阅读说明：本技术 一种用于扩增甜瓜内源病毒的引物对及其应用 (Primer pair for amplifying endogenous viruses of melons and application of primer pair ) 是由 赵立群 邱艳红 徐秀兰 田文 张海军 张晓飞 刘慧� 温常龙 于 2021-08-26 设计创作，主要内容包括：本发明公开了一种用于扩增甜瓜内源病毒基因组片段的引物对,包括第一引物和第二引物；第一引物的核酸序列如SEQ ID NO.1所示；第二引物的核酸序列如SEQ ID NO.2所示。本发明还公开了包括前述引物对的试剂与试剂盒及其应用。本发明还公开了使用前述引物对检测待测植物材料是否感染甜瓜内源病毒的方法以及检测待测病毒是否为甜瓜内源病毒的方法。前述引物对检测甜瓜内源病毒的敏感性高,特异性好。(The invention discloses a primer pair for amplifying a melon endogenous virus genome segment, which comprises a first primer and a second primer; the nucleic acid sequence of the first primer is shown as SEQ ID NO. 1; the nucleic acid sequence of the second primer is shown as SEQ ID NO. 2. The invention also discloses a reagent and a kit comprising the primer pair and application thereof. The invention also discloses a method for detecting whether the plant material to be detected is infected with the melon endogenous virus by using the primer pair and a method for detecting whether the virus to be detected is the melon endogenous virus. The primer pair has high sensitivity and good specificity in detecting the melon endogenous virus.)

1. A primer pair for amplifying a fragment of a melon endogenous viral genome, the primer pair comprising a first primer and a second primer;

the nucleic acid sequence of the first primer is shown as SEQ ID NO. 1; the nucleic acid sequence of the second primer is shown as SEQ ID NO. 2.

2. A reagent for amplifying a fragment of an endogenous viral genome of cucumis melo, the reagent comprising the primer pair of claim 1.

3. The reagent of claim 2, wherein the molar ratio of the first primer to the second primer is 1: 1;

preferably, the reagent further comprises a reverse transcriptase and/or a DNA polymerase;

preferably, the reagent further comprises a positive control sample.

4. A kit for amplifying a fragment of an endogenous viral genome of Cucumis melo, comprising the primer pair of claim 1 or the reagent of claim 2 or 3.

5. Use of the primer pair of claim 1, the reagent of claim 2 or 3, or the kit of claim 4 in a1 or a 2:

a1, detecting the endogenous viruses of the melon;

a2, preparing a preparation for detecting the endogenous viruses of the melon.

6. The use of claim 5, wherein a1 refers to:

detecting whether the plant material to be detected is infected with the melon endogenous virus; or

Detecting whether the virus to be detected is the endogenous virus of the melon;

preferably, a2 refers to:

preparing a preparation for detecting whether the plant material to be detected is infected with the melon endogenous virus; or

And preparing a preparation for detecting whether the virus to be detected is the endogenous virus of the melon.

7. A method of detecting whether a plant material to be tested is infected with an endogenous virus of cucumis melo, the method comprising:

b1, performing PCR amplification on the nucleic acid of the plant material to be detected by using the primer pair of claim 1;

b2, determining whether the plant material to be tested is infected by the melon endogenous virus according to the amplified product.

8. The method of claim 7, wherein the plant material to be tested is a melon leaf or melon seed;

preferably, the nucleic acid of the plant material to be detected is total RNA of the plant material to be detected;

preferably, the PCR amplification is RT-PCR amplification;

preferably, the step of determining is: characterizing the DNA fragment in the PCR amplification product, and judging that the plant material to be detected is not infected with the melon endogenous virus when the 755bp DNA fragment does not exist in the PCR amplification product; when the PCR amplification product contains a 755bp DNA fragment, judging that the plant material to be detected is infected with the melon endogenous virus;

preferably, said characterization is effected by agarose gel electrophoresis of said amplification products;

preferably, the temperature program adopted by the PCR amplification is as follows:

(1) reacting at 50 ℃ for 30 min;

(2) reacting at 94 ℃ for 2 min;

(3) carrying out cyclic reaction;

(4) reacting at 72 ℃ for 10 min;

the circulating reaction steps are as follows: reacting at 94 ℃ for 30 s; reacting for 30s at 55-56 ℃; the reaction is carried out for 35s at 72 ℃ and circulated for 30 times.

9. A method of detecting whether a test virus is an endogenous virus of cantaloupe, the method comprising:

c1, performing PCR amplification on the nucleic acid of the virus sample to be detected by using the primer pair of claim 1;

c2, judging whether the virus to be detected is the melon endogenous virus according to the amplified product.

10. The method of claim 9, wherein the nucleic acid of the test virus sample is total RNA of the test virus sample;

preferably, the PCR amplification is RT-PCR amplification;

preferably, the step of determining is: characterizing the DNA fragment in the amplification product, and judging that the virus sample to be tested is not the melon endogenous virus when the amplification product does not have the 755bp DNA fragment; when the amplification product contains a 755bp DNA fragment, judging that the virus sample to be detected is the melon endogenous virus;

preferably, said characterization is effected by agarose gel electrophoresis of said amplification products;

preferably, the temperature program adopted by the PCR amplification is as follows:

(1) reacting at 50 ℃ for 30 min;

(2) reacting at 94 ℃ for 2 min;

(3) carrying out cyclic reaction;

(4) reacting at 72 ℃ for 10 min;

the circulating reaction steps are as follows: reacting at 94 ℃ for 30 s; reacting for 30s at 55-56 ℃; the reaction is carried out for 35s at 72 ℃ and circulated for 30 times.

Technical Field

The invention belongs to the technical field of plant viruses, relates to a biological detection technology, and particularly relates to a primer pair, an RT-PCR reagent, an RT-PCR kit and an application and a method thereof for detecting melon endogenous viruses based on a one-step RT-PCR technology.

Background

Melon endogenous virus (CmEV) is a newly-occurring viral disease on the melon.

In recent years, melon endogenous viruses have been reported in the uk, usa, ecuador, korea and brazil. In 2020, the melon endogenous virus is reported for the first time in Shanghai of China, and the similarity of the virus sequence and the sequence of a melon endogenous virus SJ1 strain discovered in Korea is as high as 98.31%. Moreover, the melon endogenous virus found in China is proved to be capable of being transmitted through seeds. There are studies that indicate that endogenous RNA viruses can occur in plants for hundreds of generations. In addition, the melon endogenous virus can be detected in a plurality of cucurbitaceae crops such as melon, towel gourd and the like, and the virus hosts are very wide. Therefore, it is necessary to establish a sensitive, fast and accurate detection technique for the virus to detect whether important melon seeds and seedlings are infected or not, so as to prevent and control the virus from spreading and diffusing along with the seeds and seedlings in a long distance.

Disclosure of Invention

The invention aims to solve the technical problem of how to detect the melon endogenous viruses or how to detect whether a plant sample to be detected is infected with the melon endogenous viruses. In order to solve the technical problems, the invention designs the specific primer according to the virus coding region, and can quickly, accurately and sensitively detect the virus.

The invention provides a primer pair for amplifying the endogenous virus genome fragment of the melon, wherein the primer pair comprises a first primer and a second primer.

In a second aspect, the invention provides a reagent for amplifying a fragment of an endogenous viral genome of melon, the reagent comprising a primer pair according to the first aspect of the invention.

In some embodiments, the molar ratio of the first primer to the second primer is 1: 1.

In some embodiments, the reagent further comprises a reverse transcriptase and/or a DNA polymerase.

In some embodiments, the reagent further comprises a positive control sample, wherein the positive control sample can be a plant leaf infected with the melon endogenous virus, can also be the melon endogenous virus itself, and can also be a nucleotide sequence shown as SEQ ID NO.3 artificially synthesized.

In a third aspect, the invention provides a kit for amplifying endogenous viral genome fragments of melon, wherein the kit comprises the primer pair of the first aspect or the reagent of the second aspect.

In a fourth aspect, the invention provides the use of a primer pair according to the first aspect of the invention, a reagent according to the second aspect of the invention or a kit according to the third aspect of the invention in a1 or a2 as follows:

a1, detecting the endogenous viruses of the melon;

a2, preparing a preparation for detecting the endogenous viruses of the melon;

in some embodiments, a1 refers to:

detecting whether the plant material to be detected is infected with the melon endogenous virus; or

Detecting whether the virus to be detected is the endogenous virus of the melon;

in some embodiments, a2 refers to:

preparing a preparation for detecting whether the plant material to be detected is infected with the melon endogenous virus; or

And preparing a preparation for detecting whether the virus to be detected is the endogenous virus of the melon.

In a fifth aspect, the invention provides a method for detecting whether a plant material to be detected is infected with an endogenous virus of melon, the method comprising:

b1, carrying out PCR amplification on the nucleic acid of the plant material to be detected by adopting the primer pair of the first aspect of the invention;

b2, determining whether the plant material to be tested is infected by the melon endogenous virus according to the amplified product.

In some embodiments, the plant material to be tested is a cucumis melo leaf or a cucumis melo seed.

In some embodiments, the nucleic acid of the test plant material is total RNA of the test plant material.

In some embodiments, the PCR amplification is RT-PCR amplification.

In some embodiments, the determining step is: characterizing the DNA fragment in the PCR amplification product, and judging that the plant material to be detected is not infected with the melon endogenous virus when the 755bp DNA fragment does not exist in the PCR amplification product; and when the PCR amplification product contains a 755bp DNA fragment, judging that the plant material to be tested is infected with the melon endogenous virus.

In some embodiments, the characterization is by agarose gel electrophoresis of the amplification products.

In some embodiments, the temperature program used for PCR amplification is:

(1) reacting at 50 ℃ for 30 min;

(2) reacting at 94 ℃ for 2 min;

(3) carrying out cyclic reaction;

(4) reacting at 72 ℃ for 10 min;

the circulating reaction steps are as follows: reacting at 94 ℃ for 30 s; reacting for 30s at 56-58 ℃; the reaction is carried out for 35s at 72 ℃ and circulated for 30 times.

The sixth aspect of the invention provides a method for detecting whether a virus to be detected is an endogenous virus of melon, which comprises the following steps:

c1, carrying out PCR amplification on the nucleic acid of the virus sample to be detected by adopting the primer pair of the first aspect of the invention;

c2, judging whether the virus to be detected is the melon endogenous virus according to the amplified product.

In some embodiments, the nucleic acid of the test viral sample is total RNA of the test viral sample.

In some embodiments, the PCR amplification is RT-PCR amplification.

In some embodiments, the determining step is: characterizing the DNA fragment in the amplification product, and judging that the virus sample to be tested is not the melon endogenous virus when the amplification product does not have the 755bp DNA fragment; and when the amplification product contains a 755bp DNA fragment, judging that the virus sample to be detected is the melon endogenous virus.

In some embodiments, the characterization is by agarose gel electrophoresis of the amplification products.

In some embodiments, the temperature program used for PCR amplification is:

(1) reacting at 50 ℃ for 30 min;

(2) reacting at 94 ℃ for 2 min;

(3) carrying out cyclic reaction;

(4) reacting at 72 ℃ for 10 min;

the circulating reaction steps are as follows: reacting at 94 ℃ for 30 s; reacting for 30s at 55-56 ℃; the reaction is carried out for 35s at 72 ℃ and circulated for 30 times.

The primer pair for detecting the melon endogenous virus provided by the invention consists of a primer CmEV-755-F (namely a first primer) and a primer CmEV-755-R (namely a second primer). The primer pair can specifically amplify CmEV, the length of an amplified fragment is 755bp, and Cucumber Green Mottle Mosaic Virus (CGMMV), Cucumber Mosaic Virus (CMV), watermelon latent virus (CiLCV), pepper endogenous virus (BPEV), pumpkin mosaic virus (SqMV), Watermelon Mosaic Virus (WMV) and small cucurbit yellow mosaic virus (ZYMV) cannot be amplified. Any one of the plant materials to be detected can be a melon material, and more particularly can be a leaf and a seed. Therefore, whether the melon seeds, seedlings or plants carry the virus disease can be identified, and the method has wide application prospect. The primer pair provided by the invention has the advantages of good specificity, high accuracy and high sensitivity, and the detection method provided by the invention has the advantages of simple and convenient operation, high detection efficiency and the like, and can meet the basic requirements of seed, seedling producers and users, nursery sites and muskmelon field detection.

Drawings

FIG. 1 shows the results of the specificity test of the experimental group of example 3, lane 1 corresponding to the detection of amplification products from a positive control melon sample containing melon endogenous virus; lane 2 corresponds to detection of amplification products from a watermelon sample containing cucumber green mottle mosaic virus; lane 3 corresponds to detection of amplification products from watermelon samples containing cucumber mosaic virus; lane 4 corresponds to detection of amplification products from a watermelon sample containing watermelon latent virus; lane 5 corresponds to detection of amplification products from a pepper sample containing endogenous viruses from pepper; lane 6 corresponds to detection of amplification products from a watermelon sample containing cucumovirus; lane 7 corresponds to detection of amplification products from a watermelon sample containing watermelon mosaic virus; lane 8 corresponds to detection of amplification products from a pumpkin sample containing zucchini yellow mosaic virus; lane 9 corresponds to detection of amplification products from healthy melon samples; lane M is DNA DL2000Marker, and the bands from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp in sequence.

FIG. 2 shows the results of the specificity test of control 1 of example 3, lane 1 corresponding to the detection of amplification products from a positive control melon sample containing melon endogenous virus; lane 2 corresponds to detection of amplification products from a watermelon sample containing cucumber green mottle mosaic virus; lane 3 corresponds to detection of amplification products from watermelon samples containing cucumber mosaic virus; lane 4 corresponds to detection of amplification products from a watermelon sample containing watermelon latent virus; lane 5 corresponds to detection of amplification products from a pepper sample containing endogenous viruses from pepper; lane 6 corresponds to detection of amplification products from a watermelon sample containing cucumovirus; lane 7 corresponds to detection of amplification products from a watermelon sample containing watermelon mosaic virus; lane 8 corresponds to detection of amplification products from a pumpkin sample containing zucchini yellow mosaic virus; lane 9 corresponds to detection of amplification products from healthy melon samples; lane M is DNA DL2000Marker, and the bands from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp in sequence.

FIG. 3 shows the results of the specificity test of control 2 of example 3, lane 1 corresponding to the detection of amplification products from a positive control melon sample containing melon endogenous virus; lane 2 corresponds to detection of amplification products from a watermelon sample containing cucumber green mottle mosaic virus; lane 3 corresponds to detection of amplification products from watermelon samples containing cucumber mosaic virus; lane 4 corresponds to detection of amplification products from a watermelon sample containing watermelon latent virus; lane 5 corresponds to detection of amplification products from a pepper sample containing endogenous viruses from pepper; lane 6 corresponds to detection of amplification products from a watermelon sample containing cucumovirus; lane 7 corresponds to detection of amplification products from a watermelon sample containing watermelon mosaic virus; lane 8 corresponds to detection of amplification products from a pumpkin sample containing zucchini yellow mosaic virus; lane 9 corresponds to detection of amplification products from healthy melon samples; lane M is DNA DL2000Marker, and the bands from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp in sequence.

FIG. 4 shows the results of the specificity test of control 3 of example 3, lane 1 corresponding to the detection of amplification products from a positive control melon sample containing melon endogenous virus; lane 2 corresponds to detection of amplification products from a watermelon sample containing cucumber green mottle mosaic virus; lane 3 corresponds to detection of amplification products from watermelon samples containing cucumber mosaic virus; lane 4 corresponds to detection of amplification products from a watermelon sample containing watermelon latent virus; lane 5 corresponds to detection of amplification products from a pepper sample containing endogenous viruses from pepper; lane 6 corresponds to detection of amplification products from a watermelon sample containing cucumovirus; lane 7 corresponds to detection of amplification products from a watermelon sample containing watermelon mosaic virus; lane 8 corresponds to detection of amplification products from a pumpkin sample containing zucchini yellow mosaic virus; lane 9 corresponds to detection of amplification products from healthy melon samples; lane M is DNA DL2000Marker, and the bands from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp in sequence.

FIG. 5 shows the results of the sensitivity test of example 4, lane 1 corresponding to detection of the amplification product of RNA solution 1; lane 2 corresponds to detection of amplification products from RNA solution 2; lane 3 corresponds to detection of amplification products from RNA solution 3; lane 4 corresponds to detection of amplification products from RNA solution 4; lane 5 corresponds to detection of the amplification product of RNA solution 5; lane 6 corresponds to detection of amplification products from RNA solution 6; lane 7 corresponds to detection of amplification products from RNA solution 7; lane 8 corresponds to detection of amplification products from RNA solution 8; lane 9 corresponds to a blank control, lane M corresponds to a DNA DL2000Marker, and the bands from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp in sequence.

FIG. 6 shows the results of the accuracy test of example 5, in which lanes 1-6 are shown for the detection of endogenous viruses in Cucumis melo in a test sample using the primer set of the present invention. Wherein lanes 1 and 2 correspond to the detection of amplification products from a positive control cantaloupe melon sample containing melon endogenous virus; lanes 3 and 4 correspond to detection of amplification products of the southern hainan melon sample to be detected containing the melon endogenous virus; lanes 5 and 6 correspond to the detection of amplification products from a melon sample to be tested that does not contain endogenous viruses of the melon; lane M corresponds to DNA DL2000Marker, and the bands from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp in sequence.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The following examples are provided as the basis for further modifications and applications by those skilled in the art and are not to be construed as limiting the invention in any way.

The experimental procedures in the following examples are conventional unless otherwise indicated, and may be carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. Materials, reagents and the like used in the following examples are conventionally commercially available unless otherwise specified.

Cucumber green mosaic virus (CGMMV), Cucumber Mosaic Virus (CMV), watermelon latent virus (CiLCV), pepper endogenous virus (BPEV), squash mosaic virus (SqMV), Watermelon Mosaic Virus (WMV), Zucchini Yellow Mosaic Virus (ZYMV) in the following examples are all common biological materials in the art.

Example 1: design and Synthesis of primers

Based on the muskmelon endogenous virus genome with the GenBank accession number of KT727022, a specific primer pair for identifying the muskmelon endogenous virus polyprotein gene is obtained by carrying out a large amount of sequence analysis, sequence design, manual screening optimization and effect verification on a virus coding region. The specific primer pair consists of CMEV-755-F and CMEV-755-R and is used for amplifying 623-1377 sites of the polyprotein gene. The primer sequences are as follows:

CmEV-755-F：5'-CGTGGAAATTGCACTTCCGAGAT-3'(SEQ ID NO.1)；

CmEV-755-R：5'-CTAGCAACTTGTTTGCTGGCACACG-3'(SEQ ID NO.2)；

wherein, CmEV-755-F is a forward primer, and CmEV-755-R is a reverse primer.

The theoretical amplification product of the specific primer pair has the size of 755bp, the nucleotide sequence of the theoretical amplification product is shown as SEQ ID NO.3 in a sequence table, and the corresponding RNA sequence is shown as SEQ ID NO.4 in the sequence table.

5'-CGTGGAAATTGCACTTCCGAGATGTTAGAGGTTTTAAACAAACTTGTGGCTAAATTTGGAGAATATGAGGCTTATTATAACGTTTATTTAGAAACTCAACATAGACATTTCGGATACCTACAGAAAGAAGACAATGGGAAGTACTCCTTCATCAATGCAAAATTCAATCCACTCAAGAAGTGCTCAGTGTGTAAGTACATGAACCATTATGAGGAGATGATAAGGGGTCTTAGGATGGTCCATTACAGTTTAAAACATTACAGACCAAAATCGAAGGTAGTGACTGATGAATTCTTAACCACATATAATGTGGGAGATGTAATAGAGGATGTCCCTTGGCGTTGTGGATACTGTTTTTCAGATTTAAATTTGCAAGTGTTTATGCGAGAGGATAGATTACAAGAATTAATCCAGATTGTCACTCAGCATTTGTTTGTGCTTATGGGAGACATAGAGCAACTTCTAAAGATGGGTTATGGAGACCTATATGATGATATGGACGTGGCTAGCAAAGAAAAATTAAAATCAGCTGATCAAGTGATCGTTAACGAGTATTTTTACCATTTGCAACGCAAAATAATACCAGTGGGGCGCGATTTTGATGCTGCACAGAGTAAGACTATTACAGAAGTATTGGGTGGTTGTTTCTTTAAGCCACAAAGCAAATTAAATAGTGTGAAACCCGAATTTATAGCTGAACTATCAACTTTAACTGAACTAATTGAAAAATCGTGTGCCAGCAAACAAGTTGCTAG-3'(SEQ ID NO.3)。

5'-CGUGGAAAUUGCACUUCCGAGAUGUUAGAGGUUUUAAACAAACUUGUGGCUAAAUUUGGAGAAUAUGAGGCUUAUUAUAACGUUUAUUUAGAAACUCAACAUAGACAUUUCGGAUACCUACAGAAAGAAGACAAUGGGAAGUACUCCUUCAUCAAUGCAAAAUUCAAUCCACUCAAGAAGUGCUCAGUGUGUAAGUACAUGAACCAUUAUGAGGAGAUGAUAAGGGGUCUUAGGAUGGUCCAUUACAGUUUAAAACAUUACAGACCAAAAUCGAAGGUAGUGACUGAUGAAUUCUUAACCACAUAUAAUGUGGGAGAUGUAAUAGAGGAUGUCCCUUGGCGUUGUGGAUACUGUUUUUCAGAUUUAAAUUUGCAAGUGUUUAUGCGAGAGGAUAGAUUACAAGAAUUAAUCCAGAUUGUCACUCAGCAUUUGUUUGUGCUUAUGGGAGACAUAGAGCAACUUCUAAAGAUGGGUUAUGGAGACCUAUAUGAUGAUAUGGACGUGGCUAGCAAAGAAAAAUUAAAAUCAGCUGAUCAAGUGAUCGUUAACGAGUAUUUUUACCAUUUGCAACGCAAAAUAAUACCAGUGGGGCGCGAUUUUGAUGCUGCACAGAGUAAGACUAUUACAGAAGUAUUGGGUGGUUGUUUCUUUAAGCCACAAAGCAAAUUAAAUAGUGUGAAACCCGAAUUUAUAGCUGAACUAUCAACUUUAACUGAACUAAUUGAAAAAUCGUGUGCCAGCAAACAAGUUGCUAG-3'(SEQ ID NO.4)。

Control primer pairs CmEV-C1-F and CmEV-C1-R are designed aiming at melon endogenous virus polyprotein gene, the length of an expected amplification product is 413bp, and the specific sequences are as follows:

CmEV-C1-F：5'-GGTGGAATATGGGTTGATGCTAG-3'(SEQ ID NO.5)

CmEV-C1-R：5'-CGTCGTGATGGACATCAACTCTAC-3'(SEQ ID NO.6)

a control primer pair CmEV-C2-F and CmEV-C2-R is designed aiming at melon endogenous virus polyprotein gene, the length of an expected amplification product is 710bp, and the specific sequence is as follows:

CmEV-C2-F：5'-GAGGTGAACACAGCACTTGTTG-3'(SEQ ID NO.7)

CmEV-C2-R：5'-CCAGAGAATAATGGACAGCCAC-3'(SEQ ID NO.8)

a control primer pair CmEV-C3-F and CmEV-C3-R is designed aiming at melon endogenous virus polyprotein gene, the length of an expected amplification product is 381bp, and the specific sequence is as follows:

CmEV-C3-F：5'-AAGSGAGAATWATHGTRTGGCA-3'(SEQ ID NO.9)

CmEV-C3-R：5'-CTAGWGCKGTBGTAGCTTGWCC-3'(SEQ ID NO.10)

wherein, S represents: g or c, W represents: a or t/u, H represents: a or c or t/u, R represents: g or a, K represents: g or t/u, B represents: g or c or t/u.

Example 2: establishment of an authentication method

Method for identifying whether virus to be detected is endogenous virus of melon

1. The total RNA of the sample to be detected is extracted by a TRIZOL method or other reagent kit which can be used for extracting the virus RNA according to the instructions.

2. The total RNA extracted in step 1 was used as a template, and the primer pair designed in example 1 was used for amplification by one-step RT-PCR.

The reaction system was as follows (25. mu.l): primer CmEV-755-F0.5 u L (10 u mol/L), primer CmEV-755-R0.5 u L (10 u mol/L), PrimeScript 1Step Enzyme Mix 1 u L, 2x 1Step Buffer (Dye Plus)12.5 u L, template 1 u L (i.e. Step 1 extraction of total RNA, concentration 100ng-200 ng/L), the balance with RNase free water make up 25 u L. Among them, PrimeScript 1Step Enzyme Mix and 2x 1Step Buffer (Dye Plus) were derived from PrimeScript from Takara^TMOne Step RT-PCR Kit Ver.2(Dye Plus), cat # RR 057A.

The reaction conditions were as follows:

reacting for 30min at 1.50 ℃;

reacting for 2min at 2.94 ℃;

reacting at 3.94 ℃ for 30 s;

reacting at 4.55 ℃ for 30 s;

reacting at 5.72 ℃ for 35 s;

reacting at 6.72 ℃ for 5min

In which 30 cycles of reaction were performed from steps 3-5.

3. And (3) taking the product obtained in the step (2), and detecting by adopting agarose gel electrophoresis. And melon endogenous virus was used as a positive control sample.

If the amplification products of the positive control sample and the detection sample both contain 755bp DNA fragments, the result is positive, and the virus sample to be detected contains the melon endogenous virus.

If the amplification product of the positive control sample contains the 755bp DNA fragment, and the amplification product of the detection sample does not contain the 755bp DNA fragment, the result is negative, and the virus sample to be detected does not contain the melon endogenous virus.

If the positive control sample has no specific amplification, the detection process is judged to be wrong, and the detection needs to be carried out again.

Method for identifying whether plant material to be detected is infected with melon endogenous virus

1. The total RNA of the plant to be detected is extracted by a TRIZOL method or other reagent boxes which can be used for extracting the plant RNA according to the instructions, and the sample can be plant tissues to be detected, such as leaves or seeds.

2. Step 2 of the method for identifying whether the virus to be detected is the melon endogenous virus.

3. Step 3 of the method for identifying whether the virus to be detected is the melon endogenous virus.

If the amplification products of the positive control sample and the detection sample both contain 755bp DNA fragments, the result is positive, and the plant material to be detected is infected with the melon endogenous virus.

If the amplification product of the positive control sample contains the 755bp DNA fragment, and the amplification product of the detection sample does not contain the 755bp DNA fragment, the result is negative, and the plant material to be detected is not infected with the melon endogenous virus.

If the positive control sample has no specific amplification, the detection process is judged to be wrong, and the detection needs to be carried out again.

Example 3: experiment of specificity

The viruses to be tested were as follows:

melon endogenous virus, cucumber green mottle mosaic virus, cucumber mosaic virus, watermelon latent virus, pepper endogenous virus, pumpkin mosaic virus, watermelon mosaic virus and cucurbita pepo yellow mosaic virus.

Experimental groups: test samples of plants infected with the above viruses, respectively, were determined and tested according to the "method for identifying whether the plant material to be tested is infected with the melon endogenous virus" of example 2.

The results are shown in FIG. 1. In FIG. 1, lane 1 corresponds to the detection of amplification products from a positive control melon sample containing melon endogenous virus; lane 2 corresponds to detection of amplification products from a watermelon sample containing cucumber green mottle mosaic virus; lane 3 corresponds to detection of amplification products from watermelon samples containing cucumber mosaic virus; lane 4 corresponds to detection of amplification products from a watermelon sample containing watermelon latent virus; lane 5 corresponds to detection of amplification products from a pepper sample containing endogenous viruses from pepper; lane 6 corresponds to detection of amplification products from a watermelon sample containing cucumovirus; lane 7 corresponds to detection of amplification products from a watermelon sample containing watermelon mosaic virus; lane 8 corresponds to detection of amplification products from a pumpkin sample containing zucchini yellow mosaic virus; lane 9 corresponds to detection of amplification products from healthy melon samples; lane M corresponds to DNA DL2000 Marker. Except that the amplification product of the positive control sample of the melon endogenous virus corresponding to the lane 1 contains a 755bp DNA fragment, other samples to be detected have no specific amplification band, which indicates that the RT-PCR detection method of the melon endogenous virus established by the invention has good specificity.

Control group 1: the difference from the experimental group was that the primers were replaced with CmEV-C1-F and CmEV-C1-R, and the results are shown in FIG. 2. Some non-specific amplification bands are shown in FIG. 2, indicating that the specificity of this pair of primers is not good.

Control group 2: the difference from the experimental group was that the primers were replaced with CmEV-C2-F and CmEV-C2-R, and the results are shown in FIG. 3. Some non-specific amplified bands of varying degrees are shown in FIG. 3, indicating that the specificity of this pair of primers is not good. The watermelon sample for cucumber mosaic virus also had visible amplification bands.

Control group 3: the difference from the experimental group was that the primers were replaced with CmEV-C3-F and CmEV-C3-R, and the results are shown in FIG. 4. Some non-specific amplified bands of varying degrees are shown in FIG. 4, indicating that the specificity of this pair of primers is not good. The amplification bands of different degrees are obtained for other samples except for healthy melon samples, and the specificity is poor for three bands visible to the naked eye of watermelon samples containing watermelon latent virus.

Example 4: sensitivity test

The viruses to be tested were as follows: melon endogenous virus (CmEV).

1. Extracting and determining total RNA of melon leaf containing melon endogenous virus by using a TRIZOL method.

2. Taking the total RNA obtained in the step 1, and performing tenfold gradient dilution by using sterile water to respectively obtain the following RNA solutions: RNA solution 1 with an RNA concentration of 200 ng/. mu.l, RNA solution 2 with an RNA concentration of 20 ng/. mu.l, and RNA solution with an RNA concentration of 2 ng/. mu.lSolution 3, RNA concentration 2X 10^-1ng/. mu.l RNA solution 4, RNA concentration 2X 10^-2ng/. mu.l RNA solution 5, RNA concentration 2X 10^-3ng/. mu.l RNA solution 6, RNA concentration 2X 10^-4ng/. mu.l RNA solution 7, RNA concentration 2X 10^-5ng/. mu.l RNA solution 8.

3. And (3) taking the RNA solution obtained in the step (2) as a template, wherein the dosage of the RNA solution in each reaction system is 1 mu l, and performing one-step RT-PCR amplification by adopting the primer pair designed in the embodiment 1.

Reaction System and reaction conditions with reference to step 2 of "one, method for identifying whether the virus to be tested is an endogenous virus of melon" of example 2, sterile water was used as a blank.

4. And (4) taking the amplification product in the step (3), and detecting by adopting agarose gel electrophoresis.

The results are shown in FIG. 5. In fig. 5, lane 1 corresponds to RNA solution 1, lane 2 corresponds to RNA solution 2, lane 3 corresponds to RNA solution 3, lane 4 corresponds to RNA solution 4, lane 5 corresponds to RNA solution 5, lane 6 corresponds to RNA solution 6, lane 7 corresponds to RNA solution 7, lane 8 corresponds to RNA solution 8, lane 9 corresponds to a blank control (sterile water), and lane M corresponds to DNA DL2000 Marker. The results showed that the detection results were positive at a viral RNA concentration of 2 ng/. mu.l or higher in the template (lane 3). The PCR detection method for the muskmelon endogenous viruses established by the invention is proved to have good sensitivity.

Example 5: detecting plant samples to be tested

1. Melon sample

Taking a plurality of muskmelon leaves with the disease symptoms of muskmelon endogenous virus diseases and muskmelon leaves without muskmelon endogenous virus as plant materials to be detected. Each plant material to be tested was taken from a different melon plant.

And (3) taking a plant material to be detected, extracting total RNA of the muskmelon leaves to be detected by using a TRIZOL method, and then detecting according to the second method for identifying whether the plant material to be detected is infected with the endogenous viruses of the muskmelon in the embodiment 2.

The results are shown in FIG. 6. In FIG. 6, lanes 1, 2, 3 and 4 show the results of detecting whether the melon endogenous virus is infected in the plant melon to be tested using the primers of the present invention; lanes 5 and 6 show the results of the control sample assay without melon endogenous virus; lane M corresponds to DNA DL2000 Marker; the amplification products of the four samples corresponding to lanes 1, 2, 3 and 4 were sequenced by Sanger method, and the nucleotide sequences were identical to the nucleotide sequence of SEQ ID No.3, and were determined to be endogenous viruses of melon after Blast analysis. The result shows that the melon endogenous virus RT-PCR detection method established by the invention can effectively identify the melon endogenous virus from melon samples.

The results show that each plant material to be detected containing the melon endogenous virus detects the melon endogenous virus, and the accuracy of the method provided by the invention is 100%.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> agriculture and forestry academy of sciences of Beijing City

<120> primer pair for amplifying melon endogenous viruses and application thereof

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ctagcaactt gtttgctggc acacg 25

<210> 3

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cagaaagaag acaatgggaa gtactccttc atcaatgcaa aattcaatcc actcaagaag 180

tgctcagtgt gtaagtacat gaaccattat gaggagatga taaggggtct taggatggtc 240

cattacagtt taaaacatta cagaccaaaa tcgaaggtag tgactgatga attcttaacc 300

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actcagcatt tgtttgtgct tatgggagac atagagcaac ttctaaagat gggttatgga 480

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<212> DNA

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