A multiplex PCR method and kit for detecting four cucurbit viruses transmitted by whitefly

Abstract

The present invention belongs to the technical field of plant virus detection, and specifically relates to a method and a kit for detecting four cucurbit viruses transmitted by whiteflies using multiple PCR. The technical problem to be solved by the present invention is to provide a new option for simultaneously detecting four cucurbit viruses transmitted by whiteflies. The technical scheme of the present invention is a kit for detecting four cucurbit viruses transmitted by whiteflies using multiple PCR, comprising primer pairs for amplifying ToLCNDV, SLCCNV, CYSDV and CCYV. The present invention also provides a method for detecting four cucurbit viruses transmitted by whiteflies using multiple PCR, comprising the following steps: extracting RNA of a sample to be tested, reverse transcribing it into cDNA, adding primer pairs for amplifying ToLCNDV, SLCCNV, CYSDV and CCYV, and detecting the amplified products after amplification. The present invention establishes a system for detecting these four viruses at one time, which greatly saves time and improves detection efficiency while ensuring accuracy.

Description

Technical Field

[0001] The invention belongs to the technical field of plant virus detection, and particularly relates to a method and a kit for detecting four cucurbit viruses transmitted by whiteflies using multiple PCR. Background Art

[0002] Bemisia whitefly belongs to the family Aleyrodidae in the order Hemiptera. It is a type of insect with piercing-sucking mouthparts. It is tiny, with an average body length of less than 1 mm. It is a major agricultural pest occurring worldwide, causing great harm to cash crops and food crops such as Solanaceae, Cucurbitaceae, Cruciferae, Leguminosae, and cotton. Bemisia whitefly can not only feed directly on plants, but also spread a variety of plant viruses, causing greater losses to crops. Therefore, the rapid identification of the types of viruses spread by Bemisia whitefly is of great guiding significance for the formulation of precise prevention and control measures.

[0003] There are many methods for detecting plant viruses, such as biological observation, serological reaction, electron microscopy and PCR technology. These methods can achieve good detection results when dealing with a small number of samples or a single virus target, but they are still time-consuming and laborious when facing a large number of complex samples. Multiplex PCR technology can add multiple pairs of primers to the reaction, so that multiple target viruses can be detected in one reaction, reducing the cost of detection and improving the detection efficiency. At present, the identification of virus types transmitted by whiteflies is still based on the RT-PCR detection method of a single primer. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a new option for simultaneously detecting four melon viruses transmitted by whiteflies.

[0005] The technical scheme of the present invention is a kit for multiplex PCR detection of four melon viruses transmitted by whiteflies, comprising primer pairs for amplifying ToLCNDV, SLCCNV, CYSDV and CCYV, wherein the nucleotide sequences of the primer pairs for amplifying ToLCNDV are shown as SEQ ID No.1 and SEQ ID No.2; the nucleotide sequences of the primer pairs for amplifying SLCCNV are shown as SEQ ID No.3 and SEQ ID No.4; the nucleotide sequences of the primer pairs for amplifying CYSDV are shown as SEQ ID No.5 and SEQ ID No.6; and the nucleotide sequences of the primer pairs for amplifying CCYV are shown as SEQ ID No.7 and SEQ ID No.8.

[0006] Furthermore, the kit also includes at least one of dNTP, DEPC-H2O, buffer, DNA polymerase, RNA reverse transcriptase, and RNA extraction reagent.

[0007] The present invention also provides the use of the kit in detecting cucurbit viruses transmitted by whitefly.

[0008] Specifically, the cucurbit virus transmitted by whitefly is at least one of ToLCNDV, SLCCNV, CYSDV and CCYV.

[0009] The present invention also provides a method for multiplex PCR detection of four melon viruses transmitted by whiteflies, comprising the following steps: extracting RNA of the sample to be tested, reverse transcribing it into cDNA, adding primer pairs for amplifying ToLCNDV, SLCCNV, CYSDV and CCYV, and detecting the amplified products after amplification.

[0010] Furthermore, the amplification system is 40 μL,

[0011] The concentration of each primer was 10 μM.

[0012] Specifically, the amount of each primer added is 0.2 to 0.6 mL.

[0013] Preferably, the amounts of upstream and downstream primers added for amplifying ToLCNDV, SLCCNV, CYSDV and CCYV are consistent, namely 0.2 μL, 0.4 μL, 0.6 μL and 0.3 μL, respectively.

[0014] Specifically, the amplification program is: pre-denaturation at 94°C for 8 min; denaturation at 94°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 1 min 30 s, 34 cycles; extension at 72°C for 5 min, and storage at 4°C.

[0015] Furthermore, the method for detecting the amplification product after amplification is electrophoresis or sequencing.

[0016] Specifically, if after detection, the amplified product contains a fragment of 1657bp in length, it indicates that the sample to be tested is infected with ToLCNDV; if after detection, the amplified product contains a fragment of 902bp in length, it indicates that the sample to be tested is infected with SLCCNV; if after detection, the amplified product contains a fragment of 1167bp in length, it indicates that the sample to be tested is infected with CYSDV; if after detection, the amplified product contains a fragment of 537bp in length, it indicates that the sample to be tested is infected with CCYV.

[0017] Beneficial effects of the present invention: The applicant discovered that four whitefly-transmitted viruses, ToLCNDV, SLCCNV, CYSDV, and CCYV, caused serious damage to melon crops. Based on this, the present invention designed specific detection primers for these four viruses, and the target virus sequence amplified by each pair of primers was different in length. By adjusting the primer dosage and annealing temperature, a multiplex RT-PCR detection technology system was established. The detection technology system can detect these four viruses at one time, which greatly saves time and improves detection efficiency while ensuring accuracy. It has important application value for the detection, diagnosis, and pathogenic mechanism research of melon viruses transmitted by whiteflies. The system can quickly, accurately, and efficiently identify the types of plant viruses transmitted by whiteflies that infect melon crops, and provide technical support for the early diagnosis of melon virus diseases and the formulation of prevention and control measures. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 , adjust the test results of each primer dosage. Marker: DNA marker 2000; Positive Control: Positive control, mix S1~S5, a mixed sample containing four viruses. The primer dosages of ToLCNDV, SLCCNV, CYSDV and CCYV in lane 1 are: 0.3μL, 0.5μL, 0.7μL, 0.5μL; the primer dosages of ToLCNDV, SLCCNV, CYSDV and CCYV in lane 2 are: 0.2μL, 0.4μL, 0.6μL, 0.3μL; the primer dosages of ToLCNDV, SLCCNV, CYSDV and CCYV in lane 3 are: 0.4μL, 0.6μL, 0.8μL, 0.6μL.

[0019] Figure 2 , test results of different annealing temperatures. The annealing temperatures of the PCR program in lanes 1 to 4 were 50°C, 52°C, 54°C, and 56°C, respectively. The rest of the reaction conditions were the same. Marker: DNA marker 2000; Positive Control: Positive control, mix S1 to S5, a mixed sample containing four viruses.

[0020] Figure 3 , multiple RT-PCR mixed verification results. Marker: DNA marker 2000; Positive Control: Positive control, mix S1~S5, mixed sample containing four viruses; Healthy: Healthy pumpkin sample; CucurbitSample 1-5: Cucurbit samples S1-S5.

[0021] Figure 4, ToLCNDV detection electrophoresis diagram, Marker: DNA marker 2000; Positive Control: positive control, mix S1~S5, a mixed sample containing four viruses; Healthy: healthy pumpkin sample; Cucurbit Sample1-5: melon samples S1-S5.

[0022] Figure 5 , SLCCNV detection electrophoresis diagram, Marker: DNA marker 2000; Positive Control: positive control, mix S1~S5, a mixed sample containing four viruses; Healthy: healthy pumpkin sample; Cucurbit Sample1-5: melon samples S1-S5.

[0023] Figure 6 , CYSDV detection electrophoresis diagram, Marker: DNA marker 2000; Positive Control: positive control, mix S1~S5, a mixed sample containing four viruses; Healthy: healthy pumpkin sample; Cucurbit Sample 1-5: melon samples S1-S5.

[0024] Figure 7 , CCYV detection electrophoresis diagram, Marker: DNA marker 2000; Positive Control: positive control, mix S1~S5, a mixed sample containing four viruses; Healthy: healthy pumpkin sample; Cucurbit Sample 1-5: melon samples S1-S5. DETAILED DESCRIPTION

[0025] The present invention aims to establish an efficient multiplex RT-PCR plant virus detection technology to simultaneously detect four cucurbit viruses transmitted by whiteflies, including two DNA viruses (Chinese squash leaf curl virus SLCCNV and New Delhi tomato leaf curl virus ToLCNDV of the genus Bean golden mosaic virus) and two RNA viruses (cucurbit yellow dwarf disorder virus CYSDV and cucurbit chlorotic yellows virus CCYV of the genus Trichovirus).

[0026] Example 1 Design of 4 pairs of PCR specific detection primers

[0027] 1. Search the sequences of the viruses to be detected, such as SLCCNV, ToLCNDV, CCYV, and CYSDV, in the NCBI virus database, perform Blast analysis, find several sequences with high homology, and download them. The specific sequence accession numbers are as follows:

[0028] ToLCNDV: OP903116, OP683995, AY939924, NC_004612, KF577603;

[0029] SLCCNV: MW389918, MW389922, OM238222, HM566113, NC_007338;

[0030] CYSDV: MT819949, KX120125, KT946801, KT946802, KT946803;

[0031] CCYV:KY400633, OR865221, OQ190464, OR492253, PP404048

[0032] 2. Then use DANMAN software to compare the sequences and find the conserved regions of the corresponding viruses to design primers. The primer design is completed in SnapGene software.

[0033] 3. Since the sequences of viruses of the same genus are highly homologous, it is necessary to design primers in the difference interval to distinguish different viruses of the same genus. Based on this idea, multiple pairs of primers were designed. After preliminary screening and removal of non-specific amplification bands, the four pairs of primers listed in Table 1 were finally obtained. The primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0034] Table 1 Primers for multiplex RT-PCR detection

[0035]

[0036] Note: Sequences from different isolates of the same virus may have different detection lengths.

[0037] Example 2 Optimizing and adjusting the dosage and annealing temperature of four pairs of primers

[0038] 1. Plant Total RNA Extraction

[0039] The melon crop samples suspected of carrying the virus collected from the field were classified, ground with liquid nitrogen, and the total RNA of the leaves was extracted using the Trizol method. The Trizol reagent was purchased from Takara.

[0040] 2. Reverse Transcription

[0041] The extracted plant total RNA was reverse transcribed to obtain its cDNA, and the reverse transcription reagent (PrimeScript TMIV1st strand cDNA Synthesis Mix was purchased from Takara, and cDNA was used as a template for PCR detection. The virus infection type contained in each sample has been determined by combining small RNA deep sequencing and RT-PCR detection, as follows:

[0042] S1 (cucumber): SLCCNV, CYSDV, CCYV;

[0043] S2 (Squash): SLCCNV, CCYV;

[0044] S3 (Pumpkin): SLCCNV;

[0045] S4 (Luffa): ToLCNDV;

[0046] S5 (Pumpkin): SLCCNV.

[0047] 3. Exploration of different primer addition amounts

[0048] The specific reaction system is as follows: cDNA 4 μL; Primer F (ToLCNDV, SLCCNV, CYSDV, CCYV are used in the following amounts: Lane 1, 0.3 μL, 0.5 μL, 0.7 μL, 0.5 μL; Lane 2, 0.2 μL, 0.4 μL, 0.6 μL, 0.3 μL; Lane 3, 0.4 μL, 0.6 μL, 0.8 μL, 0.6 μL); Primer R (the amounts of upstream and downstream primers are the same); 2× Ex Taq Mix Ex Taq ™ Version2.0 plus dye, purchased from Takara) 20μL; ddH2O 13μL, the total system is 40μL. The reaction procedure is: 94℃ pre-denaturation for 8 min; 94℃ denaturation for 30s, 50℃ annealing for 30s, 72℃ extension for 1min30s, 34 cycles; 72℃ extension for 5min; 4℃ storage. A mixed sample containing 4 viruses was used as a template. The test results are as follows Figure 1 As shown in the figure, the brightness of the bands varies according to the amount of primers used. Finally, the amount of primers added in lane 2 with the best amplification effect was determined as the final solution. The concentration of ToLCNDV, SLCCNV, CYSDV, and CCYV primers was 10 μM, and the amount of upstream and downstream primers added was the same, with the usage amounts being 0.2 μL, 0.4 μL, 0.6 μL, and 0.3 μL, respectively.

[0049] 4. Exploration of different annealing temperatures

[0050] The annealing temperature in the reaction program was changed according to the primer addition amount and reaction program determined in the above scheme. The specific system is: cDNA 4 μL; Primer F 1.5 μL (ToLCNDV, SLCCNV, CYSDV, CCYV are used in amounts of 0.2 μL, 0.4 μL, 0.6 μL, 0.3 μL respectively); Primer R 1.5 μL (the upstream and downstream primers are used in the same amount); 2× Ex Taq Mix 20μL; ddH2O 13μL, total system 40μL. Reaction procedure: 94℃ pre-denaturation 8 min; 94℃ denaturation 30s, 50℃ / 52℃ / 54℃ / 56℃ annealing 30s, 72℃ extension 1min30s, 34 cycles; 72℃ extension 5min; 4℃ storage. Figure 2 As shown in the figure, the brightness of the bands varies at different annealing temperatures. The amplification effect is best when the annealing temperature is 54°C, followed by 56°C. According to the test results, the annealing temperature was finally determined to be 54°C.

[0051] Example 3 Using these four pairs of primers to perform multiplex RT-PCR detection on diseased samples of melon crops

[0052] According to the scheme determined by the above method, the final reaction system and reaction procedure are as follows: the concentration of ToLCNDV, SLCCNV, CYSDV, and CCYV primers is 10 μM, the amount of upstream and downstream primers is consistent, 0.2 μL, 0.4 μL, 0.6 μL, and 0.3 μL, respectively, the reaction system is cDNA 4 μL; total primer F 1.5 μL; total primer R 1.5 μL; 2× Ex Taq Mix 20μL; ddH2O 13μL, the total system is 40μL. The reaction procedure is 94℃ pre-denaturation for 8 min; 94℃ denaturation for 30s, 54℃ annealing for 30s, 72℃ extension for 1min30s, 34 cycles; 72℃ extension for 5min; 4℃ storage. Melon samples S1-S5 were used as templates; S1~S5 were mixed, and the mixed sample containing four viruses was used as a positive control. The RT-PCR product was placed in a 1.5% agarose gel, using GL DNA Marker2000 (purchased from Acry Biotechnology Co., Ltd.), and imaged under a gel imaging system. According to the size of the band, the type of virus corresponding to the size was determined, thereby determining the rationality of the multiple RT-PCR primers. The test results are as follows Figure 3 As shown, after optimizing the primer dosage and annealing temperature, multiple viruses or a single virus type in the sample can be accurately detected.

[0053] Example 4 RT-PCR verification of each virus using specific primers

[0054] In order to verify whether the multiplex RT-PCR test results of the above four pairs of primers are correct, the four pairs of primers designed this time are verified one by one to verify the effect of the primers. Reaction system: cDNA 1μL; Primer F 0.5μL; Primer R 0.5μL; 2× ExTaq Mix 12.5μL; ddH2O 10.5μL, the total system is 25μL. The reaction procedure is 94℃ pre-denaturation for 8 min; 94℃ denaturation for 30s, 54℃ annealing for 30s, 72℃ extension for 1min30s, 34 cycles; 72℃ extension for 5min; 4℃ storage. Melon samples S1-S5 were used as templates, and a mixed sample containing 4 viruses was used as a positive control. The RT-PCR product was imaged in a 1.5% agarose gel using GL DNA Marker 2000 (purchased from Acry Biotech), and imaged under a gel imaging system. According to the size of the band, the type of virus corresponding to the size was determined, thereby determining the rationality of the multiple RT-PCR primers. The test results are shown in Figures 4 to 7 , the figure shows that the results of single primer verification are Figure 3 The results of multiplex RT-PCR were consistent.

Claims

1. A method for detecting four cucurbit viruses transmitted by whitefly by multiplex PCR, characterized in that: A kit for detecting four melon viruses transmitted by whitefly by multiple PCR was used for detection, the kit comprising primer pairs for amplifying ToLCNDV, SLCCNV, CYSDV and CCYV, the nucleotide sequences of the primer pairs for amplifying ToLCNDV are shown in SEQ ID No.1 and SEQ ID No.2; the nucleotide sequences of the primer pairs for amplifying SLCCNV are shown in SEQ ID No.3 and SEQ ID No.4; the nucleotide sequences of the primer pairs for amplifying CYSDV are shown in SEQ ID No.5 and SEQ ID No.6; the nucleotide sequences of the primer pairs for amplifying CCYV are shown in SEQ ID No.7 and SEQ ID No.8; The method comprises the following steps: extracting RNA of a sample to be tested, reverse transcribing it into cDNA, adding a primer pair for amplifying ToLCNDV, SLCCNV, CYSDV and CCYV, and detecting the amplified product after amplification.

2. The method according to claim 1, characterized in that: The amplification system is 40 μL, the concentration of each primer is 10 μM, and the amount of each primer added is 0.2-0.6 mL.

3. The method according to claim 2, characterized in that: The amounts of upstream and downstream primers added for amplifying ToLCNDV, SLCCNV, CYSDV, and CCYV were consistent, namely 0.2 μL, 0.4 μL, 0.6 μL, and 0.3 μL, respectively.

4. The method according to claim 1, characterized in that: The amplification program was as follows: pre-denaturation at 94°C for 8 min; denaturation at 94°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 1 min 30 s, 34 cycles; extension at 72°C for 5 min, and storage at 4°C.

5. The method according to claim 1, characterized in that: The method for detecting the amplification product after amplification is electrophoresis or sequencing.

6. The method according to claim 5, characterized in that: If after detection, the amplified product contains a fragment of 1657bp in length, it indicates that the sample to be tested is infected with ToLCNDV; if after detection, the amplified product contains a fragment of 902bp in length, it indicates that the sample to be tested is infected with SLCCNV; if after detection, the amplified product contains a fragment of 1167bp in length, it indicates that the sample to be tested is infected with CYSDV; if after detection, the amplified product contains a fragment of 537bp in length, it indicates that the sample to be tested is infected with CCYV.

7. The method according to claim 1, characterized in that: The kit further comprises at least one of dNTP, DEPC-H2O, buffer, DNA polymerase, RNA reverse transcriptase, and RNA extraction reagent.

8. Application of the method of claim 1 in the detection of melon viruses transmitted by whitefly, characterized in that: The cucurbit virus transmitted by whitefly is at least one of ToLCNDV, SLCCNV, CYSDV and CCYV.

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