Multiplex PCR (Polymerase Chain Reaction) detection method for simultaneously detecting three main viruses of areca catechu and application

Abstract

The invention belongs to the technical field of biology, and particularly relates to a multiplex PCR (polymerase chain reaction) detection method for simultaneously detecting three main viruses of areca catechu and application of the multiplex PCR detection method. According to the invention, a multi-virus synchronous detection technology system is constructed, three key pathogenic viruses of areca yellows can be accurately identified at one time, and compared with a traditional single-virus detection method, the detection efficiency is improved by more than 3 times. Through innovative primer design and reaction system optimization, the detection period is compressed to be within 1.5 hours from conventional several hours, meanwhile, the detection cost can be reduced by 66.7%, high-efficiency, low-cost and high-sensitivity rapid virus screening is achieved, a solid foundation is laid for formulating a targeted prevention and control strategy and constructing a scientific prevention and control system, and the method has a wide application prospect. The method has great practical value for promoting healthy development of the betel nut industry.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a multiplex PCR detection method and application for simultaneously detecting three main viruses of areca nut. Background Art

[0002] Areca nut is an important cash crop and is widely planted in tropical and subtropical regions. However, the outbreak of areca nut yellowing disease seriously threatens the sustainable and healthy development of the areca nut industry. The yield of infected areca nut decreases by 30%-50%, and even reaches zero production. Research has found that viruses play a key role in the occurrence and development of areca nut yellowing disease. Among them, it is clear that areca palm velarivirus 1 (APV1) is closely related to the yellowing of areca nut leaves. The average detection rate of APV1 in the areca nut yellowing areas across Hainan Island is above 67%, and the highest is 100%. In addition, yellow leaf-associated ormycovirus (APYLaOMV) and areca palm latent totivirus 1 (APLTV1) are newly discovered viruses related to areca nut yellowing. The detection rates of 500 samples are 32.98% and 29.22% respectively. The symptoms of areca nut yellowing disease are typical. In the initial stage, the yellowing of the leaves starts from the leaf tip of the middle or bottom leaves, and the yellow-green boundary is obvious. In the later stage, the yellowing spreads to the whole leaf, the yellow-green boundary on the older leaves becomes blurred, the new leaves are underdeveloped, the crown width is significantly reduced, and the "bunch top" symptom appears.

[0003] In recent years, areca palm necrotic ringspot virus (APNRSV) and areca palm spindle-spot virus (ANSSV) have also been discovered. They cause circular necrosis and fusiform necrosis of the leaves respectively and belong to the family Potyviridae. However, they are significantly different from the symptoms of areca nut yellowing disease, can be clearly distinguished according to the symptoms, and have not yet caused serious harm to the areca nut industry.

[0004] At present, a total of 5 viruses, namely APV1, APLTV1, APYLaOMV, APNRSV and ANSSV, have been discovered on areca nut. Among them, these 3 viruses, APV1, APLTV1 and APYLaOMV, threaten the healthy development of the areca nut industry. They can infect alone or often in combination, resulting in the aggravation of areca nut yellowing symptoms, the increasing area of disease occurrence and the continuous aggravation of economic losses. Rapid and accurate detection of viruses has become a key requirement for the development of the industry.

[0005] In the field of plant virus detection, biological detection, serological analysis, electron microscopy observation, and molecular biological methods centered on PCR technology are the main detection means, and these methods are often used alone or in combination for virus identification. However, the existing detection technologies have significant limitations: Although the traditional RT-PCR method is widely used in the detection of areca nut viruses, it has problems such as cumbersome detection procedures, large human input, and high costs; at the same time, whether it is classical detection methods or molecular biological technologies, most can only achieve single virus detection, and it is difficult to meet the need for rapid simultaneous screening of multiple virus complex infections in areca nut. Therefore, there is an urgent need to develop new high-throughput and high-efficiency areca nut virus detection technologies and supporting detection kits to provide technical support for the early diagnosis and scientific prevention and control of areca nut yellowing disease, and help the healthy and sustainable development of the areca nut industry. Summary of the Invention

[0006] The present invention provides a multiplex PCR detection method and application for simultaneously detecting three main viruses of areca nut, which can accurately identify three key pathogenic viruses of areca nut yellowing disease at one time. Compared with the traditional single virus detection method, the detection efficiency is increased by more than 3 times.

[0007] The technical solution of the present invention is realized as follows:

[0008] A multiplex primer for simultaneously detecting three main viruses of areca nut, including primer APYLaOMV-630, APV1-495 and / or APLTV1-395, and their nucleotide sequences are respectively:

[0009] APYLaOMV-630F: 5'-GAATTACCCACGCCTCCAG-3' and APYLaOMV630 R: 3'-GAGCTTTCCAGGACATGCC-5';

[0010] APV1-495 F: 5'-GAAAGATCTGGACCGAGTAATGG-3' and APV1-495 R: 3'-TACACATACAAGTTAGCAGGAGG-5';

[0011] APLTV1-395 F: 5'-CTAACCGCTCTAGCTGACAC-3' and APLTV1-395 R: 3'-GTGAACCTCGCCTGATTCTG-5'.

[0012] A kit for simultaneously detecting three main viruses of areca nut, including the above-mentioned multiplex primer.

[0013] Use of the above-mentioned multiplex primers or kit in the detection of Areca palm symptomless virus 1 (APV1), Areca palm yellowing-associated mycovirus (APYLaOMV) and / or Areca palm latent monopartite virus 1 (APLTV1).

[0014] Use of the above-mentioned multiplex primers or kit for detecting Areca palm symptomless virus 1, Areca palm yellowing-associated mycovirus and / or Areca palm latent monopartite virus 1 from Areca palm leaves.

[0015] A method for simultaneously detecting three main viruses of Areca palm, comprising the following steps:

[0016] (1) Extracting RNA of the sample to be tested using Areca palm leaves as raw materials; performing reverse transcription using the RNA of the sample to be tested as a template to obtain cDNA;

[0017] (2) Using the obtained cDNA as template DNA, performing PCR amplification using the above-mentioned multiplex primers or the said kit;

[0018] (3) Determining the virus species of the Areca palm leaves to be tested according to the size of the amplified target band;

[0019] The said viruses are respectively Areca palm symptomless virus 1, Areca palm yellowing-associated mycovirus and / or Areca palm latent monopartite virus 1.

[0020] Furthermore, the band or target fragment of Areca palm symptomless virus 1 is 495 bp, the band or target fragment of Areca palm yellowing-associated mycovirus is 630 bp, and the band or target fragment of Areca palm latent monopartite virus 1 is 395 bp.

[0021] Furthermore, in the step (2), during the PCR amplification process, the PCR system is set to 25 μL: 12.5 μL Taq enzyme Mix, 2 μL template DNA, and the addition amount of each primer for detecting Areca palm symptomless virus 1, Areca palm yellowing-associated mycovirus and Areca palm latent monopartite virus 1 is 0.3 - 0.7 μL; the annealing temperature is 51 - 57 °C, and the number of cycles is 25 - 45.

[0022] Furthermore, in the step (2), during the PCR amplification process, the addition amounts of each primer for detecting Areca palm symptomless virus 1 (APV1), Areca palm yellowing-associated mycovirus (APYLaOMV) and Areca palm latent monopartite virus 1 (APLTV1) are 0.7, 0.5 and 0.3 μL respectively.

[0023] Furthermore, in the step (2), operating on a PCR instrument, pre-denaturing at 94 °C for 3 min, denaturing at 94 °C for 30 s, annealing at 53 °C for 30 s, extending at 72 °C for 1 min, for a total of 35 cycles, and extending at 72 °C for 10 min.

[0024] Use of the above-described method in differentiating Areca palm symptomless virus 1, Areca palm yellowing-related mycovirus, and / or Areca palm latent monopartite virus 1, wherein the band or target fragment of Areca palm symptomless virus 1 is 495 bp, the band or target fragment of Areca palm yellowing-related mycovirus is 630 bp, and the band or target fragment of Areca palm latent monopartite virus 1 is 395 bp.

[0025] Advantages of the present invention:

[0026] The present invention constructs a multi-virus synchronous detection technology system, which can accurately identify three key pathogenic viruses of Areca palm yellowing disease at one time. Compared with the traditional single-virus detection method, the detection efficiency is increased by more than 3 times. Through innovative primer design and reaction system optimization, the detection cycle is compressed from several hours to within 1.5 hours (originally, it took 249 minutes to detect the three viruses separately, and now it only takes 83 minutes to detect the three viruses, saving 166 minutes). The original cost of reverse transcription and PCR for detecting one RNA virus sample was 36 yuan (excluding labor costs). Using this method, 24 yuan can be saved, and the detection cost is equivalent to 1 / 3 of the conventional method, with a 66.7% reduction in detection cost. It can achieve high-efficiency, low-cost, and high-sensitivity rapid virus screening. This method not only fills the technical gap in the joint detection of multiple Areca palm viruses, but also provides core support for the accurate tracing of Areca palm yellowing disease, lays a solid foundation for formulating targeted prevention and control strategies and constructing a scientific prevention and control system, and has great practical value for promoting the healthy development of the Areca palm industry. Description of the Drawings

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. The following drawings are only some embodiments of the present invention.

[0028] Figure 1 For the specificity of multiplex PCR; M: 100 bp DNA Marker; 1: APYLaOMV + APV1 + APLTV1; 2: APYLaOMV + APV1; 3: APYLaOMV + APLTV1; 4: APV1 + APLTV1; 5: APYLaOMV; 6: APV1; 7: APLTV1; CK: negative control.

[0029] Figure 2 For the optimization of multiplex PCR primer concentration; M: 100 bp DNA Marker; 1 - 16: different combinations of primer pair addition amounts; CK: negative control; Note: The corresponding amounts in the table are the amounts of forward and reverse primers added. For example, when amplifying APV1 - 495 in No. 1, 0.5 μL of both the forward primer and the reverse primer are added, and the same applies to others.

[0030] Figure 3For optimizing the annealing temperature of multiplex PCR; M: 100bp DNA Marker; 1: 51°C; 2: 52°C; 3: 53°C; 4: 54°C; 5: 55°C; 6: 56°C; 7: 57°C; 8: negative control.

[0031] Figure 4 For optimizing the number of cycles of multiplex PCR; M: 100bp DNA Marker; 1: 25 cycles; 2: 30 cycles; 3: 35 cycles; 4: 40 cycles; 5: 45 cycles; 6: negative control.

[0032] Figure 5 For the sensitivity of multiplex PCR; M: 100bp DNA Marker; 1: 10 0 ; 2: 10 -1 ; 3: 10 -2 ; 4: 10 -3 ; 5: 10 -4 ; 6: 10 -5 ; CK: negative control. Detailed implementation manners

[0033] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0034] Embodiment 1

[0035] 1. Extraction of RNA from areca nut leaves

[0036] Experimental materials: Areca nut yellowing leaves, sourced from the main areca nut planting cities and counties in Hainan Province.

[0037] Using the CTAB method, total RNA was extracted from the areca nut leaf samples. The specific operation steps are as follows:

[0038] (1) Prepare a blade, forceps, small steel beads for grinding, etc. and sterilize them. Cut and weigh 0.1 - 0.2 g of areca nut leaves with the blade, and put them together with 3 sterilized steel beads into a 0.2 mL RNase-free centrifuge tube. Close the lid and place it in liquid nitrogen for sufficient freezing.

[0039] (2) Put the centrifuge tube into a cell disruptor to break it until the sample becomes uniform particles or powder. Add 1 mL of CTAB solution to the centrifuge tube and mix well with a vortex oscillator for more than 15 seconds.

[0040] (3) Place the centrifuge tube in a 65 °C water bath for 5 - 10 min, and gently invert it 3 times during this period for mixing.

[0041] (4) Add 1 mL of chloroform / isoamyl alcohol (24:1) pre-cooled at 4 °C to the centrifuge tube, invert it up and down for mixing, and centrifuge at 10000×g for 15 min under 4 °C conditions.

[0042] (5) Prepare a new 2 mL RNase-free centrifuge tube, transfer the supernatant after centrifugation in the previous step into the new tube, add 200 μL of chloroform / isoamyl alcohol (24:1), invert it up and down for mixing, and centrifuge at 10000×g for 15 min under 4 °C conditions.

[0043] (6) Prepare a new 2 mL RNase-free centrifuge tube, add 4 mol / L LiCl, and place it at 4 °C for 8 h.

[0044] (7) Take out the centrifuge tube from the previous step and centrifuge at 12000×g for 15 min under 4 °C conditions.

[0045] (8) Discard the supernatant, add 1 mL of 75% ethanol solution, gently stir the precipitate at the bottom of the tube for washing, and centrifuge at 12000×g for 5 min under 4 °C conditions.

[0046] (9) Discard the supernatant, add 1 mL of absolute ethanol, gently stir the precipitate at the bottom of the tube for washing, and centrifuge at 12000×g for 5 min under 4 °C conditions.

[0047] (10) Discard the supernatant (note not to pour out the precipitate), centrifuge at 12000×g for 2 min under 4 °C conditions. Use a pipette to suck away the excess supernatant and open the lid to dry it in a fume hood.

[0048] (11) Add 30 - 50 μL of RNase-free Water to dissolve the precipitate.

[0049] (12) Store at -80 °C.

[0050] 2. cDNA synthesis

[0051] Use the reverse transcription kit from Gen Star, Beijing Kangrun Chengye Biotechnology Co., Ltd. The specific operation steps are as follows:

[0052] (1) Prepare the following reaction system in a 0.2 mL RNase-free centrifuge tube:

[0053]

[0054] (2) Gently pipette up and down for mixing and briefly centrifuge.

[0055] (3) Place the centrifuge tube into a PCR instrument, set the program as follows and run: incubate at 37 °C for 2 min, heat at 55 °C for 15 min, and inactivate at 85 °C for 2 min.

[0056] (4) Take out the cDNA of the reverse transcription product and store it at -20 °C.

[0057] 3 Multiplex PCR primer design

[0058] Based on the assembled and sequenced APYLaOMV whole genome sequence, the CP region of the APLTV1 sequence TCWP531, and the CP region of the APV1 reference sequence MN296265.1, specific primers were designed using Primer 5.0, and the primers were screened and evaluated using DNAman. Finally, the primers are shown in Table 1.

[0059] Table 1 Multiplex PCR primers

[0060]

[0061] 4 Optimization of multiplex PCR reaction conditions and system

[0062] 4.1 Primer specificity detection

[0063] Specificity detection was performed on three pairs of primers. The cDNAs of the positive samples of APV1, APYLaOMV, and APLTV1 were mixed in equal volumes and pairwise, corresponding to a total of 7 different infection situations, namely APV1 + APYLaOMV + APLTV1, APV1 + APYLaOMV, APV1 + APLTV1, APYLaOMV + APLTV1, APV1, APYLaOMV, and APLTV1. Amplification reactions were carried out on the above templates according to the reference reaction conditions and system to test the specificity of multiplex PCR.

[0064] Table 2 PCR amplification system

[0065]

[0066] Table 3 PCR amplification program

[0067]

[0068] The amplified products were electrophoresed on a 1.5% agarose gel, and the reaction results were judged by observing the darkness of the bands.

[0069] 4.2 Optimization of multiplex PCR amplification conditions

[0070] 4.2.1 Annealing temperature optimization: A cDNA equal-volume mixture of viruses APV1, APYLaOMV, and APLTV1 was used as a template. Annealing temperatures were set at 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, and 57°C respectively to screen for the optimal annealing temperature.

[0071] 4.2.2 Cycle number optimization: The cycle numbers for the denaturation, annealing, and extension steps were set at 25, 30, 35, 40, and 45 respectively to screen for the optimal cycle number.

[0072] 4.2.3 Primer concentration ratio optimization: For the optimization of the multiplex PCR amplification system, based on the results of adding three pairs of primers in equal ratios, the addition amounts of the three primers were set at 0.3 μL, 0.5 μL, and 0.7 μL respectively, and permutations and combinations were carried out. By observing the gel electrophoresis results, the optimal primer concentration ratio was screened.

[0073] 4.2.4 Multiplex PCR sensitivity detection: An equal-volume mixture was diluted in a 10-fold gradient and mixed evenly as a template to test the sensitivity of the multiplex PCR method under the optimal reaction conditions and system. And a batch of betel nut field samples were detected to evaluate its actual application effect.

[0074] 5. Experimental results

[0075] 5.1 Specificity of multiplex PCR

[0076] One to three positive samples of viruses APYLaOMV, APV1, and APLTV1 were added to the system respectively, and amplification was carried out under the initial reaction system and conditions. The amplification results are shown in the electrophoresis Figure 1 as follows. The number and size of the amplified fragments in each sample were consistent with the types of viruses added to the template cDNA. No bands were amplified in the negative control results, indicating good primer specificity. Conclusion: The three target bands in sample No. 5 are relatively clear, and the bands in sample No. 11 are relatively uniform.

[0077] 5.2 Primer ratio optimization

[0078] The final concentrations of the primers for APYLaOMV, APV1, and APLTV1 were set at 0.12 μM, 0.2 μM, and 0.28 μM respectively (i.e., the addition amounts of the forward and reverse primers were both 0.3 μL, 0.5 μL, and 0.7 μL), and permutations and combinations were carried out. When the final concentrations of the primers for APYLaOMV, APV1, and APLTV1 were 0.2 μM, 0.28 μM, and 0.12 μM respectively, the three bands were all relatively clear, so this was the optimal primer concentration ( Figure 2 ). The amplification system and procedure were the same as in 4.1.

[0079] Table 4 Addition amounts of each primer

[0080]

[0081] 5.3 Annealing temperature optimization

[0082] Equal amounts of cDNA from single virus infection samples of the three viruses were added to the system, with the added amount being 2 L. The primer addition amount was based on treatment 5 in Table 4, and PCR reaction was performed. The PCR amplification products were separated by polyacrylamide gel electrophoresis.

[0083] The amplification was performed at 51-57℃ annealing temperature. The electrophoresis results showed that with the increase of annealing temperature, the brightness of the 630bp band of APYLaOMV gradually decreased, while the bands of APV1 and APLTV1 gradually became brighter. When the annealing temperature was 53℃, the bands were relatively clear. Therefore, the optimal annealing temperature was 53℃ ( Figure 3 ).

[0084] 5.4 Cycle Number Optimization

[0085] Equal amounts of cDNA from single virus infection samples of the three viruses were added to the system, with the added amount being 2 L. The primer addition amount was based on treatment 5 in Table 4, and PCR reaction was performed. The PCR amplification products were separated by polyacrylamide gel electrophoresis.

[0086] Amplification was performed at cycle numbers ranging from 25 to 45. The results are shown in Figure 4. When the cycle number is 30 or less, the bands are faint and difficult to observe. When the cycle number is greater than 40, miscellaneous bands begin to appear, and 35 cycles is a suitable number.

[0087] 5.5 Sensitivity Detection

[0088] Viral cDNA was diluted 10-fold as amplification template. -3 When the template cDNA is diluted to 10 -2 When the brightness of the three target fragments reaches the lowest brightness that can be identified, the detection sensitivity of multiplex PCR is 10 times that of the cDNA stock solution. -2 Times. The result is as follows Figure 5 As shown, diluted to 10 -3 There are also faint bands, indicating that the primers have good sensitivity.

[0089] According to the above results, the optimal conditions are finally determined to be:

[0090] The multiplex PCR system was set to 25 μL: 12.5 μL Taq enzyme Mix, 2 μL template DNA, 0.5 μL each of the forward and reverse primers of APYLaOMV-630, 0.7 μL each of the forward and reverse primers of APV1-495, 0.3 μL each of the forward and reverse primers of APLTV1-395. After adding the primers, water was added to make up to 25 μL; the PCR amplification program was: pre-denaturation at 94 °C for 3 min, denaturation at 94 °C for 30 s, annealing at 53 °C for 30 s, extension at 72 °C for 1 min, for a total of 35 cycles, and extension at 72 °C for 10 min.

[0091] Example 2

[0092] Batch detection of areca nut field samples was carried out to evaluate the practical application effect of the detection method described in the present invention.

[0093] Randomly collect 26 areca nut leaf samples (including diseased and non-diseased ones), extract the RNA of each sample, and perform reverse transcription using the RNA of the sample to be tested as a template to obtain cDNA. PCR amplification was carried out using the primers in Table 1 according to the method in Example 1 for the detection of areca nut yellowing virus. At the same time, RT-PCR was carried out for the three viruses respectively, and the results are shown in Table 5.

[0094] Detection rate = number of detected samples / total number of samples × 100%

[0095] Table 5 Simultaneous detection of 3 viruses by multiplex PCR method

[0096]

[0097] The RT-PCR system was set to 25 μL: 14.5 μL Taq enzyme Mix, 2 μL template DNA, 1.0 μL each of the forward and reverse primers of APYLaOMV-630; 1.5 μL each of the forward and reverse primers of APV1-495; 1.0 μL each of the forward and reverse primers of APLTV1-395; then ddH2O was added respectively to make up to 25 μL; PCR amplification was carried out separately. The program was: pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 s, annealing at 53 °C for 45 s, extension at 72 °C for 50 s, with the number of repeated cycles being 35; extension at 72 °C for 10 min.

[0098] Multiplex PCR reaction system:

[0099] The multiplex PCR system was set to 25 μL: 12.5 μL Taq enzyme Mix, 2 μL template DNA, 0.5 μL each of the forward and reverse primers of APYLaOMV-630, 0.7 μL each of the forward and reverse primers of APV1-495, 0.3 μL each of the forward and reverse primers of APLTV1-395. After adding the primers, make up to 25 μL with water; pre-denature at 94 °C for 3 min, denature at 94 °C for 30 s, anneal at 53 °C for 30 s, extend at 72 °C for 1 min, for a total of 35 cycles, and extend at 72 °C for 10 min.

[0100] As can be seen from Table 5, by using the multiplex PCR detection method of the present invention to detect the above samples, the detection rates of the three viruses have all been improved to varying degrees. At the same time, by using the multiplex PCR detection method of the present invention, it is possible to detect three viruses, namely Areca palm potyvirus 1 (APV1), Areca palm latent virus 1 (APLTV1), and Areca palm yellowing-associated mycovirus (APYLaOMV), at one time. Among them, there are 12 samples infected with three viruses, 6 samples infected with only two viruses, 3 samples infected with only one virus, and 2 samples with no virus detected. When using the RT-PCR method to detect the three viruses separately, there are 9 samples infected with three viruses, 8 samples infected with two viruses, 4 samples infected with one virus, and 5 samples with no virus detected. For the three samples QJ4-1, HK25, and HKHQ, RT-PCR did not detect any virus, and the plants did not show any disease symptoms. However, when using the multiplex PCR of the present invention for inspection, virus infections were detected in all of them, and three viruses were simultaneously detected in the sample HK25. Thus, compared with the RT-PCR detection method, the multiplex PCR not only has a higher detection rate, but also the detection results are more accurate.

[0101] The above results show that after systematic optimization of the primer combination and reaction system of the present invention, it can accurately identify and simultaneously amplify the characteristic nucleic acid fragments of the three viruses, significantly improving the detection efficiency and accuracy of the viruses related to Areca palm yellowing disease, and providing key technical support for the rapid diagnosis and precise prevention and control of Areca palm yellowing disease.

[0102] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A multiplex primer for simultaneously detecting three main viruses of areca nut, characterized in that, Comprising primers APYLaOMV-630, APV1-495 and / or APLTV1-395, whose nucleotide sequences are respectively: APYLaOMV-630F: 5'-GAATTACCCACGCCTCCAG-3' and PvlaO-630R: 3'-GAGCTTTCCAGGACATGCC-5'; APV1-495 F: 5'-GAAAGATCTGGACCGAGTAATGG-3' and APV1-495 R: 3'-TACACATACAAGTTAGCAGGAGG-5'; APLTV1-395 F: 5'-CTAACCGCTCTAGCTGACAC-3' and APLT-395R: 3'-GTGAACCTCGCCTGATTCTG-5'.

2. A kit for simultaneously detecting three main viruses of areca nut, characterized in that, Comprising the multiplex primers described in claim 1.

3. Use of the multiplex primers described in claim 1 or the kit described in claim 2 for detecting Areca catechu cryptic virus 1, Areca yellowing-related mycovirus and / or Areca latent monopartite virus 1.

4. Use of the multiplex primers described in claim 1 or the kit described in claim 2 for detecting Areca catechu cryptic virus 1, Areca yellowing-related mycovirus and / or Areca latent monopartite virus 1 from Areca catechu leaves.

5. A method for simultaneously detecting three main viruses of areca nut, characterized in that, Comprising the following steps: (1) Extracting RNA of the sample to be tested using Areca catechu leaves as raw materials; performing reverse transcription using the RNA of the sample to be tested as a template to obtain cDNA; (2) Using the obtained cDNA as template DNA, performing PCR amplification using the multiplex primers described in claim 1 or the kit described in claim 2; (3) Determining the virus species of the Areca catechu leaves to be tested according to the size of the amplified target band; The viruses are respectively Areca catechu cryptic virus 1, Areca yellowing-related mycovirus and / or Areca latent monopartite virus 1.

6. The method according to claim 5, characterized in that The band or target fragment of Areca catechu cryptic virus 1 is 495bp, the band or target fragment of Areca yellowing-related mycovirus is 630bp, and the band or target fragment of Areca latent monopartite virus 1 is 395bp.

7. The method according to claim 5, characterized in that In the step (2), during the PCR amplification process, the PCR system is set to 25 μL: 12.5 μL Taq enzyme Mix, 2 μL template DNA, and the addition amount of each primer for detecting Areca catechu cryptic virus 1, Areca yellowing-related mycovirus and Areca latent monopartite virus 1 is 0.3 - 0.7 μL; the annealing temperature is 51 - 57 °C, and the number of cycles is 25 - 45.

8. The method according to claim 7, wherein The addition amounts of each primer for detecting Areca catechu cryptic virus 1, Areca yellowing-related mycovirus and Areca latent monopartite virus 1 are 0.7, 0.5 and 0.3 μL respectively.

9. The method according to claim 7, characterized in that In the step (2), operating on a PCR instrument, pre-denaturing at 94 °C for 3 min, denaturing at 94 °C for 30 s, annealing at 53 °C for 30 s, extending at 72 °C for 1 min, for a total of 35 cycles, and extending at 72 °C for 10 min.

10. Use of the method according to claim 5 in differentiating Areca catechu cryptic virus 1, Areca yellowing-associated mycovirus, and / or Areca latent monopartite virus 1, characterized in that, The band or target fragment of betel nut latent virus No. 1 is 495bp, the band or target fragment of betel nut yellowing-related fungal virus is 630bp, and the band or target fragment of betel nut latent single component virus 1 is 395bp.

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