Primers and detection methods for detecting three kiwifruit viruses: AcVA, ACVB and AcCRaV

Abstract

The present invention discloses a primer and a detection method for detecting three kiwifruit viruses, AcVA, ACVB and AcCRaV, and belongs to the field of genetic engineering technology. The present invention uses three pairs of primers to simultaneously detect three viruses of kiwifruit through multiple RT-PCR: kiwifruit virus A (AcVA), kiwifruit virus B (AcVB) and kiwifruit chlorotic ringspot-associated virus (AcCRaV). The nucleotide sequences of the three pairs of primers are shown in SEQ ID NO.1-6. The present invention also optimizes the annealing temperature, extension time, number of cycles, primer type and concentration of the multiple RT-PCR. The method of the present invention can be successfully applied to virus detection of field samples, effectively improving detection efficiency, shortening detection time, and saving detection costs.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and specifically designs a primer and a detection method for detecting three kiwifruit viruses, namely, AcVA, ACVB and AcCRaV. Background Art

[0002] Kiwifruit (Actinidia chinensis Planch) is a perennial vine of the Actinidia genus of the Actinidia family. It originated in China and has developed rapidly worldwide due to the unique flavor and nutritional value of its fruit, and has international economic importance. Kiwifruit is one of the characteristic industries of Sichuan Province, but the introduction and seedling sales activities between regions have promoted the spread of kiwifruit diseases, seriously affecting the yield and quality of kiwifruit. The spread and spread of viral diseases have also become one of the hidden dangers threatening the safe production of kiwifruit. There are many types of kiwifruit viruses, and they often manifest as mixed infections.

[0003] At present, 13 viruses have been reported in my country, including Actinidia chlorotic ringspot-associated virus (AcCRaV), Actinidia virus A (AcVA) and Actinidia virus B (AcVB). Among them, AcCRaV, AcVA and AcVB are mainly host to kiwifruit, often infecting at the same time, with symptoms such as chlorosis, yellowing and ring spots. AcCRaV belongs to the family Fimoviridae and is a negative-sense single-stranded RNA virus of the genus Emaravirus; AcVA and AcVB are both members of the genus Vitivirus of the family Betaflexiviridae.

[0004] Several plant virus detection methods have been developed, such as transmission electron microscopy, next-generation sequencing, and enzyme-linked immunosorbent assay (ELISA). These methods are accurate and effective, but time-consuming and require certain detection skills. Since mixed viral infection is a common phenomenon in plants, it is necessary to develop methods for the simultaneous detection of multiple viruses for routine disease diagnosis and quarantine. Currently, the following methods are available for diagnosis and quarantine of routine diseases: a highly sensitive method for detecting AcCRaV and efficient elimination of kiwifruit chlorotic ringspot-associated virus from in vitro cultured kiwifruit branches, both of which are limited to one virus; in recent years, there have been many reports on the use of multiplex RT-PCR to detect multiple viruses on woody plants, such as citrus, cotton, and grapes; using RT-PCR, two sets of primers were used to detect four viruses on peach and apricot trees to improve detection efficiency; a one-step multiplex real-time reverse transcription polymerase chain reaction method was used to rapidly detect three pear tree viruses; the use of multiplex RT-PCR to simultaneously detect stone fruit tree viruses can save time and materials; and a multiplex reverse transcription polymerase chain reaction method was used to successfully detect four viruses in kiwifruit field samples.

[0005] Based on previous experiments, multiplex RT-PCR has shown the characteristics of being fast, time-saving, labor-saving and highly accurate, and has broad application prospects. The difference between multiplex PCR and single PCR is that two or more target DNAs or RNAs are placed in a single reaction to detect multiple viruses at the same time. AcCRaV, AcVB and AcVA are common viruses on kiwifruit. Traditional single RT-PCR is more cumbersome and time-consuming. Multiplex RT-PCR has high sensitivity, is fast and simple, and can be used to diagnose kiwifruit infection with various virus combinations, which has important application value.

[0006] So far, there have been no reports of using multiplex RT-PCR to simultaneously detect kiwifruit virus A, kiwifruit virus B and kiwifruit chlorotic ringspot-associated virus at home and abroad. Therefore, it is of vital importance to provide a method for simultaneously detecting the three viruses AcCRaV, AcVB and AcVA. Summary of the invention

[0007] In view of the above-mentioned deficiencies in the prior art, the present invention provides a primer and a detection method for detecting three kiwifruit viruses, AcVA, ACVB and AcCRaV. Primers are designed for mixed infection, which simplifies the operation of kiwifruit virus detection. When applied in practice, it is expected to improve detection efficiency, shorten detection time, save detection costs, and provide a feasible method for the detection of kiwifruit viruses.

[0008] The present invention adopts the following technical solutions:

[0009] A primer for detecting three kiwifruit viruses, AcVA, ACVB and AcCRaV, comprises three pairs of primers, and the nucleotide sequences of the three pairs of primers are:

[0010] AcVA-F: 5'-CATGGCAAAGAATATCTCAAG-3' (SEQ ID NO. 1);

[0011] AcVA-R: 5'-AGATCCAACCCAGAGTTGAAA-3' (SEQ ID NO. 2);

[0012] AcVB-F: 5'-GTTATGCCAAACGTTTATGA-3' (SEQ ID NO. 3);

[0013] AcVB-R: 5'-CTCCTCGCAAACGAACTC-3' (SEQ ID NO. 4);

[0014] AcCRaV-F: 5'-TTAAGTAGAAGAACCACAATAT-3' (SEQ ID NO. 5);

[0015] AcCRaV-R: 5'-ATGCCAAAGCCTATGCAAG-3' (SEQ ID NO. 6).

[0016] The primers are used to detect three kiwifruit viruses: AcVA, ACVB and AcCRaV.

[0017] The primers are used in preparing a kit for detecting three kiwifruit viruses, namely AcVA, ACVB and AcCRaV.

[0018] A kit for detecting three kiwifruit viruses, AcVA, ACVB and AcCRaV, comprising the upstream primer and downstream primer mentioned in the claim.

[0019] Furthermore, the upstream primers are a mixture of AcVA / F, ACVB / F and AcCRaV / F in equal volume ratios, and the downstream primers are a mixture of AcVA / R, ACVB / R and AcCRaV / R in equal volume ratios.

[0020] A method for detecting three kiwifruit viruses, AcVA, ACVB and AcCRaV, comprises the following steps:

[0021] (1) using the total RNA of the sample to be tested as a template and using the composition of the three downstream primers of claim 1 mixed in equal volumes as a primer for reverse transcription to obtain cDNA;

[0022] (2) Using the cDNA obtained in step (1) as a template, PCR amplification is performed using the primers described in claim 1, and the amplified product is detected by agarose gel electrophoresis. Whether the virus is infected is determined based on whether the corresponding band is amplified.

[0023] Furthermore, in the above step (1), the reverse transcription is performed using a composition of equal volumes of the above three downstream primers as a primer, and the concentration of the reverse transcription primer is 10 to 30 μM.

[0024] Furthermore, the upstream primer used for PCR amplification in the above step (2) is a mixture of the above three upstream primers in an equal volume ratio, and the downstream primer is a mixture of the above three downstream primers in an equal volume ratio.

[0025] Furthermore, the PCR reaction system is 2×Taq PCR Master Mix, 12-13 μL; cDNA, 0.8-1.2 μL; upstream and downstream primers, 0.8-1.2 μL each, supplemented with ddH2O to 24-26 μL; the PCR reaction system is preferably 2×Taq PCR Master Mix, 12.5 μL; cDNA, 1.0 μL; upstream and downstream primers, 1.0 μL each, supplemented with ddH2O to 25 μL

[0026] Furthermore, the PCR reaction program is: pre-denaturation at 93-95°C for 2-4 min; denaturation at 93-95°C for 25-35 s; annealing at 44.5-49°C for 25-35 s; extension at 70-74°C for 35-60 s; 28-38 cycles; extension at 70-74°C for 8-12 min; the PCR program is preferably pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s; annealing at 46.3°C for 30 s; extension at 72°C for 50 s; 35 cycles; extension at 72°C for 10 min.

[0027] By adopting the above technical solution, the beneficial effects of the present invention are:

[0028] 1. The present invention designs specific primer pairs for three viruses based on the viral coat protein genes of three kiwifruit viruses, AcVA, ACVB and AcCRaV, and confirms the specificity of the primers by sequencing. In the traditional single RT-PCR, a pair of primers is used for detection, which has high sensitivity but only targets one virus. Among the three pairs of primers designed by the present invention, each pair of primers can detect the corresponding virus separately, ensuring high sensitivity. The three pairs of primers designed by the present invention can also detect three viruses at the same time, and save time and reagents. The Tm values ​​of the three pairs of primers are close, and the sizes of the amplified fragments are obviously different, which is convenient for distinguishing them in agarose gel electrophoresis analysis.

[0029] 2. The multiplex RT-PCR procedure was optimized to obtain the best reaction conditions. There are multiple pairs of primers in multiplex RT-PCR, and the formation of primer dimers will lead to increased nonspecific amplification. The optimization of multiplex PCR conditions aims to minimize such nonspecific amplification; the present invention determines the optimal annealing temperature, extension time and number of amplification cycles for multiplex RT-PCR of three viruses, AcVA, AcVB and AcCRaV. Annealing temperature is generally considered to be a key factor in PCR specificity and amplification efficiency. The three pairs of primers were selected with similar Tm values ​​to allow the use of a common annealing temperature, and it was experimentally confirmed that an annealing temperature of 46.3°C can effectively optimize the amplification of all viral targets; changes in extension time and number of amplification cycles can effectively improve amplification efficiency. When the number of cycles is less than 32, no effective amplification is obtained for gel electrophoresis detection, and too many amplification times are not conducive to improving amplification efficiency, but increase detection time; increasing extension time is conducive to increasing the number of longer products, which was also confirmed in this experiment; the sensitivity of the reaction is affected by the number of different primer pairs, rather than the total amount of primers present in the mixture, among which AcVB and AcCRaV showed high sensitivity in single RT-PCR, and AcVB was least affected in multiple RT-PCR; 10μM specific primers were selected for reverse transcription to save reagents.

[0030] 3. The primers designed by the present invention combined with RT-PCR can successfully detect single and mixed infections of AcVA, AcVB and AcCRaV in field samples, which saves nearly half the time and about two-thirds of the reagents compared with the traditional single RT-PCR detection method. The results of the detection of 100 kiwifruit leaf samples collected showed that 52% (52 samples) of the samples were infected with one or more viruses, of which 37% (37 samples) were infected with a single virus and 15% (15 samples) were mixed infections; among all the positive samples, the detection rate of AcVA was 13 .5% (7 samples), AcVB detection rate was 34.6% (18 samples), and AcCRaV detection rate was 73.1% (38 samples); in single virus infection samples, AcVA accounted for 10.8% (4 samples), AcVB accounted for 21.6% (8 samples), and AcCRaV accounted for 67.6% (25 samples); in mixed infection samples, AcVB and AcCRaV accounted for the largest proportion of simultaneous infection, accounting for 66.7% (10 samples), 20% (3 samples) were co-infected with AcVA and AcCRaV, and the three viruses accounted for only 13.3% (2 samples). The test results showed that AcCRaV was the most seriously infected in kiwifruit leaf samples collected in Qionglai City, Pujiang County and Wenjiang District, Sichuan Province, and was the dominant virus, followed by AcVB, and AcVA was less.

[0031] In addition, the PCR buffer composition, dNTP, enzyme concentration and DNA template can be optimized in multiplex RT-PCR. PCR additives, such as dimethyl sulfoxide, glycerol, bovine serum albumin or betaine, are also helpful in improving the amplification efficiency of multiplex PCR. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Agarose gel electrophoresis analysis of single PCR experiments of AcVA, AcVB and AcCRaV.

[0033] Figure 2 Symptoms on kiwifruit leaves with single infection and mixed infection.

[0034] Figure 3 This is an agarose gel analysis of the amplified products obtained by reverse transcription and RT-PCR amplification using different primers.

[0035] Figure 4 Agarose gel electrophoresis analysis of multiple RT-PCR products at different annealing temperatures.

[0036] Figure 5 Agarose gel electrophoresis analysis of multiple RT-PCR products with different extension times.

[0037] Figure 6 Agarose gel electrophoresis analysis of multiple RT-PCR products at different cycle numbers.

[0038] Figure 7 Agarose gel electrophoresis analysis of multiplex RT-PCR products of different combinations of viral RNA.

[0039] Figure 8 Agarose gel electrophoresis analysis of multiple RT-PCR products at different primer concentrations.

[0040] Fig. 9 Agarose gel electrophoresis analysis of the sensitivity of single RT-PCR detection of AcVA.

[0041] Fig.10 Agarose gel electrophoresis analysis of the sensitivity of single RT-PCR detection of AcVB.

[0042] Fig.11 Agarose gel electrophoresis analysis of the sensitivity of single RT-PCR detection of AcCRaV.

[0043] Fig.12 Agarose gel electrophoresis analysis of multiplex RT-PCR of 20 virus-infected kiwifruit leaves. DETAILED DESCRIPTION

[0044] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0045] Example 1 RT-PCR establishment and optimization

[0046] 1. Obtain kiwifruit leaves: Collect leaves suspected of being infected with the virus with typical symptoms such as blister spots, wrinkles, ring spots, chlorosis, narrow and long deformation, yellowing and necrosis of leaf veins from the main kiwifruit growing areas in the southwest region, including Dujiangyan City, Qionglai City, Pujiang County and Wenjiang District of Chengdu City. Store the diseased leaves in dry bottles together with anhydrous calcium chloride at 4°C. Store fresh leaf samples in a refrigerator at -80°C for RT-PCR analysis.

[0047] 2. Extraction of total RNA from kiwifruit leaves: Take kiwifruit leaf samples (0.1 g) and grind them in liquid nitrogen with 2 mL sterile centrifuge tubes, extract with phenol, chloroform and isoamyl alcohol (v:v:v=25:24:1) (500 μL) and RNA buffer (500 μL); after centrifugation, mix the supernatant (about 400 μL) with 2 volumes of 4M lithium chloride solution, incubate at -20°C for at least 8 h, then centrifuge at 4°C at 12000 rpm for 10 min to recover the particles; the obtained particles are repeatedly centrifuged with 1000 μL 70% ethanol at 4°C, air-dried and then suspended in 30 μL ddH2O, stored and incubated at -20°C for 8 h, then centrifuged at 4°C at 12000 r / min for 10 min, discard the supernatant, recover the RNA in the particles, and obtain a total RNA preparation;

[0048] 3. Primer design: Virus-specific primers were designed from the conserved regions of the viral coat protein (CP) genes of AcCRaV, AcVB, and AcVA obtained from GenBank using DNAMAN 8.0 and MEGA 7.0, as shown in Table 1. The specificity of the primers was verified by BLASTN on NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). All primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and diluted to 10 μM and 50 μM with ddH2O for PCR amplification.

[0049] Table 1 Specific primers used for respective single and multiplex RT-PCR analysis

[0050]

[0051] 4. Single RT-PCR amplification: First use BeyoRT TM ⅡFirst Strand cDNA Synthesis Kit (RNase H-) was used to prepare 20 μL reaction system, and reverse transcription was performed to obtain cDNAs of three viruses, AcCRaV, AcVB and AcVA, and healthy kiwifruit leaves: Total RNA, 1 μL; Oligo(dT 18 )primer, 1μL; Reaction Buffer (5×), 4μL; Rnase inhibitor, 1μL; dNTP Mix, 2μL; eyoRT TM ⅡM-MLV reverse transcriptase, 1 μL; supplemented with DEPC-treated water to 20 μL; the reaction procedure was: incubate at 42°C for 1 h; incubate at 80°C for 10 min to obtain cDNA, and the reverse transcription reagents were purchased from Sangon Biotech (Shanghai) Co., Ltd.;

[0052] The cDNA obtained above was used as a template, and single PCR amplification was performed using random primers and the virus-specific primers in Table 1 of step 3; the 25 μL PCR reaction system was: 2×Taq PCR Master Mix, 12.5 μL, cDNA, 1.0 μL, 1.0 μL of upstream and downstream primers, and ddH2O was added to 25 μL; the PCR reaction program was: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s; 48°C annealing for 30 s; 72°C extension for 40 s; 35 cycles; 72°C extension for 10 min. The obtained PCR amplification product was detected by 1.5% agarose gel electrophoresis to determine its specificity. The results are as follows: Figure 1 As shown, lane M is a 2000 bp molecular marker; lanes 1, 2, and 3 are single RT-PCR experimental products of AcVA, AcVB, and AcCRaV, respectively; H is a negative control of simulated infection; Figure 2 The leaf symptom diagrams are single infection and mixed infection;

[0053] Depend on Figure 1 It can be seen that three viruses were successfully detected in virus-infected kiwifruit leaf samples, and amplicons of expected sizes were amplified in their respective specific PCR experiments, including AcVA of 471 bp, AcVB of 690 bp, and AcCRaV of 933 bp. No products were obtained using virus-negative sample controls;

[0054] 5. Multiplex RT-PCR Experiment

[0055] (1) Optimize reverse transcription conditions

[0056] RT-PCR experiments were performed using a mixture of random primers and specific reverse primers of the three viruses. The total RNA of the three viruses, AcVA, AcVB and AcCRaV, was evenly mixed in a volume ratio of 1:1:1 as a reverse transcription template; the primers in the reverse transcription were a mixture of random primers and specific reverse primers of the three viruses (AcVA / R, AcVB / R and AcCRaV / R were mixed in a volume ratio of 1:1:1, 2 μL); three primer concentrations (10 μM, 20 μM and 30 μM) were used respectively. The cDNA transcribed at 1 μM was subjected to PCR experiment (other conditions and operation methods of reverse transcription were the same as those in step 4). During PCR amplification, the upstream primer was replaced with a composition of AcVA / F, AcVB / F and AcCRaV / F mixed evenly in a volume ratio of 1:1:1, and the downstream primer was replaced with a composition of AcVA / R, AcVB / R and AcCRaV / R mixed evenly in a volume ratio of 1:1:1. 1 μL of each upstream and downstream primer was used for PCR amplification. The experimental results are shown in the figure. Figure 3 As shown, M is a 2000 bp molecular marker; H is a negative control of simulated infection; Random is a random primer; Specific is a virus-specific reverse primer; 10, 20 and 30 represent 10 μM, 20 μM and 30 μM, respectively;

[0057] Depend on Figure 3 It can be seen that the effect of using specific reverse primers is better than random primers, and the optimal concentration of primers is 10μM;

[0058] (2) Optimizing PCR parameters

[0059] a. After reverse transcription using 10 μM specific reverse primer, PCR amplification was performed using the upstream and downstream primers used in step A PCR amplification, and the annealing temperatures were set to 44.5°C, 44.8°C, 45.4°C, 46.3°C, 47.4°C, 48.8°C and 49.0°C for PCR amplification. The amplified products were detected by 1.5% agarose gel electrophoresis. The results are as follows: Figure 4 As shown, lanes 1-8 represent annealing at 44.5°C, 44.8°C, 45.4°C, 46.3°C, 47.4°C, 48.8°C, 48.8°C and 49.0°C, respectively; lane M represents a 2000 bp molecular marker; H represents a negative control of mock infection;

[0060] Depend on Figure 4It can be seen from the experimental results at different annealing temperatures that the changes of AcCRaV and AcVB viruses are not obvious at 44.5℃ to 48.8℃, and the main difference is reflected in the effect of AcVA. On the whole, the PCR products of the three viruses under the treatment of lanes 1 to 2 have the best effect, and the difference between different temperature treatments is small. Therefore, 46.3℃ is selected as the optimal annealing temperature of multiple RT-PCR for subsequent experiments;

[0061] b. Carry out RT-PCR experiment under the optimal reverse transcription conditions and optimal annealing temperature parameters, optimize the PCR amplification extension time, set the extension time to 35s, 40s, 45s, 50s, 55s and 60s respectively, and the other conditions are the same as the PCR amplification in step 4. The amplified product is detected by 1.5% agarose gel electrophoresis. The results are as follows Figure 5 As shown, the extension times of lanes 1-8 are 35s, 40s, 45s, 50s, 55s and 60s, respectively; lane M represents a 2000bp molecular marker; H represents a negative control of mock infection;

[0062] Depend on Figure 5 It can be seen that the virus-specific RT-PCR products were effectively amplified in all treatments, and the effect was significantly improved from 45s. When the extension time reached 60s, the number of products of the three viruses decreased. The best effect was achieved in the treatments of 50s and 55s, so 50s was selected as the optimal extension time for PCR.

[0063] c. Perform RT-PCR experiment under the optimal reverse transcription conditions, optimal annealing temperature and optimal extension time parameters, optimize the number of PCR amplification cycles, set the number of cycles to 26, 28, 30, 32, 35 and 38 times, and the other conditions are the same as the PCR amplification in step 4. The amplified product is detected by 1.5% agarose gel electrophoresis. The results are as follows: Figure 6 As shown, the cycle numbers of lanes 1-8 are 26, 28, 30, 32, 35 and 38, respectively; lane M represents a 2000 bp molecular marker; H represents a negative control of mock infection;

[0064] Depend on Figure 6 It can be seen that when PCR was performed with 26 to 40 amplification cycles, the results showed that there were very few three viral products in the amplification products with amplification times of 26, 28 and 30, which increased significantly at 32 times, and the effect was best at 35 times, and there was no obvious increase at higher times; in addition, the RT-PCR product of AcVA was the least at different amplification cycle numbers, and was not easily detected at low amplification cycle numbers, while the product of AcVB increased most significantly at higher cycle numbers. On the whole, 35 amplification cycles were selected as the optimal number of multiple RT-PCR for subsequent experiments.

[0065] In all the above experiments, virus positive and negative controls were included, healthy plants were used as negative controls, PCR amplification products were detected by 1.5% agarose gel electrophoresis, and subsequent multiplex PCR experiments were performed using optimized parameters.

[0066] Example 2 Multiplex RT-PCR analysis of different combinations of viral total RNA species

[0067] Total RNA of plants infected with AcVA, AcVB and AcCRaV was used as the experimental template, and four mixed combinations of AcVA / R+AcVB / R+AcCRaV / R, AcVB / R+AcCRaV / R, AcVA / R+AcCRaV / R and AcVA / R+AcVB / R were used as primers, 2.0 μL of primers (each 10 μM), and cDNA was obtained by reverse transcription. Each reaction contained 1 μL of RNA template mixture (700 ng / μL). PCR amplification was carried out under the above-mentioned optimized annealing temperature, extension time and number of amplification cycles. The reaction system was 25 μL, and the obtained amplified products were detected by 1.5% agarose gel electrophoresis. The results are shown in FIG. Figure 7 As shown; lanes 1–7 are from kiwifruit leaves infected with AcVA, kiwifruit leaves infected with AcVB, kiwifruit leaves infected with AcCRaV, a mixture of RNA from kiwifruit leaves infected with AcVA and AcVB, a mixture of RNA from kiwifruit leaves infected with AcVA and AcCRaV, a mixture of RNA from kiwifruit leaves infected with AcVB and AcCRaV, and a mixture of RNA from kiwifruit leaves infected with the three viruses, respectively; lane M represents a 2000bp molecular marker; H represents a negative control of mock infection.

[0068] Depend on Figure 7 It can be seen that the four primer combinations corresponding to different viruses can effectively obtain virus-specific amplicons and successfully detect the presence of viruses. The types and quantities of viral RNA templates used in the seven different situations simulating field infections are different (infection with one virus or simultaneous infection with two or three viruses); the total RNA content in the combination of simultaneous infection with two viruses is half of that in the combination of simultaneous infection with three viruses is one-third of that in the combination of simultaneous infection with three viruses, so the amount of cDNA used in PCR also varies with the types of viruses tested; the amplification effect of only one virus is better than other combinations, among which AcCRaV is the least obvious; the two virus mixture combinations show that one virus is better and the other is second, and in the combination of AcVA and AcVB, AcVA has the best effect, and AcCRaV is less affected; AcVB performed best among the three virus mixtures (in all the above experiments, no product was obtained in the negative control, and no non-specific DNA products were observed).

[0069] Example 3 Sensitivity detection of multiplex RT-PCR

[0070] The total RNA concentrations in the three virus-infected samples were determined using a Nanodrop spectrophotometer (Thermo science, USA). The total RNA concentrations of all extracted mixed infection samples were adjusted to 700 ng / μL, and then the total RNA was diluted 10-fold in series (10 0 -10 8 ) was used as a reverse transcription template, and 4 μL of three kinds of total RNA and optimized reverse primers were used to synthesize cDNA. The synthesized cDNA was diluted 10 times in a series of gradients (10 0 -10 8 ), and PCR amplification was performed according to the optimal parameters of other conditions obtained by optimization. The primers were the same as the PCR amplification primers in step 5 (1) of Example 1. The amplified products were detected by 1.5% agarose gel electrophoresis. The results were as follows: Figure 8 As shown, Figure 8 Lanes 1-9 are 10 0 -10 8 10-fold serial dilutions; Lane M: 2000 bp molecular marker; Lane H: negative control of mock infection;

[0071] Depend on Figure 8 It can be seen that the sensitivity of AcVA is 10 3 , the sensitivity of AcVB is 10 8 , the sensitivity of AcCRaV is 10 3 ; AcVB can still show high sensitivity when the cDNA concentration is extremely low;

[0072] Using cDNA of AcVA 10 0 -10 8 The serial dilutions were used as single PCR templates and amplified with specific primers of AcVA. The amplified products were detected by 1.5% agarose gel electrophoresis. The results were as follows: Fig. 9 As shown; Lanes 1-9 are 10 0 -10 8 10-fold serial dilutions; Lane M represents a 2000 bp molecular marker; Lane H represents a negative control of mock infection;

[0073] Depend on Fig. 9 It can be seen that the sensitivity of AcVA in single PCR detection is 10 3 ;

[0074] Using cDNA of AcVB 10 0 -10 8The serial dilutions were used as single PCR templates and amplified with specific primers of AcVB. The amplified products were detected by 1.5% agarose gel electrophoresis. Fig.10 As shown; Lanes 1-9 are 10 0 -10 8 10-fold serial dilutions; Lane M represents a 2000 bp molecular marker; Lane H represents a negative control of mock infection;

[0075] Depend on Fig.10 It can be seen that the sensitivity of AcVB in single PCR sensitivity detection is 10 7 ;

[0076] Using cDNA of AcCRaV 10 0 -10 8 The serial dilutions were used as single PCR templates and amplified with specific primers of AcCRaV. The amplified products were detected by 1.5% agarose gel electrophoresis. Fig.11 As shown; Lanes 1-9 are 10 0 -10 8 10-fold serial dilutions; Lane M represents a 2000 bp molecular marker; Lane H represents a negative control of mock infection;

[0077] Depend on Fig.11 It can be seen that the sensitivity of AcCRaV in single PCR sensitivity detection is 10 8 , AcVB and AcCRaV both showed high sensitivity; due to the inconsistency of the initial cDNA content in single PCR and multiplex PCR, the experimental results of the two cannot be directly compared.

[0078] Example 4 Field sample detection

[0079] Based on previous studies, optimized multiplex RT-PCR parameters were used to test 100 kiwifruit leaves from plants suspected of being infected with the virus collected from four cities and counties in Sichuan Province, China. The samples were stored at -80°C. The confirmed virus-negative samples were used as negative controls, and the total RNA of field samples infected with the three viruses alone was mixed equally as a positive control. The results of the single RT-PCR experiment were used to verify the accuracy of the multiplex RT-PCR experimental results. All the above experiments were performed three times independently.

[0080] The experimental results showed that 52 samples were infected with one or more viruses; among the 37 samples infected with a single virus, 4 were AcVA, 8 were AcVB, and the remaining 25 were AcCRaV; among the 13 samples infected with two viruses, there was no co-infection of AcVA and AcVB, 10 were co-infected with AcVB and AcCRaV, and 3 were co-infected with AcVA and AcCRaV; only two cases were infected with three viruses at the same time.

[0081] The above results were verified by single PCR respectively, and 20 representative kiwifruit leaf samples infected with single virus or multiple viruses were selected from 52 samples for multiple RT-PCR experiments. Fig.12 As shown, lane M represents a 2000 bp molecular marker; H represents a field sample from a virus-free field; P represents a mixture of total RNA from field samples infected with three single viruses, confirming the accuracy of the multiplex RT-PCR results. The experiments were performed independently three times, and their respective single PCR results are shown in Table 2;

[0082] Table 2 Results of testing 20 field samples using a single RT-PCR

[0083]

[0084]

[0085] From Table 2 and Fig.12 It can be seen that the three pairs of primers of AcVA, AcVB and AcCRaV provided by the present invention, combined with the highly sensitive multiplex RT-PCR method, can successfully detect samples with single virus infection or mixed infection, simplify the operation of kiwi virus detection, improve detection efficiency, shorten detection time, save detection costs, and provide a new means for the detection of three major viruses in kiwi fruit.

Claims

1. A method for detecting three kiwifruit viruses, AcVA, ACVB and AcCRaV, characterized in that: The following steps are involved: (1) Using the total RNA of the sample to be tested as a template, reverse transcription is performed to obtain cDNA; (2) Using the cDNA obtained in step (1) as a template, performing PCR amplification using primers, detecting the amplified product by agarose gel electrophoresis, and determining whether the virus is infected based on whether the corresponding band is amplified; Wherein, the nucleotide sequences of the primers are: AcVA-F: 5'-CATGGCAAAGAATATCTCAAG-3'; AcVA-R: 5'-AGATCCAACCCAGAGTTGAAA-3'; AcVB-F: 5'-GTTATGCCAAACGTTTATGA-3'; AcVB-R: 5'-CTCCTCGCAAACGAACTC-3'; AcCRaV-F: 5'-TTAAGTAGAAGAACCACAATAT-3'; AcCRaV-R: 5'-ATGCCAAAGCCTATGCAAG-3'; In step (1), the reverse transcription is performed using a composition of three downstream primers of the primer mixed in equal volumes as a primer, and the primer concentration is 10-30 μM; The upstream primers used for PCR amplification in step (2) are three upstream primers of the primers mixed in equal volume ratio, and the downstream primers are three downstream primers of the primers mixed in equal volume ratio; The PCR reaction system is 2 × Taq PCR Master Mix, 12-13 μL; cDNA, 0.8-1.2 μL; upstream and downstream primers, 0.8-1.2 μL each, supplemented with ddH2O to 24-26 μL; The PCR reaction procedure is: pre-denaturation at 93-95°C for 2-4 min; denaturation at 93-95°C for 25-35 s; Anneal at 46.3°C for 25-35s; extend at 70-74°C for 50s; 35 cycles; extend at 70-74°C for 8-12min.

2. The use of primers of three kiwifruit viruses, AcVA, ACVB and AcCRaV, in detecting three kiwifruit viruses, AcVA, ACVB and AcCRaV, is characterized in that: The nucleotide sequences of the primers are: AcVA-F: 5'-CATGGCAAAGAATATCTCAAG-3'; AcVA-R: 5'-AGATCCAACCCAGAGTTGAAA-3'; AcVB-F: 5'-GTTATGCCAAACGTTTATGA-3'; AcVB-R: 5'-CTCCTCGCAAACGAACTC-3'; AcCRaV-F: 5'-TTAAGTAGAAGAACCACAATAT-3'; AcCRaV-R: 5'-ATGCCAAAGCCTATGCAAG-3'.

3. A kit for detecting three kiwifruit viruses, AcVA, ACVB and AcCRaV, characterized in that: The invention comprises primers of three kiwifruit viruses, AcVA, ACVB and AcCRaV; the nucleotide sequences of the primers are respectively: AcVA-F: 5'-CATGGCAAAGAATATCTCAAG-3'; AcVA-R: 5'-AGATCCAACCCAGAGTTGAAA-3'; AcVB-F: 5'-GTTATGCCAAACGTTTATGA-3'; AcVB-R: 5'-CTCCTCGCAAACGAACTC-3'; AcCRaV-F: 5'-TTAAGTAGAAGAACCACAATAT-3'; AcCRaV-R: 5'-ATGCCAAAGCCTATGCAAG-3'.

4. The kit according to claim 3, characterized in that The upstream primers among the three pairs of primers were mixed in equal volumes, and the downstream primers were mixed in equal volumes.

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