. mu.L of upstream and downstream primers (TYLCV-ddPCR-F and TYLCV-ddPCR-R) 1.6. mu.L each, 10. mu. mol/. mu.L of Probe (TYLCV-ddPCR-Probe) 0.4. mu.L, sample DNA 1. mu.L, RNase Free Water 5.4. mu.L.

The specific operation of the step (3) is as follows: adding the 20. mu.L reaction system and 70. mu.L droplet-forming oil into the corresponding wells of the droplet-generating card, automatically generating droplets on a droplet generator, carefully transferring the generated 40. mu.L water-in-oil droplets to a 96-well reaction plate, sealing the 96-well reaction plate on a membrane sealing instrument, and finally performing amplification reaction on a gradient PCR instrument.

The procedure of the amplification reaction of step (4) is: pre-denaturation at 95 ℃ for 10 min; denaturation at 94 ℃ for 30s, annealing and extension at 60 ℃ for 1min, and 40 cycles; keeping the temperature at 98 ℃ for 10 min. After the amplification is finished, fluorescence analysis is carried out in a micro-drop digital PCR fluorescence analysis system, and the following settings are carried out in software connected with an instrument: and selecting an absolute quantitative method, setting the specific gene channel as a FAM fluorescence channel, and analyzing fluorescence data.

Example 4

Sensitive detection

The DNA samples prepared in example 2 were diluted to 5 concentration gradients of 200 pg/. mu.L, 40 pg/. mu.L, 8 pg/. mu.L, 1.6 pg/. mu.L, 0.32 pg/. mu.L, and the detection was performed by the method of example 3, and a negative control using Bemisia tabaci honeydew DNA not infected with virus as a template was added.

Since the sample DNA occupies one twentieth of the volume of the reaction system, the template amount concentration in the reaction system is one twentieth of the sample DNA. The copy number of each reaction is counted, and the sensitivity of the method system is tested. The sensitivity measurements are shown in Table 1, and the data fit standard curve in the linear range is shown in FIG. 2

TABLE 1

The data fitting standard curve of fig. 2 is plotted from the actual copy number detection results in table 1. As can be seen from FIG. 2, the copy number of the TYLCV gene was in the interval of 0.39-65.1 copies/. mu.LAll exhibit good linearity, R²Is 0.9954. The result shows that the method has good quantitative detection capability when the template amount concentration is 0.32-200 pg/mu L. When the template amount is 0.32 pg/mu L, the TYLCV can be detected, and the result shows that the method can successfully detect the target gene with extremely low content in the sample and has extremely high sensitivity.

Example 5

Quantitative detection

And (3) quantitatively detecting the concentration of the DNA which can be stably amplified in the linear range verification, detecting the proper template quantity of the linear range verification result, carrying out 8 parallel micro-drop digital PCR amplifications on the DNA of the bemisia tabaci honeydew sample with the template quantity of about 1.6 pg/mu L, and calculating the copy number of each parallel specific gene, wherein the test result is shown in a table 2.

TABLE 2

As can be seen from table 2, the average copy number of 8 parallel amplifications of the specific gene TYLCV was 1.175/. mu.l, and the detected copy number of the TYLCV gene was 23.5 (the copy number of TYLCV was 20 × 1.175 ═ 23.5 due to the reaction system of 20. mu.l). FIG. 2 shows the PCR accuracy detection results of TYLCV. According to the method, the TYLCV can be accurately detected through the bemisia tabaci honeydew.

The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.

Claims (10)

1. A specific primer of a micro-drop digital PCR for detecting tomato yellow leaf curl virus is characterized in that the nucleotide sequence of the specific primer is as follows:

5’-CCGAAAGCCCAGAATATACAGA-3’(SEQ ID NO.1)，

5’-CCAGATCCACGAGTAACATCAC-3’(SEQ ID NO.2)。

2. a specific probe for detecting the micro-drop digital PCR of tomato yellow leaf curl virus is characterized in that the nucleotide sequence of the specific probe is as follows:

5’-CCCATGTAAAGTCCAGTCTTATGAGCAACG-3’(SEQ ID NO.3)。

3. the specific probe of the droplet-type digital PCR for detecting the tomato yellow leaf curl virus of claim 2, wherein the 5 'end of the specific probe is labeled with a fluorescent group, and the 3' end of the probe is labeled with a quenching group;

the fluorescent group is one of FAM, TET, HEX, CY3 or JOE;

the quenching group is one of BHQ1, BHQ2, TAMRA, MGB or DABCYL.

4. A kit for detecting tomato yellow leaf curl virus, comprising the primer of claim 1 and/or the probe of any one of claims 2 to 3.

5. A kit for quantitative detection of tomato yellow leaf curl virus, comprising the detection means of claim 4.

6. A method for non-invasive rapid determination of whether a plant is infected with tomato yellow leaf curl virus,

performing detection confirmation by using the specific primer of claim 1 and the specific probe of claim 2 or 3;

or

Carrying out detection confirmation by using the tool for detecting tomato yellow leaf curl virus as claimed in claim 4;

or

Detection confirmation using the kit of claim 5.

7. The method of non-invasive rapid determination of whether a plant is infected with tomato yellow leaf curl virus according to claim 6, wherein the test substance is DNA extracted from honeydew secreted by vector insects.

8. The method for non-invasive rapid determination of whether a plant is infected with tomato yellow leaf curl virus according to claim 7, wherein the vector insect is bemisia tabaci.

9. The method for non-invasive rapid determination of whether a plant is infected with tomato yellow leaf curl virus according to claim 8, comprising the steps of:

(1) collecting whitefly honeydew on plant leaves by using a cotton swab stained with buffer ASL and extracting DNA from the honeydew;

(2) taking the DNA extracted in the step (1) as a template, adding primers with sequences of SEQ ID NO.1 and SEQ ID NO.2 and a probe with sequence of SEQ ID NO.3, and 2 XddPCR Super Mix For Probes and RNase free water to prepare a reaction system;

(3) adding the system prepared in the step (2) and the droplet generating oil into a droplet generating card, and placing the droplet generating card into a droplet generator to generate droplets;

(4) transferring the microdroplets generated in the step (3) to a ddPCR 96 pore plate for amplification reaction, and analyzing microdroplet fluorescence data after the reaction is finished;

(5) analyzing the data result, calculating the copy number contained in the sample by copies/mu L to obtain whether the collected sample is infected by TYLCV, and judging that the sample is infected by TYLCV when the number of the positive drops is more than 5.

10. The method of claim 9, wherein said plant is selected from the group consisting of tomato, pepper, and eggplant.

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