Primer combination, kit for simultaneously detecting multiple plant viruses infecting tomato and application thereof

Abstract

The application discloses a primer combination for simultaneously detecting multiple plant viruses invading tomatoes, a kit and application thereof, and belongs to the technical field of plant virus detection. In view of papaya virus E, a newly discovered RNA virus invading tomatoes in production, and tomato yellow leaf curl virus, a DNA virus also transmitted by whitefly, the application designs a kit for simultaneously detecting papaya virus E and tomato yellow leaf curl virus in whitefly, so that the whitefly virus-carrying condition can be detected, and early warning and early prevention and treatment of the two viruses can be realized. In view of a tomato sample, the application also designs a kit capable of simultaneously detecting papaya virus E, tomato yellow leaf curl virus, tomato spotted wilt virus and tomato brown rugose fruit virus in tomatoes, so that accurate and efficient detection can be performed on the four virus complex invasions and actual occurrence conditions of tomatoes.

Description

Technical Field

[0001] This invention relates to the field of plant virus detection technology, specifically to primer combinations, reagent kits, and their applications for the simultaneous detection of multiple plant viruses infecting tomatoes. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Viruses severely damage important economic crops such as tomatoes, often leading to reduced yields, decreased quality, or even total crop failure, causing huge economic losses to agricultural production.

[0004] In 2025, new tomato plants exhibiting virus-like symptoms were discovered in vegetable greenhouses in Dezhou City, Shandong Province. These plants showed obvious yellowing of the leaves, and on the surface of immature green fruits, dark green spots of varying sizes were visible; mature red fruits showed green and yellow spots, accompanied by discoloration. The incidence rate reached 10-60%, severely impacting tomato yield and quality. It was identified as being caused by a newly emerging papaya virus E (PpVE), for which effective detection and control methods are currently lacking.

[0005] Tomato yellow leaf curl virus (TYLCV) is an important geminitrovirus that threatens tomato production. It can systemically infect tomatoes and cause typical yellow leaf curl symptoms.

[0006] PpVE (RNA virus) and TYLCV (DNA virus) share the common vector, the whitefly, making them highly susceptible to co-infection. Accurately assessing the virus-carrying status of whitefly populations in the field is crucial for early warning of viral diseases, elucidation of transmission mechanisms, and formulation of control strategies. However, in practice, efficiently extracting and accurately detecting these two different nucleic acid types of viruses from single or trace vector samples faces technical bottlenecks such as low nucleic acid abundance and cumbersome extraction procedures. More importantly, traditional direct detection methods for DNA viruses like TYLCV cannot effectively distinguish between substantial infection and inactive DNA contamination on the body surface.

[0007] Tomato spotted wilt virus (TSWV), tomato brown wrinkled fruit virus (ToBRFV), and PpVE can all cause obvious fruit symptoms, but these symptoms are difficult to distinguish. Therefore, timely, accurate, and efficient detection of these four plant viruses is a crucial prerequisite for carrying out viral disease control, preventing the spread of viruses, and ensuring the safety of agricultural production.

[0008] Currently, detection methods for plant viruses mainly include immunoassays (such as ELISA and test strips) and molecular assays (such as PCR, RT-PCR, and qPCR). Among these, molecular assays have become the mainstream technology for plant virus detection due to their high sensitivity and specificity. However, the genomic structure, conserved region distribution, and variation characteristics of four viruses—PpVE, TYLCV, TSWV, and ToBRFV—differ significantly. TYLCV is a DNA virus with a small genome (approximately 2.7 kb), concentrated conserved regions, but prone to recombination mutations. TSWV is a segmented negative-sense RNA virus with a high mutation rate and limited conserved sequences. ToBRFV is a positive-sense single-stranded RNA virus, highly homologous to closely related viruses such as tobacco mosaic virus and tomato mosaic virus, making specific differentiation difficult. PpVE is a newly emerging plant virus, and related research is rarely reported. The aforementioned characteristics make primer design for the detection of the four viruses extremely difficult. Primers must simultaneously meet multiple requirements, including high specificity, no cross-reactivity, Tm value matching, no primer dimer formation, and no template competition. The screening of primers is a huge undertaking, and any genomic variation of any virus (especially variations in the primer binding region) may lead to deviations in the detection results. Summary of the Invention

[0009] To address the shortcomings of the existing technologies, the purpose of this invention is to provide primer combinations, kits, and applications for the simultaneous detection of multiple plant viruses infecting tomatoes.

[0010] Specifically, the present invention relates to the following technical solutions:

[0011] In a first aspect, the present invention provides primer pairs for the simultaneous detection of multiple plant viruses infecting tomatoes, including primer pair A for detecting papaya virus E and primer pair B for detecting tomato yellow leaf curl virus.

[0012] Primer pair A consists of the upstream primer shown in SEQ ID NO.1 and the downstream primer shown in SEQ ID NO.2; primer pair B consists of the upstream primer shown in SEQ ID NO.3 and the downstream primer shown in SEQ ID NO.4.

[0013] The papain virus E is an isolate of PpVE-Tomato-1. This virus strain was deposited on February 6, 2026, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing), with the accession number CGMCC No. 47097; and classified as: Papain Virus E. Betacytorhabdovirus papaya virus E.

[0014] The PpVE-Tomato-1 of this invention can infect tomatoes and cause the following new symptoms in tomatoes:

[0015] The leaves turn yellow, dark green spots appear on the surface of immature green fruits, and green and yellow spots appear on the surface of mature red fruits, accompanied by discoloration.

[0016] Furthermore, the primer pair also includes primer pair C for detecting tomato spotted wilt virus and primer pair D for detecting tomato brown wrinkled fruit virus;

[0017] Primer pair C consists of the upstream primer shown in SEQ ID NO.5 and the downstream primer shown in SEQ ID NO.6; primer pair D consists of the upstream primer shown in SEQ ID NO.7 and the downstream primer shown in SEQ ID NO.8.

[0018] Furthermore, the primer combination containing primer pair A and primer pair B also includes: a whitefly internal reference primer;

[0019] The whitefly internal reference primer consists of the upstream primer shown in SEQ ID NO.9 and the downstream primer shown in SEQ ID NO.10;

[0020] Furthermore, the primer combination comprising primer pair A, primer pair B, primer pair C and primer pair D also includes: tomato internal reference primer;

[0021] The tomato internal reference primer consists of the upstream primer shown in SEQ ID NO.11 and the downstream primer shown in SEQ ID NO.12.

[0022] A second aspect of the invention provides the application of the above primer combination in (1) or (2) as follows:

[0023] (1) Prepare a kit for simultaneous detection of papaya virus E and tomato yellow leaf curl virus in whiteflies;

[0024] (2) Prepare a kit for simultaneous detection of papain virus E, tomato yellow leaf curl virus, tomato wilt virus and tomato brown wrinkled fruit virus in tomatoes.

[0025] A third aspect of the present invention provides a kit for simultaneously detecting papaya virus E and tomato yellow leaf curl virus in whiteflies;

[0026] The kit contains: primer pair A for detecting papaya virus E, primer pair B for detecting tomato yellow leaf curl virus, and whitefly internal control primers.

[0027] Furthermore, the kit also contains: papaya virus E-positive plasmid and tomato yellow leaf curl virus positive plasmid;

[0028] The papaya virus E-positive plasmid is formed by ligating the PpVE target amplification fragment shown in SEQ ID NO.13 into the pCB301 vector; the tomato yellow leaf curl virus positive plasmid is formed by ligating the TYLCV target amplification fragment shown in SEQ ID NO.14 into the pCB301 vector.

[0029] In a fourth aspect, the present invention provides a kit for simultaneously detecting papain virus E, tomato yellow leaf curl virus, tomato wilt virus and tomato brown wrinkle virus in tomatoes.

[0030] The kit contains: primer pair A for detecting papaya virus E, primer pair B for detecting tomato yellow leaf curl virus, primer pair C for detecting tomato spotted wilt virus, primer pair D for detecting tomato brown wrinkled fruit virus, and tomato internal control primers.

[0031] Furthermore, the kit also contains: papaya virus E positive plasmid, tomato yellow leaf curl virus positive plasmid, tomato wilt virus positive plasmid and tomato brown wrinkled fruit virus positive plasmid.

[0032] The papaya virus E-positive plasmid is formed by ligating the PpVE target amplification fragment shown in SEQ ID NO. 13 into the pCB301 vector; the tomato yellow leaf curl virus-positive plasmid is formed by ligating the TYLCV target amplification fragment shown in SEQ ID NO. 14 into the pCB301 vector; the tomato spotted wilt virus-positive plasmid is formed by ligating the TSWV target amplification fragment shown in SEQ ID NO. 15 into the pCB301 vector; and the tomato brown wrinkled fruit virus-positive plasmid is formed by ligating the ToBRFV target amplification fragment shown in SEQ ID NO. 16 into the pCB301 vector.

[0033] The beneficial effects of this invention are:

[0034] (1) In response to the newly discovered papaya virus E and tomato yellow leaf curl virus, which are plant viruses transmitted by whiteflies, this invention designs a kit for simultaneously detecting papaya virus E and tomato yellow leaf curl virus in whiteflies. This kit can detect the virus-carrying status of whiteflies and formulate corresponding prevention and control measures to achieve early warning and early prevention and control of these two viruses.

[0035] (2) For tomato samples, the present invention also designed a kit that can simultaneously detect papaya virus E, tomato yellow leaf curl virus, tomato wilt virus and tomato brown wrinkled fruit virus in tomatoes, which can accurately and effectively detect the actual disease situation of tomatoes.

[0036] (3) This invention innovatively establishes a single RNA extraction and simultaneous detection system. By targeting and detecting the transcripts (mRNA) of DNA viruses such as TYLCV, false positives caused by environmental or surface inactive DNA contamination are fundamentally eliminated. This strategy not only confirms the activity and infection status of the virus, but also improves the detection throughput and accuracy of trace samples (such as single whiteflies and tomato tissue). Attached Figure Description

[0037] Figure 1 Symptoms appearing on tomato leaves and fruits in the field; in the image, AD shows yellowing of leaves; EG shows dark green spots on the surface of green fruits accompanied by yellowing; HJ shows green and yellow patches on red fruits accompanied by yellowing.

[0038] Figure 2 Schematic diagram of the genome structure of PpVE-Tomato-1.

[0039] Figure 3 Results of the specificity study of primer pair A for detecting papain virus E.

[0040] Figure 4 The results of RT-PCR specificity investigation of two tomato viruses in whiteflies using the kit of Example 3 were shown in the figure. 1. Whitefly carrying PpVE alone; 2. Whitefly carrying TYLCV alone; 3. Whitefly carrying both PpVE and TYLCV.

[0041] Figure 5 The results of PCR sensitivity assessment using the kit in Example 3 to simultaneously detect plasmids containing two tomato virus fragments were shown in the figure. In the figure, 1 represents 200 pg of each positive plasmid; 2 represents 20 pg of each positive plasmid; 3 represents 2 pg of each positive plasmid; 4 represents 0.2 pg of each positive plasmid; 5 represents 0.02 pg of each positive plasmid; 6 represents 0.002 pg of each positive plasmid; 7 represents 0.0002 pg of each positive plasmid; and 8 represents 0.00002 pg of each positive plasmid.

[0042] Figure 6 The results of RT-PCR specificity investigation of four tomato pathogens in tomatoes were simultaneously detected using the kit in Example 4. In the figure, 1 is tomato cDNA infected with PpVE; 2 is tomato cDNA infected with TYLCV; 3 is tomato cDNA infected with TSWV; 4 is tomato cDNA infected with ToBRFV; and 5 is tomato cDNA of a mixture of PpVE, TYLCV, TSWV and ToBRFV.

[0043] Figure 7The results of PCR sensitivity assessment for simultaneous detection of four tomato pathogens in tomatoes using the kit in Example 4 are shown in the figure. In the figure, 1 represents 200 pg of each positive plasmid; 2 represents 20 pg of each positive plasmid; 3 represents 2 pg of each positive plasmid; 4 represents 0.2 pg of each positive plasmid; 5 represents 0.02 pg of each positive plasmid; 6 represents 0.002 pg of each positive plasmid; 7 represents 0.0002 pg of each positive plasmid; and 8 represents 0.00002 pg of each positive plasmid.

[0044] Figure 8 The PCR bands were quantified using ImageJ software. In the figure, A represents the quantification result of PpVE amplification product, where F1 / R1, F2 / R2, F3 / R3, and F4 / R4 correspond to the four primer pairs in Table 1, and F / R represents primer pair A used to detect PpVE in Example 1. B represents the quantification result of TYLCV amplification product, where F1 / R1 and F2 / R2 correspond to the two primer pairs in Table 2, and F / R represents primer pair B used to detect TYLCV in Example 1. Detailed Implementation

[0045] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0046] To enable those skilled in the art to more clearly understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments. If specific experimental conditions are not specified in the embodiments, they are generally based on conventional conditions or conditions recommended by the reagent company; the reagents, consumables, etc. used in the following embodiments, unless otherwise specified, can be obtained commercially. Wherein:

[0047] The NCBI designation for Tomato Yellow Leaf Curl Virus (TYLCV) is MN432609.1, for Tomato Spotted Wilt Virus (TSWV) it is MK986670.1, and for Tomato Brown Fruit Curl Virus (ToBRFV) it is MT018320.1.

[0048] Papaya virus E (PpVE) originated in a greenhouse in Dezhou City, Shandong Province. Figure 1 It was isolated from tomato plants exhibiting the symptoms shown. The genome structure is as follows: Figure 2As shown, it contains seven open reading frames (ORFs). The nucleotide sequence of the 5' untranslated region (UTR) is shown in SEQ ID NO.17, with a length of 152 nucleotides; the nucleotide sequence of ORF1 (positions 153-1508) is shown in SEQ ID NO.18, encoding a 51 kDalton nucleoprotein (N); the spacer sequence between ORF1 and ORF2 is shown in SEQ ID NO.19; the nucleotide sequence of ORF2 (positions 1700-3019) is shown in SEQ ID NO.20, encoding a 48.4 kDalton phosphoprotein (P); the spacer sequence between ORF2 and ORF3 is shown in SEQ ID NO.21; the nucleotide sequence of ORF3 (positions 3182-3751) is shown in SEQ ID NO.22, encoding a 21.1 kDalton putative protein (P3); the spacer sequence between ORF3 and ORF4 is shown in SEQ ID NO.23; the nucleotide sequence of ORF4 (positions 3768-4007) is shown in SEQ ID NO.18. As shown in SEQ ID NO. 24, a putative protein of 9.3 kilodaltons (P4) is encoded; the spacer sequence between ORF4 and ORF5 is shown in SEQ ID NO. 25; the nucleotide sequence of ORF5 (positions 4295-4939) is shown in SEQ ID NO. 26, encoding a matrix protein of 24.5 kilodaltons (M); the spacer sequence between ORF5 and ORF6 is shown in SEQ ID NO. 27; the nucleotide sequence of ORF6 (positions 5158-6717) is shown in SEQ ID NO. 28, encoding a glycoprotein of 58 kilodaltons (G); the spacer sequence between ORF6 and ORF7 is shown in SEQ ID NO. 29; the nucleotide sequence of ORF7 (positions 6975-13316) is shown in SEQ ID NO. 30, encoding a large RNA polymerase of 241.2 kilodaltons (L); the nucleotide sequence of the 3' untranslated region (UTR) is shown in SEQ ID NO. 31, with a length of 158 nucleotides.

[0049] The sequences shown in SEQ ID NO.17-SEQ ID NO.31 together constitute the whole genome sequence of PpVE-Tomato-1.

[0050] Example 1: Primer combination for simultaneous detection of papaya virus E and tomato yellow leaf curl virus in whiteflies

[0051] Based on the specific sequences of papain virus E and tomato yellow leaf curl virus, primer sequences for amplifying papain virus E and tomato yellow leaf curl virus, respectively, were designed. During primer design, primer pairs for amplifying papain virus E and tomato yellow leaf curl virus were prevented from forming primer dimers in the same reaction system; furthermore, the two viruses were designed to produce amplified fragments of different sizes to ensure clear band separation and easy interpretation during electrophoresis.

[0052] After repeated optimization, the final primer combination is as follows:

[0053] Primer pair A for detecting papain virus E:

[0054] PpVE-F: 5′-ATTCTTGCAGTCCTCCCTTTCG-3′; (SEQ ID NO.1)

[0055] PpVE-R: 5′-CATAATCTTAGCCATTCTTTTCCCGA-3′. (SEQ ID NO.2)

[0056] Primer pair B for detecting tomato yellow leaf curl virus:

[0057] TYLCV-F: 5′-ATGCCTCTAATCCAGTGTATGC-3′; (SEQ ID NO.3)

[0058] TYLCV-R: 5′-ACAAATGTTTTCTCAACTTCCGA-3′. (SEQ ID NO.4)

[0059] Example 2: Primer combination for simultaneous detection of papain virus E, tomato yellow leaf curl virus, tomato wilt virus and tomato brown fruit wrinkle virus in tomatoes.

[0060] Based on the specific sequences of papaya virus E, tomato yellow leaf curl virus, tomato spotted wilt virus, and tomato brown wrinkled fruit virus, primer sequences for amplifying each antigen were designed.

[0061] Primer pair A, used to detect papain virus E:

[0062] PpVE-F: 5′-ATTCTTGCAGTCCTCCCTTTCG-3′; (SEQ ID NO.1)

[0063] PpVE-R: 5′-CATAATCTTAGCCATTCTTTTCCCGA-3′. (SEQ ID NO.2)

[0064] Primer pair B for detecting tomato yellow leaf curl virus:

[0065] TYLCV-F: 5′-ATGCCTCTAATCCAGTGTATGC-3′; (SEQ ID NO.3)

[0066] TYLCV-R: 5′-ACAAATGTTTTCTCAACTTCCGA-3′. (SEQ ID NO.4)

[0067] Primer pair C for detecting tomato spotted wilt virus:

[0068] TSWV-F: 5′-CTGCACAATCCCAAGACA-3′; (SEQ ID NO.5)

[0069] TSWV-R: 5′-GCTAAGAGATTGAGRAATGGTATA-3′. (SEQ ID NO.6)

[0070] Primer pair D used for detecting tomato brown wrinkled fruit virus:

[0071] ToBRFV-F: 5′-CTTCCAAACGTGTACGCAC-3′; (SEQ ID NO.7)

[0072] ToBRFV-R: 5′-GTCGAGAGATATGTCGAATAGA-3′. (SEQ ID NO.8)

[0073] Example 3: Kit for simultaneous detection of papaya virus E and tomato yellow leaf curl virus in whiteflies

[0074] The kit provided in this embodiment includes the following substances:

[0075] (1) Primer combination for simultaneous detection of papaya virus E and tomato yellow leaf curl virus in whiteflies in Example 1;

[0076] (2) Whitefly internal control primer:

[0077] RPL29-F: 5′-TCGGAAAATTACCGTGAG-3′; (SEQ ID NO.9)

[0078] RPL29-R: 5′-GAACTTGTGATCTACTCCTCTCGT-3′. (SEQ ID NO.10)

[0079] The amplification length of the whitefly internal reference primer was 144 bp.

[0080] (3) Papaya virus E-positive plasmid and tomato yellow leaf curl virus positive plasmid:

[0081] The papaya virus E-positive plasmid is formed by ligating the PpVE target amplification fragment shown in SEQ ID NO.13 into the pCB301 vector; the tomato yellow leaf curl virus positive plasmid is formed by ligating the TYLCV target amplification fragment shown in SEQ ID NO.14 into the pCB301 vector.

[0082] (4) RT-PCR reaction reagents:

[0083] Random primers, HiScript II Q RT SuperMix for qPCR (+gDNAwiper), 2× rTaq Master Mix (Novizan, Nanjing), and RNase-free water.

[0084] Example 4: A kit for simultaneous detection of papain virus E, tomato yellow leaf curl virus, tomato wilt virus and tomato brown wrinkled fruit virus in tomatoes.

[0085] The kit provided in this embodiment includes the following substances:

[0086] (1) Primer combination for simultaneous detection of papain virus E, tomato yellow leaf curl virus, tomato wilt virus and tomato brown wrinkled fruit virus in tomatoes in Example 2;

[0087] (2) Tomato internal control primer:

[0088] UBI-F: 5′-CGTGGTGGTGCTAAGAAG-3′; (SEQ ID NO.11)

[0089] UBI-R: 5′-TAGGTGAGCCCACACTTACC-3′. (SEQ ID NO.12)

[0090] The amplification length of the tomato internal reference primer was 224 bp.

[0091] (3) Papaya virus E positive plasmid, tomato yellow leaf curl virus positive plasmid, tomato spotted wilt virus positive plasmid and tomato brown wrinkled fruit virus positive plasmid;

[0092] The papaya virus E-positive plasmid is formed by ligating the PpVE target amplification fragment shown in SEQ ID NO. 13 into the pCB301 vector; the tomato yellow leaf curl virus-positive plasmid is formed by ligating the TYLCV target amplification fragment shown in SEQ ID NO. 14 into the pCB301 vector; the tomato spotted wilt virus-positive plasmid is formed by ligating the TSWV target amplification fragment shown in SEQ ID NO. 15 into the pCB301 vector; and the tomato brown wrinkled fruit virus-positive plasmid is formed by ligating the ToBRFV target amplification fragment shown in SEQ ID NO. 16 into the pCB301 vector.

[0093] (4) RT-PCR reaction reagents:

[0094] Random primers, HiScript II Q RT SuperMix for qPCR (+gDNAwiper), 2× rTaq Master Mix (Novizan, Nanjing), and RNase-free water.

[0095] Example 5: Method for simultaneous detection of two tomato pathogens in whiteflies using the kit from Example 3

[0096] 1. RNA extraction:

[0097] Take 1-3 whiteflies and put them into a sampling tube to break them up. Add 500 μL of Trizol and lyse them thoroughly. Add chloroform to separate the layers. Take the upper aqueous phase and add an equal volume of isopropanol to precipitate RNA. Wash with 75% ethanol, dry, and dissolve in RNase-free water.

[0098] 2. Reverse transcription:

[0099] Reverse transcription was performed using random primers and the HiScript II Q RT SuperMix for qPCR (+gDNAwiper) reverse transcription kit. The reaction program was 50℃ for 15 min, then 85℃ for 5 s. cDNA template was obtained.

[0100] 3. Composition of the PCR system (20 μL):

[0101] The system contains: 10 μL 2× rTaq Master Mix (Novazia, Nanjing), 0.5 μL each of PpVE / TYLCV specific primer pair and RPL29 internal control primer pair (F / R, 10 μM), 2 μL cDNA template, and RNase-free water to a final volume of 20 μL.

[0102] 4. PCR amplification program:

[0103] Pre-denaturation at 94℃ for 5 min; 94℃ for 30 s → 55℃ for 30 s → 72℃ for 1 min, for a total of 35 cycles; final extension at 72℃ for 10 min.

[0104] 5. Result determination:

[0105] PCR products were separated and observed by electrophoresis on a 2% agarose gel and staining with ethidium. Excluding the internal control, a 401 bp band in the amplified product indicated that the whitefly carried only PpVE; a 282 bp band indicated that the whitefly carried only TYLCV; and the presence of both 401 bp and 282 bp bands in the amplified product indicated that the whitefly carried both PpVE and TYLCV.

[0106] Example 6: Method for simultaneous detection of four tomato pathogens in tomatoes using the kit from Example 4

[0107] 1. RNA extraction:

[0108] Take 0.2 g of tomato leaves and put them into a sample tube to break them up. Add 500 μL of Trizol and lyse them thoroughly. Add chloroform to separate the layers. Take the upper aqueous phase and add an equal volume of isopropanol to precipitate RNA. Wash with 75% ethanol, dry and dissolve in RNase-free water.

[0109] 2. Reverse transcription:

[0110] Reverse transcription was performed using random primers and the HiScript II Q RT SuperMix for qPCR (+gDNAwiper) reverse transcription kit. The reaction program was 50℃ for 15 min, then 85℃ for 5 s. cDNA template was obtained.

[0111] 3. PCR system composition (20 μL):

[0112] The system contains: 10 μL 2×rTaq Master Mix (Novazia, Nanjing), 0.5 μL each of the PpVE / TYLCV / TSWV / ToBRFV specific primer pair and the UBI internal control primer pair (F / R, 10 μM), 2 μL cDNA template, and RNase-free water to a final volume of 20 μL.

[0113] 4. PCR amplification program:

[0114] Pre-denaturation at 94℃ for 5 min; 94℃ for 30 s → 55℃ for 30 s → 72℃ for 1 min, for a total of 35 cycles; final extension at 72℃ for 10 min.

[0115] 5. Result determination:

[0116] PCR products were separated and observed by electrophoresis on a 2% agarose gel and staining with ethidium. Excluding the internal control, a 401 bp band indicated PpVE infection in tomatoes; a 282 bp band indicated TYLCV infection; an 841 bp band indicated TSWV infection; and a 604 bp band indicated ToBRFV infection. The presence of two or more bands indicated multiple virus co-infection in tomatoes, and the specific viruses were determined based on the band size.

[0117] Example 7: Specificity and sensitivity of the kit from Example 3 for simultaneous detection of two tomato pathogens in whiteflies.

[0118] 1. Specificity assessment:

[0119] First, the specificity of primer pair A for detecting papain virus E was evaluated as follows:

[0120] RNA was extracted from viral samples using Trizol, and reverse transcriptase was used to reverse transcribe the RNA into cDNA. PCR was then performed using PpVE-specific primers for specific detection. Virus samples included: papaya virus E (PpVE), tobacco mosaic virus (TMV), tomato mosaic virus (ToMV), tomato mottle mosaic virus (ToMMV), tomato brownrugose fruit virus (ToBRFV), cucumber mosaic virus (CMV), pepper mild mottle virus (PMMoV), potato virus X (PVX), potato virus Y (PVY), tomato chlorosis virus (ToCV), tomato spotted wilt virus (TSWV), and tomato yellow leafcurl virus (TYLCV).

[0121] The results are as follows Figure 3 As shown, the results indicate that primer pair A can only specifically detect PpVE and will not produce interfering amplification in the presence of viruses such as TMV, ToMV, and ToMMV.

[0122] Then, using the kit from Example 3, the whiteflies carrying PpVE alone, the whiteflies carrying TYLCV alone, and the whiteflies carrying both PpVE and TYLCV were detected according to the method in Example 5.

[0123] The results are as follows Figure 4 As shown, the results indicate that the detection kit of the present invention can specifically detect two plant viruses, PpVE and TYLCV, in whiteflies.

[0124] 2. Sensitivity test:

[0125] The positive plasmids of papaya virus E and tomato yellow leaf curl virus were quantified and prepared in 10-fold dilution series, ranging from 200 pg to 0.00002 pg.

[0126] Then, using the kit from Example 3, the mixed papaya virus E-positive plasmid and tomato yellow leaf curl virus positive plasmid were detected according to the method in Example 5 to examine their sensitivity.

[0127] The results are as follows Figure 5 As shown, the results indicate that the kit in Example 3 has a detection sensitivity of 0.002 pg (1×10⁻⁶) for papaya virus E and tomato yellow leaf curl virus. 2 (copies / μL).

[0128] Example 8: Specificity and sensitivity of simultaneous detection of four tomato pathogens in tomatoes using the kit from Example 4.

[0129] 1. Specificity assessment:

[0130] Then, using the kit from Example 4, the methods from Example 6 were used to detect tomatoes infected with PpVE, TYLCV, TSWV, ToBRFV, and co-infected with PpVE, TYLCV, TSWV, and ToBRFV.

[0131] The results are as follows Figure 6 As shown, the results indicate that the detection kit of the present invention can specifically detect four plant viruses in tomatoes: PpVE, TYLCV, TSWV, and ToBRFV.

[0132] 2. Sensitivity test:

[0133] The positive plasmids of papaya virus E, tomato yellow leaf curl virus, tomato spotted wilt virus, and tomato brown wrinkled fruit virus were quantified and prepared into a 10-fold dilution series, ranging from 200 pg to 0.00002 pg.

[0134] Then, using the kit from Example 4, the mixed papaya virus E-positive plasmid, tomato yellow leaf curl virus positive plasmid, tomato spotted wilt virus positive plasmid, and tomato brown wrinkled fruit virus positive plasmid were detected according to the method of Example 6, and their sensitivity was examined.

[0135] The results are as follows Figure 7 As shown, the results indicate that the kit in Example 4 has a detection sensitivity of 0.002 pg (1 × 10⁻⁶) for PpVE and TYLCV. 2 The detection sensitivity for TSWV and ToBRFV was 0.02 pg (1×10⁻⁶ copies / μL). 3 (copies / μL).

[0136] Comparative example:

[0137] For different conserved regions of the PpVE and TYLCV genome sequences, primer pairs were designed as shown in Tables 1 and 2 below:

[0138] Table 1: Primer pairs for PpVE

[0139]

[0140] Table 2: Primer pairs for TYLCV

[0141]

[0142] The primer pairs for detecting PpVE in Table 1 and the primer pairs for detecting TYLCV in Table 2 were combined and paired to obtain primer combinations for simultaneous detection of PpVE and TYLCV. The detection sensitivity of PpVE and TYLCV was investigated according to the method in Example 7.

[0143] The results showed that the detection sensitivity of the primer combinations in Tables 1 and 2 for PpVE and TYLCV was only 1000 copies / μL (or higher concentration); the detection sensitivity was significantly lower than that of the primer combination in Example 1 of this invention (the limit of detection was 100 copies / μL).

[0144] respectively with 1×10 7 Using copies / μL of PpVE and TYLCV plasmids as templates, the amplification effects of the primer pairs in Example 1, Table 1, and Table 2 were verified. PCR bands were quantified using ImageJ software.

[0145] The results are as follows Figure 8 As shown, the results indicate that, compared to the primer pairs in Tables 1 and 2, primer pair A (F / R) for detecting papaya virus E and primer pair B (F / R) for detecting tomato yellow leaf curl virus in Example 1 have the best amplification effects.

[0146] The above description is merely a preferred embodiment of this application and is not intended to limit this application. For those skilled in the art, any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A primer combination for simultaneous detection of multiple plant viruses infecting tomatoes, characterized in that, include: Primer pair A for detecting papaya virus E and primer pair B for detecting tomato yellow leaf curl virus; Primer pair A consists of the upstream primer shown in SEQ ID NO.1 and the downstream primer shown in SEQ ID NO.2; primer pair B consists of the upstream primer shown in SEQ ID NO.3 and the downstream primer shown in SEQ ID NO.

4. The preservation number of the papaya virus E is: CGMCC No. 47097.

2. The primer combination according to claim 1, characterized in that, The primer pair also includes primer pair C for detecting tomato spotted wilt virus and primer pair D for detecting tomato brown wrinkled fruit virus; Primer pair C consists of the upstream primer shown in SEQ ID NO.5 and the downstream primer shown in SEQ ID NO.6; primer pair D consists of the upstream primer shown in SEQ ID NO.7 and the downstream primer shown in SEQ ID NO.

8.

3. The primer combination according to claim 1, characterized in that, The primer set also includes: a whitefly internal control primer; The whitefly internal reference primer consists of the upstream primer shown in SEQ ID NO.9 and the downstream primer shown in SEQ ID NO.

10.

4. The primer combination according to claim 2, characterized in that, The primer combination also includes: tomato internal control primer; The tomato internal reference primer consists of the upstream primer shown in SEQ ID NO.11 and the downstream primer shown in SEQ ID NO.

12.

5. The use of the primer combination according to claim 1 or 3 in the preparation of a kit for simultaneous detection of papaya virus E and tomato yellow leaf curl virus in whiteflies, characterized in that, The preservation number of the papain virus E is: CGMCC No. 47097.

6. The application of the primer combination according to claim 2 or 4 in the preparation of a kit for simultaneous detection of papain virus E, tomato yellow leaf curl virus, tomato wilt virus, and tomato brown wrinkled fruit virus in tomatoes, characterized in that, The preservation number of the papaya virus E is: CGMCC No. 47097.

7. A reagent kit, characterized in that, The kit is used to simultaneously detect papaya virus E and tomato yellow leaf curl virus in whiteflies; The kit contains: primer pair A for detecting papaya virus E, primer pair B for detecting tomato yellow leaf curl virus, and whitefly internal control primers; Primer pair A consists of the upstream primer shown in SEQ ID NO.1 and the downstream primer shown in SEQ ID NO.2; primer pair B consists of the upstream primer shown in SEQ ID NO.3 and the downstream primer shown in SEQ ID NO.

4. The whitefly internal reference primer consists of the upstream primer shown in SEQ ID NO.9 and the downstream primer shown in SEQ ID NO.10; The preservation number of the papaya virus E is: CGMCC No. 47097.

8. The reagent kit according to claim 7, characterized in that, The kit also contains: papaya virus E positive plasmid and tomato yellow leaf curl virus positive plasmid; The papaya virus E-positive plasmid is formed by ligating the PpVE target amplification fragment shown in SEQ ID NO.13 into the pCB301 vector; the tomato yellow leaf curl virus positive plasmid is formed by ligating the TYLCV target amplification fragment shown in SEQ ID NO.14 into the pCB301 vector.

9. A reagent kit, characterized in that, The kit is used to simultaneously detect papain virus E, tomato yellow leaf curl virus, tomato wilt virus and tomato brown wrinkle virus in tomatoes. The kit contains: primer pair A for detecting papaya virus E, primer pair B for detecting tomato yellow leaf curl virus, primer pair C for detecting tomato spotted wilt virus, primer pair D for detecting tomato brown wrinkled fruit virus, and tomato internal control primers. Primer pair A consists of the upstream primer shown in SEQ ID NO.1 and the downstream primer shown in SEQ ID NO.2; primer pair B consists of the upstream primer shown in SEQ ID NO.3 and the downstream primer shown in SEQ ID NO.4; primer pair C consists of the upstream primer shown in SEQ ID NO.5 and the downstream primer shown in SEQ ID NO.6; primer pair D consists of the upstream primer shown in SEQ ID NO.7 and the downstream primer shown in SEQ ID NO.

8. The tomato internal reference primer consists of the upstream primer shown in SEQ ID NO.11 and the downstream primer shown in SEQ ID NO.12; The preservation number of the papaya virus E is: CGMCC No. 47097.

10. The reagent kit according to claim 9, characterized in that, The kit also contains: papaya virus E positive plasmid, tomato yellow leaf curl virus positive plasmid, tomato wilt virus positive plasmid and tomato brown wrinkled fruit virus positive plasmid. The papaya virus E-positive plasmid is formed by ligating the PpVE target amplification fragment shown in SEQ ID NO. 13 into the pCB301 vector; the tomato yellow leaf curl virus-positive plasmid is formed by ligating the TYLCV target amplification fragment shown in SEQ ID NO. 14 into the pCB301 vector; the tomato spotted wilt virus-positive plasmid is formed by ligating the TSWV target amplification fragment shown in SEQ ID NO. 15 into the pCB301 vector; and the tomato brown wrinkled fruit virus-positive plasmid is formed by ligating the ToBRFV target amplification fragment shown in SEQ ID NO. 16 into the pCB301 vector.

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