TWI488971B - Primer kit and method for detecting if cucurbitaceae plant is infected by zucchini yellow mosaic virus - Google Patents

Description

A primer group and method for detecting whether a cucurbit crop is infected by a dwarf pumpkin yellow mosaic virus law

This case relates to a primer group and method for detecting whether a cucurbit crop is infected by a virus, and more particularly to a primer group and method for detecting whether a cucurbit crop is infected by a dwarf pumpkin yellow mosaic virus.

Melons are the world's major cash crops and are susceptible to a variety of pests and viruses that cause significant economic losses. Taiwan's melons are cultivated in a large area and a wide variety, especially in the Cucurbitaceae crops such as watermelon, pumpkin and melon. Such cucurbit crops are vulnerable to the Zucchini yellow mosaic virus (ZYMV). Due to the lack of disease-resistant materials, the traditional methods are no good (Chang et al. 1987; Huang et al. 1993) . ZYMV is one of the most common viruses in most melon crops in the world, and it is also one of the most frequently occurring viruses in China.

Dwarf squash yellow mosaic virus (ZYMV) is a member of the genus Potyvirus of the Potyviridae family. ZYMV was first discovered in Italy in 1973 and soon spread to Europe, the United States, the Middle East, Japan and Taiwan. The virus particle size is about 750x12 nm, contains a positive single-stranded RNA, about 9.5 kb, and the molecular weight of the sheath protein is about 36 kDa. The virus has a wide range of hosts, including Amaranthaceae, Chenopodiaceae, Compositae, Cucurbitaceae, Labiatae, Leguminosae, Ranunculaceae. ,mysterious Scrophulariaceae, Solanaceae, and Umbelliferae, especially in Cucurbita melons such as Cucurbita pepo, Cucumis melo, C. sativus, and Watermelon (Citrullus lanatus) It often causes signs of dysplasia, yellowing of the heart, fruit malformation or cracking, which seriously affects fruit quality and yield.

Therefore, the present invention proposes a method for detecting whether a Cucurbitaceae crop is infected by the ZYMV virus to obtain a test result quickly and accurately.

The purpose of this case is to propose a method for detecting whether the Cucurbitaceae crop is infected by the dwarf pumpkin yellow mosaic virus (ZYMV), to obtain the test results quickly and accurately, and to use the early removal of the diseased plants to prevent the harm caused by the rapid infection of the virus. .

Another purpose of the present invention is to propose a primer set for detecting whether the cucurbit crop is infected by the dwarf squash yellow mosaic virus (ZYMV), and to specifically identify the RNA sequence of the yellow squash mosaic virus through the primer set and the dwarf squash yellow mosaic virus. Reverse transcription loop-mediated Isothermal Amplification (RT-LAMP) is used to establish a rapid diagnostic method for whether or not Z. sinensis crops are infected with ZYMV.

In order to achieve the above object, a preferred embodiment of the present invention provides a primer set for detecting whether a Cucurbitaceae crop is infected by a dwarf squash yellow mosaic virus, comprising four primers, the primer sequences of which are respectively SEQ ID Nos. 1 to 4. Show.

According to the concept of the present case, the primer set is subjected to an amplification reaction in conjunction with a reverse transcription constant temperature circular nucleic acid amplification method.

According to the concept of the case, the cucurbit crop contains melon, cantaloupe, courgette, watermelon, melon, pumpkin, flat palm and chayote.

In order to achieve the above object, a preferred embodiment of the present invention provides a method for detecting whether a Cucurbitaceae crop is infected by a dwarf pumpkin yellow mosaic virus, comprising the steps of: extracting RNA of a tissue to be tested; adding a primer set and reverse transcription The constant temperature circular nucleic acid amplification method reactant is subjected to a reverse transcription constant temperature circular nucleic acid amplification reaction, wherein the primer set comprises four primers, the sequences of which are respectively shown as SEQ ID NOs: 1 to 4; and the detection of whether or not a target product is produced.

According to the concept of the case, the cucurbit crop contains melon, cantaloupe, courgette, watermelon, melon, pumpkin, flat palm and chayote.

According to the concept of the present case, the tissue to be tested is a leaf.

According to the concept of the present invention, the reverse transcription thermoregular nucleic acid amplification reagent comprises deoxynucleotides, reverse transcriptase and DNA polymerase.

According to the concept of the present case, the step of detecting whether or not the target product is produced is by fluorescence detection.

S10‧‧‧Step S10

S20‧‧‧Step S20

S30‧‧‧Step S30

Figure 1 shows the sequence of the primer set for detecting whether the cucurbit crop is infected with the dwarf pumpkin yellow mosaic virus.

Figure 2 shows a flow chart of the method for detecting whether the cucurbit crop is infected by the dwarf pumpkin yellow mosaic virus.

Figure 3 shows the detection effect of the method for detecting whether the cucurbit crop is infected by the dwarf pumpkin yellow mosaic virus.

Some exemplary embodiments embodying the features and advantages of the present invention are described in detail in the following description. It should be understood that the case can have different aspects. Various changes are not excluded from the scope of the present invention, and the descriptions and drawings are used for illustrative purposes in nature and are not intended to limit the present invention.

The present invention provides a method for detecting whether a Cucurbitaceae crop is infected by Zucchini yellow mosaic virus (ZYMV), wherein the Cucurbitaceae crop system comprises melon, cantaloupe, courgette, watermelon, melon, pumpkin, Flat pupa and chayote.

In order to quickly detect whether the Cucurbitaceae crop was infected by the Zucchini yellow mosaic virus (ZYMV), four primers specific to ZYMV were developed in this case, and the reverse transcription constant temperature nucleic acid amplification method was used. Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) is used to increase the representative nucleic acid fragment as a molecular marker of ZYMV to establish a rapid diagnostic method for whether the Cucurbitaceae crop is infected with ZYMV.

The primer set used in this case is designed to compare the RNA sequence of ZYMV with the RT-LAMP method, and contains the following four primers (as shown in Figure 1):

(1) ZYMV-EU-b-F3 : 5'-TTAAGGGCAAGTGTTCCTT-3' (SEQ ID NO: 1), which corresponds to the sequence of the 7908-7100 nucleotide position of the RNA of ZYMV.

(2) ZYMV-EU-b-B3 : 5'-CGTATGACCCCATCCACT-3' (SEQ ID NO: 2), which corresponds to the sequence of 7288-7269 nucleotide positions of the RNA of ZYMV.

(3) ZYMV-EU-b-FIP : 5'-TGCTCTCCCATTAAGGGTCTGACTTTGAAGAATATTTGCAAACACAC-3' (SEQ ID NO: 3), wherein the anterior sequence 5'-TGCTCTCCCATTAAGGGTCTGA-3' corresponds to the sequence of 7166-7145 nucleotide positions of ZYMV RNA. The latter sequence 5'-CTTTGAAGAATATTTGCAAACACAC-3' corresponds to the sequence of the 7101-7125 nucleotide position of the RNA of ZYMV.

(4) ZYMV-EU-b-BIP : 5'-CAGCCGAGCAAGTTGAACAAAATCCAATTGATTAACAGTGACG-3' (SEQ ID NO: 4), wherein the anterior sequence 5'-CAGCCGAGCAAGTTGAACAAA-3' corresponds to the sequence of 7168-7188 nucleotide positions of ZYMV RNA. The latter sequence 5'-ATCCAATTGATTAACAGTGACG-3' corresponds to the sequence of 7248-7227 nucleotide positions of the RNA of ZYMV.

Please refer to Fig. 2, which is a flow chart showing the method for detecting whether a Cucurbitaceae crop is infected with ZYMV. First, the RNA of the tissue to be tested is extracted (step S10), wherein the tissue to be tested may be a leaf, but not limited thereto. Next, the primer set and the RT-LAMP reactant were added for the RT-LAMP reaction (step S20), wherein the primer set contained 4 primers, the sequences of which are shown in SEQ ID Nos. 1 to 4, respectively. After the reaction is completed, it is detected whether or not a target product is produced (step S30).

The following is an example of the detailed steps of the method for detecting whether a Cucurbitaceae crop is infected by ZYMV in this case, but the description herein is for illustrative purposes only and is not intended to limit the invention.

First, in step S10, 0.1 g of fresh leaves were ground in liquid nitrogen, and total RNA was extracted with reference to the Trizol Reagent manual, and the extracted whole RNA extract was placed on ice.

Next, in step S20, the aforementioned RNA extract is added to the above four primers and the RT-LAMP reactant, including 0.8 μl of 25 μM FIP primer, 0.8 μl of 25 μM BIP primer, 0.2 μl of 25 μM F3 primer, and 0.2 μl of 25 μM. B3 primer, 2.5 μl of buffer, 2 μl of 10 mM deoxynucleotide (dNTP), 4 μl of 50 mM MgSO ₄ , 4 μl of 5 M betaine (Betain), 0.8 μl of reverse transcriptase, 1 μl of DNA polymerase, and Water was added to 25 μl, and the above reaction was subjected to RT-LAMP reaction at 64 ° C for 60 minutes.

After that, after the reaction is completed, it is detected whether there is a target product (step) Step S30). For example, the detection method may be a fluorescence detection method. Since the target product has a double-stranded DNA structure, SYBR Green can be added, which can be chimeric with double-stranded DNA to release fluorescence, so that it can be used to detect whether there is a target product. Produce, you can test whether the plant is infected with the virus. Of course, other fluorescent substances which can be chimeric with the double-stranded DNA can be added for detection, or other methods for detecting whether or not the target product is produced can be used without being limited to those described in the present embodiment.

In order to further confirm whether the primer set and the detection method designed by the present invention can specifically test whether the plant is infected with the ZYMV virus, the present invention uses a tissue sample known to infect various viruses to be tested. Please refer to Figure 3, which shows the detection effect of the detection method in this case. In Fig. 3, the first tube NTC has no RNA, which is a negative control group, the second tube is an uninfected healthy pumpkin tissue sample, and the third tube is an uninfected healthy melon tissue sample, the fourth tube to the fourth tube. The 6 tubes are samples of common melon-like viruses infected with PRSV, CGMMV and CMV, and the 7th tube and the 8th tube are samples infected with ZYMV virus. It can be seen from the test results that only the 7th tube and the 8th tube have fluorescence generation, which is a positive reaction, while other tubes have no fluorescence, which is a negative reaction, indicating that the detection method of this case is highly specific to the ZYMV virus, so it can be used Test whether the plant is infected with the ZYMV virus.

In summary, the case developed four highly specific primers for ZYMV, and provided a method for detecting whether the cucurbit crop was infected with ZYMV, that is, using the reverse transcription constant temperature nucleic acid amplification method (RT-LAMP) to increase the output. The representative nucleic acid fragment is used as a molecular marker of ZYMV to establish a diagnostic method for whether the Cucurbitaceae crop is infected with ZYMV, thereby obtaining the detection result quickly and accurately, and utilizing the early removal of the diseased plant to prevent the harm caused by rapid infection of the virus. Therefore, the primer group and method for detecting whether the cucurbit crop is infected by the dwarf squash yellow mosaic virus in this case is of great industrial value, and the application is made according to law.

This case has to be made by people who are familiar with this technology. Modifications are not excluded as intended by the scope of the patent application.

<110> Agricultural Research Institute of the Agricultural Committee of the Executive Yuan

<120> Introduction and method for detecting whether a cucurbit crop is infected by a dwarf pumpkin yellow mosaic virus

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<212> DNA

<213> Artificial sequence

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<211> 18

<212> DNA

<213> Artificial sequence

<400> 2

<210> 3

<211> 47

<212> DNA

<213> Artificial sequence

<400> 3

<210> 4

<211> 43

<212> DNA

<213> Artificial sequence

<400> 4

Claims (8)

A primer set for detecting whether a Cucurbitaceae crop is infected by Zucchini yellow mosaic virus, comprising four primers whose primer sequences are shown in SEQ ID NOs: 1 to 4, respectively. The primer group for detecting whether the Cucurbitaceae crop is infected with Zucchini yellow mosaic virus according to the first item of the patent application scope, wherein the primer group is subjected to an amplification reaction by using a reverse transcription constant temperature circular nucleic acid amplification method. . For example, if the cucurbit crop is tested by the Zucchini yellow mosaic virus as described in the first paragraph of the patent application, the cucurbit crop is melon, cantaloupe, courgette, watermelon, and alfalfa. Melon, pumpkin, flat rose or chayote. A method for detecting whether a Cucurbitaceae crop is infected by a Zucchini yellow mosaic virus comprises the steps of: extracting RNA of a tissue to be tested; adding a primer set and a reverse transcription constant temperature nucleic acid amplification method for performing the reaction A reverse transcription thermostated circular nucleic acid amplification reaction, wherein the primer set comprises four primers, the sequences of which are shown in SEQ ID NO: 1 to 4, respectively; and the detection of whether or not a target product is produced. Is the cucurbit crop detected by the Zucchini yellow mosaic virus as described in the fourth paragraph of the patent application? The method, wherein the cucurbit crop is melon, cantaloupe, courgette, watermelon, melon, pumpkin, flat stalk or chayote. A method for detecting whether a Cucurbitaceae crop is infected with a Zucchini yellow mosaic virus as described in claim 4, wherein the tissue to be tested is a leaf. A method for detecting whether a Cucurbitaceae crop is infected with a Zucchini yellow mosaic virus according to the fourth aspect of the patent application, wherein the reverse transcription constant temperature nucleic acid amplification method comprises a deoxynucleotide , reverse transcriptase and DNA polymerase. A method for detecting whether a Cucurbitaceae crop is infected with Zucchini yellow mosaic virus as described in claim 5, wherein the step of detecting whether or not the target product is produced is by fluorescence detection.