Multiplex PCR (polymerase chain reaction) detection method for three potato Y viruses on passionflower

Abstract

The invention belongs to the technical field of plant virus detection, and particularly relates to a multiplex PCR (Polymerase Chain Reaction) detection method for three potato Y viruses on passionflower, which is a multiplex RT-PCR detection method for simultaneously detecting the three potato Y viruses of TeMV, EAPV and PFSMoV. The multiplex PCR detection method comprises the following steps: genome extraction: extracting total RNA of a sample to be detected, and then performing reverse transcription to obtain cDNA; multiple RT-PCR amplification reaction: taking the obtained cDNA as a detection template, and carrying out multiple RT-PCR amplification on the obtained cDNA and specific primers of TeMV, EAPV and PFSMoV viruses under the condition of a reaction system; and result detection: determining the result of the amplification product by means of agarose gel electrophoresis. The invention successfully establishes a multiplex PCR detection method for three potato Y viruses on passionflower, and the detection method can simultaneously detect three potato Y viruses of TeMV, EAPV and PFSMoV.

Description

Technical Field

[0001] The invention belongs to the technical field of plant virus detection, and particularly relates to a multiplex PCR detection method for three potato virus Ys on passion fruit. Background Art

[0002] Passion fruit is a highly valuable crop widely distributed across tropical and subtropical regions of my country. It is sweet and sour when eaten fresh and can also be processed into a variety of products, including juice, preserved fruit, and wine. Its unique taste and nutritious properties make it a popular choice. Passion fruit also boasts numerous medicinal benefits, including relieving anxiety, strengthening the spleen and digestion, promoting metabolism, and boosting immunity. Consequently, its cultivation has been expanding in southern my country in recent years. Viral diseases are a major constraint on the development of the passion fruit industry. Currently, over 30 virus species have been reported in passion fruit, with potato virus Y (Potyvirus) being a particularly common virus. These viruses, including Telosmamosaic virus (TeMV), East Asian passiflora virus (EAPV), and Passion fruit severe mottle virus (PFSMoV), can cause severe damage to passion fruit, either alone or in combination. EAPV is one of the pathogens of passion fruit stone fruit disease, causing the fruit's skin to become thicker and harder, with less or no flesh, and a loss of flavor. TeMV alone can cause yellowing, mosaic, shrinkage, and deformity of passion fruit leaves, weakening the plant. PFSMoV alone can cause severe mottled and shrunken leaves. These three viruses often infect the field in combination, one, two, or even three different strains. Mixed infections can cause more severe symptoms in the host.

[0003] Current prevention and control strategies for viral diseases in production primarily rely on planting virus-free seedlings, cutting off virus transmission pathways in the field, and promptly removing diseased plants. These strategies require rapid, accurate, and sensitive detection methods. While rapid detection methods have been established for one or two of the three viruses, TeMV, EAPV, and PFSMoV, using PCR, serology, or isothermal amplification techniques, no method currently exists that can simultaneously detect the presence of all three. Summary of the Invention

[0004] In order to solve the problem that there is no method in the prior art that can simultaneously detect the presence of TeMV, EAPV, and PFSMoV, the present invention intends to establish a rapid detection method based on multiplex PCR amplification technology that can simultaneously detect TeMV, EAPV, and PFSMoV three potato viruses Y.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides a multiplex PCR detection method for three potato viruses Y on passion fruit, wherein the method is a multiplex RT-PCR detection method for simultaneously detecting three potato viruses Y, TeMV, EAPV, and PFSMoV;

[0007] The multiplex PCR detection method comprises the following steps:

[0008] Genome extraction: extract total RNA from the sample to be tested, and then reverse transcribe to obtain cDNA;

[0009] Multiplex RT-PCR amplification reaction: The obtained cDNA is used as a detection template and specific primers for TeMV, EAPV, and PFSMoV viruses are used to perform multiplex PCR amplification under the reaction system conditions;

[0010] The specific primers include primers TeMV-F / R, EAPV-F / R and PFSMoV-F / R; the nucleotide sequence of the TeMVCP-F is shown in SEQ ID NO.1; the nucleotide sequence of the TeMVCP-R is shown in SEQ ID NO.2; the nucleotide sequence of the EAPV-F is shown in SEQ ID NO.3; the nucleotide sequence of the EAPV-R is shown in SEQ ID NO.4; the nucleotide sequence of the PFSMoV-F is shown in SEQ ID NO.5; the nucleotide sequence of the PFSMoV-R is shown in SEQ ID NO.6;

[0011] Result determination.

[0012] Preferably, the sample to be tested is a passion fruit plant infected or suspected of being infected with three potato virus Y: TeMV, EAPV, and PFSMoV; or the sample to be tested is a passion fruit plant infected or suspected of being infected with one of the potato virus Y alone.

[0013] Preferably, fresh leaf tissue of the sample to be tested is minced and ground into powder under liquid nitrogen pre-cooling conditions, and total RNA of the sample is extracted using a total RNA extraction reagent, and the extracted total RNA of the sample is stored at -20°C for later use.

[0014] Preferably, the reverse transcription system is: 1.0 μL of random primer 6N 10 μM, 4.0 μL of dNTP Mixture 25 mM, 2.0 μL of template RNA, 3.0 μL of RNase Free double-distilled water, and the total reaction system is 10 μL;

[0015] The solution of the reverse transcription system was denatured at 65°C for 5 minutes and then quickly cooled on ice. The reverse transcription reaction solution was added and reverse transcription was completed at 30°C for 10 minutes, 42°C for 50 minutes, and 75°C for 15 minutes to obtain cDNA.

[0016] The reverse transcription reaction solution system is: 5×Reverse Transcriptase M-MLV Buffer 4.0 μL, Recombinant RNase Inhabitar 0.5 μL, RNase nuclease M-MLV 1.0 μL, RNase Free double-distilled water 4.5 μL, making up a total of 20 μL reaction system.

[0017] Preferably, the amplification reaction system solution during the multiple PCR amplification is specifically composed of the following volumes of components: 2×Taq Master Mix: 10 μl of near-shore protein, 3 p mol of primers TeMVCP-F and TeMVCP-R, 1.5 p mol of primers EAPV-F and EAPV-R, 1.5 p mol of primers PFSMoV-F and PFSMoV-R, 1.0 μl of template cDNA, and double distilled water is added to make up the system to 20 μl.

[0018] Preferably, the reaction procedure for the multiplex PCR amplification is: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 20 s, annealing at 56°C for 20 s, extension at 72°C for 1 min, 35 cycles, and extension at 72°C for 5 min.

[0019] Preferably, the result is determined using the amplified product after PCR reaction.

[0020] Preferably, after the PCR reaction is completed, the reaction tube is taken out for agarose gel electrophoresis, and the result is determined based on whether specific amplified fragments can be observed on the gel.

[0021] Preferably, the result determination method is as follows: if there is a band at 685bp on the gel, it means that the sample to be tested contains only TeMV; if there is only a band at 405bp on the gel, it means that the sample to be tested contains only PFSMoV; if there is only a band at 248bp on the gel, it means that the sample to be tested contains only EAPV; if there is no band on the gel, it means that the sample to be tested is a healthy plant; if there are two bands at the corresponding position on the gel; it means that the sample to be tested contains double viruses; if there are three bands at the corresponding position on the gel, it means that the sample to be tested contains triple viruses.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides a multiplex PCR detection method for three potato viruses Y on passion fruit. The detection method can simultaneously detect three potato viruses Y: Telosma mosaic virus (TeMV), East Asian passiflora virus (EAPV), and Passion fruit severe mottle virus (PFSMoV). The present invention designs specific amplification primers for the coat protein gene (CP) region of TeMV, the coat protein gene (CP) region of EAPV, and the coat protein gene (CP) region of PFSMoV, and establishes a multiplex RT-PCR detection method that can rapidly detect and identify these three viruses, providing important experimental technical support for the early detection and prevention and control of potato viruses Y in the future.

[0024] The detection method established by the present invention specifically amplifies TeMV, EAPV, and PFSMoV, and has no cross-reaction with other related viruses, thus showing high specificity. The detection method has high sensitivity and adaptability for detecting PEDV, PEAV, PDCoV, and TGEV. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 are the primer screening results of the present invention; wherein, A is the TeMV primer screening result; B is the PFSMoV primer screening result; C is the EAPV primer screening result;

[0026] Figure 2 The results of primer screening in the present invention are shown in Table 2; A is the electrophoresis result of primer sets 1 and 2; B is the electrophoresis result of primer sets 3 and 4; C is the electrophoresis result of primer sets 5 and 6; and D is the electrophoresis result of primer sets 7 and 8. The specific information of the primer pairs contained in the primer sets is shown in Table 2.

[0027] Figure 3The electrophoresis test results of the three viruses TeMV, EAPV, and PFSMoV in the present invention are shown; wherein, lane M is a DNA ladder marker; lane 1 is a sample infected with TeMV, EAPV, and PFSMoV; lane 2 is a sample infected only with TeMV; lane 3 is a sample infected only with EAPV; lane 4 is a sample infected only with PFSMoV; lane N is a negative control sample;

[0028] Figure 4 The sensitivity comparison results of multiplex PCR and single PCR in the present invention are shown; A is the detection result of TeMV single PCR; B is the detection result of EAPV single PCR; C is the detection result of PFSMoV single PCR; D is the detection result of three viruses multiplex PCR;

[0029] Figure 5 The figure shows the consistency comparison of the detection results of multiplex PCR and singleplex PCR in the present invention; lane M is a DNA ladder marker; lane N is a negative control; lanes XW1-XW7 are Xunwu field samples; lanes ZG1-ZG5 are field samples in Zhanggong District, Ganzhou City; lanes GX1-GX3 are field samples in Ganxian County; lane P is a positive control sample. DETAILED DESCRIPTION

[0030] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0031] Below are the samples and reagents used in the examples:

[0032] (1) Sample and virus sources

[0033] The positive passion fruit sample used to establish the detection method was simultaneously infected with night-blooming jasmine mosaic virus (TeMV), East Asian passion fruit virus (EAPV), and passion fruit severe mottle virus (PFSMoV). The plant sample is currently stored in the greenhouse of the Golden Campus of Gannan Normal University, Ganzhou City, Jiangxi Province.

[0034] The passion fruit plants suspected of being infected with three potato viruses Y of TeMV, EAPV, and PFSMoV at the same time or infected or suspected of being infected with one of the potato viruses Y used in the embodiment were collected from passion fruit orchards in Xunwu County, Gan County, and Zhanggong District, Ganzhou City, Jiangxi Province in 2021-2022. The samples were collected from about 10 leaves of suspected diseased plants, stored in a low-temperature sample collection box, and brought back to the laboratory for nucleic acid extraction.

[0035] (2) Main reagents

[0036] TransZol Up reagent was purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0037] Isopropyl alcohol and ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0038] 75% alcohol is prepared in the laboratory (DEPC water).

[0039] DEPC water (enzyme-free water) was purchased from Biosharp.

[0040] Random primers (6N) were ordered from Nanjing GenScript Biotech Co., Ltd.

[0041] dNTP Mixture (25 mM) was purchased from TaKaRa.

[0042] Recombinant RNase Inhabitar was purchased from TaKaRa.

[0043] RNase reverse transcriptase M-MLV was purchased from TaKaRa.

[0044] 2×Taq MasterMix was purchased from Novoprotein, catalog number: E005-01.

[0045] Agarose G-10 was purchased from BIOWEST, product number: 111860.

[0046] (3) Equipment

[0047] The ProFlexPCR instrument was purchased from Thermo Fisher Scientific.

[0048] DYY-6C electrophoresis apparatus was purchased from Beijing Liuyi Instrument Factory.

[0049] JS-2012 gel imager was purchased from Shanghai Peiqing Technology Co., Ltd.

[0050] Example 1

[0051] A multiplex PCR method for detecting three types of potato virus Y on passion fruit was constructed, comprising the following steps:

[0052] 1. Primer design and synthesis

[0053] The TeMV sequences MK340755, MK340754, and MG944249, the EAPV sequences NC_007728, KP114136, KT724930, and KP114137, and the PFSMoV sequence MK449340 were downloaded from GenBank. Three pairs of specific amplification primers were designed for TeMV, EAPV, and PFSMoV, respectively, targeting the coat protein (CP) gene regions of TeMV, EAPV, and PFSMoV. All primers were synthesized by Nanjing GenScript Biotechnology Co., Ltd. Primer sequence information is shown in Table 1.

[0054] Table 1. Primer sequence information used

[0055]

[0056]

[0057] The total RNA of the passion fruit plant samples containing the three viruses stored in the laboratory was extracted using the TransZolUp (Full Gold) reagent. The operation was carried out according to the instructions. The specific steps are as follows:

[0058] (1) Weigh 0.1 g of fresh leaf tissue from passion fruit plants and chop it into small pieces. Then, immediately place it in a mortar pre-cooled with liquid nitrogen and grind the tissue until it becomes powder. During this period, liquid nitrogen needs to be added to the mortar to keep it at a low temperature.

[0059] (2) Transfer the ground sample to a 1.5 mL enzyme-free EP tube. Add 1 mL TransZol UP and 0.2 mL RNA Extraction Agent to the EP tube and swirl repeatedly to mix thoroughly. EP tube: LABSELECT.

[0060] (3) Centrifuge at 4°C, 12,000 rpm for 15 min. The sample then separates into three layers: an upper colorless aqueous phase, a middle layer, and a lower pink organic phase. RNA is primarily contained in the aqueous phase.

[0061] (4) Transfer the upper colorless aqueous phase to a new 1.5 mL enzyme-free EP tube, add 0.5 mL of isopropanol, mix gently, and incubate at room temperature for 10 min.

[0062] (5) Centrifuge at 4°C, 12,000 rpm for 10 min, remove the supernatant, add 1 mL of pre-cooled 75% alcohol for washing, centrifuge at 4°C, 8,000 rpm for 5 min, remove the supernatant, and repeat this operation twice.

[0063] (6) Centrifuge briefly at 4°C for 10 seconds, aspirate excess alcohol with a 200 μL pipette tip, and air-dry the precipitate at room temperature for 5 minutes.

[0064] (7) The precipitate was dissolved in 75 μL RNA dissolution solution and incubated at 57°C for 10 min.

[0065] (8) The concentration and purity of total RNA were detected using Nanodrop 2000. 1 μL of extracted total RNA was added as a reaction template. The extracted total RNA sample was stored at -20°C for future use.

[0066] The total RNA sample was reverse transcribed, amplified by single-plex PCR, and detected by electrophoresis according to the following procedures. The specific steps are as follows:

[0067] The reverse transcription system was as follows: 1.0 μL of random primer 6N (10 μM), 4.0 μL of dNTP Mixture (25 mM), 2.0 μL of template RNA, and 3.0 μL of RNase-free double-distilled water, with a total reaction system of 10 μL.

[0068] The solution of the above reverse transcription system was denatured at 65°C for 5 min and then quickly cooled on ice. The reverse transcription reaction solution was added and reverse transcription was completed under the reaction conditions of 30°C for 10 min, 42°C for 50 min, and 75°C for 15 min to obtain cDNA. The cDNA obtained by the reaction was stored in a refrigerator at -20°C for later use.

[0069] The reverse transcription reaction solution system is: 5×Reverse Transcriptase M-MLV Buffer 4.0 μL, Recombinant RNase Inhabitar 0.5 μL, RNase nuclease M-MLV 1.0 μL, RNase Free double-distilled water 4.5 μL, making up a total of 20 μL reaction system.

[0070] The reaction system solution for PCR amplification is specifically composed of the following volumes of components: 2×TaqMasterMix: 10 μl Novoprotein, 1.5 p mol each of primers TeMVCP-F and TeMVCP-R, or 1.5 p mol each of primers EAPV-F and EAPV-R, or 1.5 p mol each of primers PFSMoV-F and PFSMoV-R, 1.0 μl of template cDNA, and double-distilled water is added to make up the system to 20 μl.

[0071] The reaction program for PCR amplification was as follows: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 20 s, annealing at 56°C for 20 s, extension at 72°C for 1 min, 35 cycles, and extension at 72°C for 5 min.

[0072] The PCR amplification products were electrophoresed in a 1.3% agarose gel at 100 V for 30 minutes, and the results were observed on a gel imager.

[0073] result Figure 1 shown.

[0074] Based on singleplex PCR amplification and electrophoresis results, we screened the following primer pairs for TeMV: TeMVCP135F / TeMVCP708R, TeMVCP135F / TeMVCP820R; for PFSMoV: PFVCP5F / PFVCP449R, PFVCP44F / PFVCP449R; and for EAPV: EAPVCP142F / EAPVCP377R, EAPVCP142F / EAPVCP390R. Based on these primer pairs, we then proceeded to multiplex PCR amplification. A total of eight primer crossover tests were conducted using these primer pairs. Specific primer combinations are shown in Table 2.

[0075] Table 2. Multiple primer combinations

[0076] G1 G2 G3 G4 G5 G6 G7 G8 TeMV 135F / 708R 135F / 820R 135F / 708R 135F / 820R 135F / 708R 135F / 820R 135F / 708R 135F / 820R PFSMoV 5F / 449R 5F / 449R 44F / 449R 44F / 449R 5F / 449R 5F / 449R 44F / 449R 44F / 449R EAPV 142F / 377R 142F / 377R 142F / 377R 142F / 377R 142F / 390R 142F / 390R 142F / 390R 142F / 390R

[0077] The specific steps are as follows:

[0078] Total RNA was extracted from laboratory-stored passion fruit plant samples containing the three viruses using TransZol Up (Full Gold) reagent, following the manufacturer's instructions. Multiplex PCR amplification was performed using the eight primer pairs listed in Table 2 to assess the amplification performance of the different primer sets. The specific multiplex RT-PCR procedure is as follows:

[0079] The total RNA obtained was reverse transcribed, amplified by multiplex PCR, and detected by electrophoresis according to the following procedures. The specific steps are as follows:

[0080] The reverse transcription system is as follows: 10 μM random primer 6N 1.0 μL, 25 mM dNTP Mixture 4.0 μL, template RNA 2.0 μL, RNase-free double-distilled water 3.0 μL, the total reaction system is 10 μL;

[0081] The solution of the above reverse transcription system was denatured at 65°C for 5 min and then quickly cooled on ice. The reverse transcription reaction solution was added and reverse transcription was completed under the reaction conditions of 30°C for 10 min, 42°C for 50 min, and 75°C for 15 min to obtain cDNA. The cDNA obtained by the reaction was stored in a refrigerator at -20°C for later use.

[0082] The reverse transcription reaction solution system is: 5×Reverse Transcriptase M-MLV Buffer 4.0 μL, Recombinant RNase Inhabitar 0.5 μL, RNase nuclease M-MLV 1.0 μL, RNase Free double-distilled water 4.5 μL, making up a total of 20 μL reaction system.

[0083] The reaction system solution for PCR amplification is specifically composed of the following components in the following volumes: 2×TaqMasterMix: 10 μl (Novoprotein), 3 p mol each of primers TeMVCP-F and TeMVCP-R, 1.5 p mol each of primers EAPV-F and EAPV-R, 1.5 p mol each of primers PFSMoV-F and PFSMoV-R, 1.0 μl of template cDNA, and double-distilled water is added to make the system up to 20 μl.

[0084] The reaction program for PCR amplification was as follows: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 20 s, annealing at 56°C for 20 s, extension at 72°C for 1 min, 35 cycles, and extension at 72°C for 5 min.

[0085] The PCR amplification products were electrophoresed in 1.3% agarose gel at 100V for 30 minutes and the results were observed on a gel imager. Figure 2 .

[0086] Figure 2 The results showed that primer pairs G3, G4, and G8 can amplify three viral target fragments with specificity, clarity, and good discrimination. Further amplification tests were conducted on the three primer pairs G3, G4, and G8 using multiple field samples. The methods and steps used were the same as those in the multiple primer set screening section. Figure 2 The results showed no nonspecific amplification with primer pair G8, with clear bands and good discrimination. Therefore, primer set G8 was selected for further optimization to establish a multiplex RT-PCR assay for three viruses. The resulting primer set G8 is specific for TeMV, EAPV, and PFSMoV. Its sequences are shown in Table 2.

[0087] Table 2 Specific primer sequences of TeMV, EAPV, and PFSMoV

[0088]

[0089] 2. Genome extraction

[0090] The total RNA of the passionflower samples to be tested was extracted using the TransZol Up reagent from Quanshijin Company. The operation was carried out according to the instructions. The specific steps are as follows:

[0091] (1) Weigh 0.1 g of fresh, clean passion fruit leaves, chop them, place them in a mortar pre-cooled with liquid nitrogen, and grind the tissue thoroughly with a pestle until it becomes powder. Liquid nitrogen can be added during this period.

[0092] (2) Transfer the ground sample into a 1.5 mL enzyme-free EP tube (LABSELECT). Add 1 mL TransZolUP and 0.2 mL RNA Extraction Agent for every 0.05-0.1 g of tissue and swirl repeatedly to mix thoroughly.

[0093] (3) Centrifuge at 12,000 rpm for 15 minutes at 4°C. The sample will then separate into three layers: a colorless aqueous phase (upper layer), a middle layer, and a pink organic phase (lower layer). The RNA is primarily in the aqueous phase.

[0094] (4) Transfer the colorless aqueous phase (supernatant) to a new 1.5 mL enzyme-free EP tube, add 0.5 mL of isopropanol, mix gently, and incubate at room temperature for 10 min.

[0095] (5) Centrifuge at 4°C, 12,000 rpm for 10 min, remove the supernatant, add 1 mL of pre-cooled 75% alcohol for washing, centrifuge at 4°C, 8,000 rpm for 5 min, remove the supernatant, and repeat this operation twice.

[0096] (6) Centrifuge briefly at 4°C for 10 seconds, aspirate excess alcohol with a 200 μL pipette tip, and air-dry the precipitate at room temperature for 5 minutes.

[0097] (7) Dissolve the precipitate in 50-100 μL RNA dissolution solution and incubate at 55°C-60°C for 10 min.

[0098] (8) The concentration and purity of total RNA were detected using Nanodrop 2000. 1 μL of extracted total RNA was added as a reaction template and the extracted total RNA was stored at -20°C for future use.

[0099] The cDNA obtained by reverse transcription of the total RNA extracted above was prepared using TaKaRa's reverse transcription reagent. The operation was carried out according to the instructions. The specific steps are as follows:

[0100] (1) Prepare the reaction system in a 10 μL PCR tube: 1.0 μL of random primer 6N, 4.0 μL of dNTP Mixture (25 mM), 2.0 μL of total RNA extracted above, and 3.0 μL of RNase-free double-distilled water to make up the 10 μL reaction system.

[0101] (2) Place the above reaction tube in the reaction chamber of the PCR instrument, denature at 65°C for 5 minutes, and then quickly cool on ice;

[0102] (3) Add the reverse transcription reaction solution to the above premix. The reverse transcription reaction solution system is: 5×ReverseTranscriptase M-MLV Buffer 4.0μL, Recombinant RNase Inhibitor 0.5μL, RNase Nuclease M-MLV 1.0μL, RNase Free double-distilled water 4.5μL to make up 10μL reaction system.

[0103] (4) The program was set as follows: 30°C, 10 min; 40°C, 50 min; 75°C, 15 min. The cDNA obtained from the reaction was stored in a -20°C refrigerator for later use.

[0104] 3. Preparation of multiplex RT-PCR reaction system:

[0105] (1) 2× TaqMaster Mix from Nearshore Protein was used for the reaction. 10.0 μL of 2× TaqMaster Mix solution and 6.6 μL of RNase-free ddH2O were added to the reaction solution in sequence.

[0106] (2) Primer solution: including 3 pmol primers TeMV-F, TeMV-R, 1.5 pmol primers EAPV-F, EAPV-R and PFSMoV-F, PFSMoV-R. The sequences of the six primers are shown in Table 1.

[0107] 4. Multiplex RT-PCR Amplification Procedure

[0108] Add the reaction mixtures of the prepared multiplex RT-PCR system to a 20 μL PCR tube. Place the tube on a PCR instrument and set the reaction program to: 35 cycles of pre-denaturation at 94°C for 3 minutes, denaturation at 94°C for 20 seconds, annealing at 56°C for 20 seconds, extension at 72°C for 1 minute, and an additional extension at 72°C for 5 minutes. After the reaction is complete, remove the tube and perform agarose gel electrophoresis.

[0109] 5. Result determination:

[0110] 5 μL of the reaction product was drawn, mixed evenly with 1 μL of 6×DNALoadingBuffer, and then spotted into a well of a prepared agarose gel with a concentration of 1.4%. After electrophoresis for 32 minutes at 130V, the electrophoresis results were observed under the UV band of a gel imager, and the results were determined by whether specific amplified fragments could be observed on the gel. The result determination method is based on the electrophoresis detection results, observing whether amplified bands appear at characteristic positions to determine whether there are three viruses. Specifically, the judgment method is: if there is a band at 685bp on the gel, it means that the passion fruit sample to be tested contains only TeMV; if there is only a band at 405bp on the gel, it means that the sample to be tested contains only PFSMoV; if there is only a band at 248bp on the gel, it means that the passion fruit sample to be tested contains only EAPV; if there is no band on the gel, it means that the passion fruit sample to be tested is a healthy plant. If there are two bands at the corresponding positions on the gel, it means that the passion fruit sample to be tested contains double viruses; if there are three bands at the corresponding positions on the gel, it means that the passion fruit sample to be tested contains triple viruses. Figure 3 .

[0111] It should be noted that negative and positive controls must be set up during the above reaction process. The negative control is used to detect whether the reaction process is contaminated, and the positive control is used to check the experimental operation process and whether there are problems with the reagents. It is also used to compare whether amplified bands appear at the size position of the target fragment when observing the results of gel electrophoresis.

[0112] Depend on Figure 3 It can be seen that the sizes of the target fragments amplified by the three virus primers are 685bp, 405bp and 248bp respectively. Observation of the electrophoresis test results shows that corresponding amplified bands appeared at the corresponding target fragment positions, regardless of whether the sample was infected with three viruses or infected with only one virus. The bands were specific and clear, and the different bands were clearly distinguished, indicating that the primers designed and screened in this study have a good discrimination effect for the three viruses.

[0113] The present invention conducts sensitivity and experimental analysis on a multiplex PCR detection method for three types of potato virus Y on passion fruit. The specific experimental process is as follows:

[0114] Sensitivity test:

[0115] The single-plex RT-PCR amplification products of primer pairs TEMVCP-F / R, PFSMoVCP-F / R, and EAPV-F / R were purified, cloned, and sequenced. The bacterial solution with the correct sequencing was selected for expansion and plasmid extraction. The plasmid concentration was determined using the formula: copy number = (mass concentration × 6.022 × 10 23 ) / (plasmid molecular weight × 10 -9 ) The concentration was converted to copy number. The actual copy number of the three plasmid sample standards was: 1.17×1011 , using this as a standard for a 10-fold gradient, and using the multiplex PCR detection method and singleplex PCR method established by the present invention for the three types of potato virus Y on passion fruit for amplification, electrophoresis detection, and comparison of the sensitivity of the two methods. The specific operation steps are the same as the singleplex and multiplex RT-PCR operation steps mentioned above in this study. Results Figure 4 shown.

[0116] Depend on Figure 4 The results showed that the multiplex PCR detection method for three potato virus Y on passion fruit established by the present invention could detect TeMV to 1.17×10 6 , EAPV and PFSMoV can be detected to 1.17×10 7 Compared with the single-plex PCR detection method, the detection sensitivity of TeMV and EAPV was lower than that of the single-plex PCR detection method. The reason why the multiplex RT-PCR method in this study was less sensitive than the single-plex RT-PCR method may be that there is competition between different primer pairs during the amplification process, resulting in its reduced sensitivity. Although the sensitivity is slightly lower, the established multiplex RT-PCR method can detect three viruses simultaneously compared to single-plex RT-PCR, which improves detection efficiency, saves detection time and costs, and has good application prospects.

[0117] In order to verify the multiplex PCR detection method for three types of potato virus Y on passion fruit established in the present invention, the following sample detection test was carried out:

[0118] Passionflower samples collected from Xunwu County, Yudu County, Zhanggong District, Gan County, and Ruijin City, Ganzhou City, Jiangxi Province, were tested using the multiplex PCR and singleplex PCR methods developed by the present invention for detecting three types of potato virus Y on passionflower. The results of the two methods were compared for consistency. The specific procedures were as follows:

[0119] Suspected diseased samples were collected from the field, RNA was extracted from the samples, and the multiplex PCR method and singleplex PCR method established in this study were used for detection, and the consistency of the detection results was compared. Figure 5 Among them, RNA extraction was performed using TransZol Up (Full Gold) reagent, and the operation was carried out according to the instructions.

[0120] Figure 5 The results showed that the multiplex PCR detection method for three types of potato virus Y on passion fruit established by the present invention was consistent with the single-plex PCR detection results, indicating the accuracy of the detection method.

[0121] The results indicate that the results of the multiplex PCR method for detecting three types of potato virus Y on passion fruit developed in this study are consistent with those of the single-plex PCR method, indicating that the multiplex PCR method developed in this study can be used to rapidly detect three types of potato virus Y in a large number of field samples.

[0122] The present invention provides a multiplex PCR detection method for three potato viruses Y on passion fruit. The detection method can simultaneously detect three potato viruses Y: Telosma mosaic virus (TeMV), East Asian passiflora virus (EAPV), and Passion fruit severe mottle virus (PFSMoV). The present invention designs specific amplification primers for the coat protein gene (CP) region of TeMV, the coat protein gene (CP) region of EAPV, and the coat protein gene (CP) region of PFSMoV, and establishes a multiplex RT-PCR detection method that can rapidly detect and identify these three viruses, providing important experimental technical support for the early detection and prevention and control of potato viruses Y in the future.

[0123] This invention references Chinese patent number ZL 202210166250.7, which discloses a multiplex RT-PCR method capable of simultaneously detecting four viruses: EAPV (East Asian passionflower virus), TeMV (Evening jasmine mosaic virus), PLV (Passionflower latent virus), and CiaRV (Citrus rhabdovirus). Furthermore, using the primers, reaction system, and detection method, we tested passionflower plants simultaneously infected with TeMV, EAPV, and PFSMoV. However, we only detected TeMV and EAPV, but not all three potato viruses Y: TeMV, EAPV, and PFSMoV.

[0124] The present invention refers to the Chinese document "Molecular Identification and Specific Detection of Passionflower-Infecting Tuberose Mosaic Virus." The RT-PCR method disclosed in the document can only detect one TeMV virus at a time. Furthermore, using the primers, reaction system, and detection method described in the document to test passionflower plants simultaneously infected with TeMV, EAPV, and PFSMoV, we detected only TeMV, but not all three potato viruses Y.

[0125] The present invention refers to the Chinese document "Full Genome Sequence Characteristics of East Asian Passionfruit Virus Infecting Passionfruit and TC-RT-PCR Detection Technology." The TC-RT-PCR method disclosed in the document can only detect one virus at a time. Simultaneously, using its primers, reaction system, and detection method, we tested passionfruit plants simultaneously infected with TeMV, EAPV, and PFSMoV. However, we only detected EAPV, but not all three potato viruses Y: TeMV, EAPV, and PFSMoV.

[0126] The present invention references Chinese patent number CN116769969A, which discloses a multiplex LAMP assay capable of simultaneously detecting three viruses: EAPV (East Asian passionflower virus), TeMV (Tuberose mosaic virus), and CMV (Cucumber mosaic virus). Furthermore, using the primers, reaction system, and assay, we tested passionflower plants simultaneously infected with TeMV, EAPV, and PFSMoV. However, we only detected TeMV and EAPV, but not all three viruses: potato virus Y.

[0127] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes preferred embodiments.

[0128] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0129] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A multiplex PCR detection method for three types of potato virus Y on passion fruit, characterized in that: The method described is a multiplex RT-PCR assay for the simultaneous detection of three potato viruses Y: TeMV, EAPV, and PFSMoV; The multiplex PCR detection method comprises the following steps: Genome extraction: extract total RNA from the sample to be tested, and then reverse transcribe to obtain cDNA; Multiplex RT-PCR amplification reaction: The obtained cDNA is used as a detection template and specific primers for TeMV, EAPV, and PFSMoV viruses are used to perform multiplex PCR amplification under the reaction system conditions; The specific primers include primers TeMV-F / R, EAPV-F / R and PFSMoV-F / R; the nucleotide sequence of the TeMVCP-F is shown in SEQ ID NO.1; the nucleotide sequence of the TeMVCP-R is shown in SEQ ID NO.2; the nucleotide sequence of the EAPV-F is shown in SEQ ID NO.3; the nucleotide sequence of the EAPV-R is shown in SEQ ID NO.4; the nucleotide sequence of the PFSMoV-F is shown in SEQ ID NO.5; the nucleotide sequence of the PFSMoV-R is shown in SEQ ID NO.6; Result determination.

2. The multiplex PCR detection method for three kinds of potato virus Y on passion fruit according to claim 1, wherein The sample to be tested is a passion fruit plant that is simultaneously infected or suspected of being simultaneously infected with TeMV, EAPV, and PFSMoV potato virus Y; or the sample to be tested is a passion fruit plant that is solely infected or suspected of being solely infected with one of the potato virus Y.

3. The multiplex PCR detection method for three kinds of potato virus Y on passion fruit according to claim 2, characterized in that, Fresh leaf tissue of the sample to be tested was minced and ground into powder under liquid nitrogen pre-cooling conditions. Total RNA of the sample was extracted using a total RNA extraction reagent and the extracted total RNA of the sample was stored at -20°C for later use.

4. The multiplex PCR detection method for three kinds of potato virus Y on passion fruit according to claim 1, wherein The reverse transcription system is as follows: 1.0 μL of random primer 6N 10 μM, 4.0 μL of dNTP Mixture 25 mM, 2.0 μL of template RNA, and 3.0 μL of RNase Free double-distilled water, with a total reaction system of 10 μL. The solution of the reverse transcription system was denatured at 65°C for 5 minutes and then quickly cooled on ice. The reverse transcription reaction solution was added and reverse transcription was completed at 30°C for 10 minutes, 42°C for 50 minutes, and 75°C for 15 minutes to obtain cDNA. The reverse transcription reaction solution system is: 5×Reverse Transcriptase M-MLV Buffer 4.0 μL, Recombinant RNase Inhabitar 0.5 μL, RNase nuclease M-MLV 1.0 μL, RNase Free double-distilled water 4.5 μL, making up a total of 20 μL reaction system.

5. The multiplex PCR detection method for three kinds of potato virus Y on passion fruit according to claim 1, wherein The amplification reaction system solution during the multiple PCR amplification reaction is specifically composed of the following volumes of components: 2×Taq Master Mix: 10 μl of nearshore protein, 3 p mol of primers TeMVCP-F and TeMVCP-R, 1.5 p mol of primers EAPV-F and EAPV-R, 1.5 p mol of primers PFSMoV-F and PFSMoV-R, 1.0 μl of template cDNA, and double distilled water is added to make up the system to 20 μl.

6. The multiplex PCR detection method for three kinds of potato virus Y on passion fruit according to claim 1, characterized in that, The reaction program for the multiplex PCR amplification was as follows: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 20 s, annealing at 56°C for 20 s, extension at 72°C for 1 min, 35 cycles, and further extension at 72°C for 5 min.

7. The multiplex PCR detection method for three kinds of potato virus Y on passion fruit according to claim 1, characterized in that, The result is determined by using the amplified product after PCR reaction.

8. The multiplex PCR detection method for three kinds of potato virus Y on passion fruit according to claim 7, characterized in that, After the PCR reaction is completed, the reaction tube is taken out and subjected to agarose gel electrophoresis. The result is determined based on whether specific amplified fragments can be observed on the gel.

9. The multiplex PCR detection method for three types of potato virus Y on passion fruit according to claim 8, characterized in that, The result determination method is as follows: if there is a band at 685 bp on the gel, it means that the sample to be tested contains only TeMV; if there is only a band at 405 bp on the gel, it means that the sample to be tested contains only PFSMoV; if there is only a band at 248 bp on the gel, it means that the sample to be tested contains only EAPV; If there are no bands on the gel, it means that the sample to be tested is a healthy plant; if there are two bands at the corresponding positions on the gel, it means that the sample to be tested contains double viruses; if there are three bands at the corresponding positions on the gel, it means that the sample to be tested contains triple viruses.

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