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CN-122012818-A - Primer combination and kit for simultaneously detecting various plant viruses infecting tomatoes and application of primer combination and kit

CN122012818ACN 122012818 ACN122012818 ACN 122012818ACN-122012818-A

Abstract

The invention discloses a primer combination and a kit for simultaneously detecting a plurality of tomato-infected plant viruses and application thereof, and belongs to the technical field of plant virus detection. Aiming at the newly discovered RNA virus papaya virus E infecting tomatoes and DNA virus tomato yellow leaf curl virus transmitted by bemisia tabaci in production, the invention designs a kit for simultaneously detecting the papaya virus E and the tomato yellow leaf curl virus in the bemisia tabaci, and can detect the virus carrying condition of the bemisia tabaci and realize early warning and early prevention and treatment of the two viruses. Aiming at tomato samples, the invention also designs a kit capable of simultaneously detecting papaya virus E, tomato yellow leaf curl virus, tomato spotted wilt virus and tomato brown wrinkle fruit virus in tomatoes, and can accurately and efficiently detect the compound infection and actual occurrence of four tomato viruses.

Inventors

  • YAN ZHIYONG
  • LIU SHUO
  • LI XIANGDONG
  • Mou Xiuqi
  • Yue Yiheng
  • ZHANG YANLI
  • TIAN YANPING
  • Zang Bonan
  • WANG JIALE
  • WU YANG

Assignees

  • 山东农业大学

Dates

Publication Date
20260512
Application Date
20260403

Claims (10)

  1. 1. The primer combination for simultaneously detecting a plurality of plant viruses infecting tomatoes is characterized by comprising a primer pair A for detecting papaya virus E and a primer pair B for detecting tomato yellow leaf curl virus; The primer pair A consists of an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.2, and the primer pair B consists of an upstream primer shown in SEQ ID NO.3 and a downstream primer shown in SEQ ID NO. 4; The preservation number of the papaya virus E is CGMCC No. 47097.
  2. 2. The primer combination according to claim 1, wherein the primer combination further comprises a primer pair C for detecting tomato spotted wilt virus and a primer pair D for detecting tomato brown wrinkle fruit virus; The primer pair C consists of an upstream primer shown as SEQ ID NO.5 and a downstream primer shown as SEQ ID NO.6, and the primer pair D consists of an upstream primer shown as SEQ ID NO.7 and a downstream primer shown as SEQ ID NO. 8.
  3. 3. The primer combination of claim 1, wherein the primer combination further comprises a bemisia tabaci internal reference primer; The bemisia tabaci internal reference primer consists of an upstream primer shown in SEQ ID NO.9 and a downstream primer shown in SEQ ID NO. 10.
  4. 4. The primer combination of claim 2, wherein the primer combination further comprises a tomato inner reference primer; The tomato inner reference primer consists of an upstream primer shown as SEQ ID NO.11 and a downstream primer shown as SEQ ID NO. 12.
  5. 5. Use of a primer combination according to claim 1 or 3 for the preparation of a kit for simultaneous detection of papaya virus E and tomato yellow leaf curl virus in bemisia tabaci.
  6. 6. Use of a primer combination according to claim 2 or 4 for the preparation of a kit for simultaneous detection of papaya virus E, tomato yellow leaf curl virus, tomato spotted wilt virus and tomato brown wrinkle virus in tomatoes.
  7. 7. A kit for simultaneous detection of papaya virus E and tomato yellow leaf curl virus in bemisia tabaci; the kit comprises a primer pair A for detecting papaya virus E, a primer pair B for detecting tomato yellow leaf curl virus and a bemisia tabaci internal reference primer; The primer pair A consists of an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.2, and the primer pair B consists of an upstream primer shown in SEQ ID NO.3 and a downstream primer shown in SEQ ID NO. 4; The bemisia tabaci internal reference primer consists of an upstream primer shown in SEQ ID NO.9 and a downstream primer shown in SEQ ID NO. 10.
  8. 8. The kit according to claim 7, wherein the kit further comprises papaya virus E positive plasmid and tomato yellow leaf curl virus positive plasmid; The papaya virus E positive plasmid is a PpVE target amplified fragment shown in SEQ ID NO.13 and is formed by ligating a PpVE target amplified fragment shown in SEQ ID NO.14 into a pCB301 vector, and the tomato yellow leaf curl virus positive plasmid is a TYLCV target amplified fragment shown in SEQ ID NO.14 and is formed by ligating a pCB301 vector.
  9. 9. A kit for simultaneous detection of papaya virus E, tomato yellow leaf curl virus, tomato spotted wilt virus and tomato brown wrinkle virus in tomatoes; the kit comprises a primer pair A for detecting papaya virus E, a primer pair B for detecting tomato yellow leaf curl virus, a primer pair C for detecting tomato spotted wilt virus, a primer pair D for detecting tomato brown wrinkle virus and a tomato inner reference primer; The primer pair A consists of an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.2, the primer pair B consists of an upstream primer shown in SEQ ID NO.3 and a downstream primer shown in SEQ ID NO.4, the primer pair C consists of an upstream primer shown in SEQ ID NO.5 and a downstream primer shown in SEQ ID NO.6, and the primer pair D consists of an upstream primer shown in SEQ ID NO.7 and a downstream primer shown in SEQ ID NO. 8; The tomato inner reference primer consists of an upstream primer shown as SEQ ID NO.11 and a downstream primer shown as SEQ ID NO. 12.
  10. 10. The kit according to claim 9, wherein the kit further comprises papaya virus E positive plasmid, tomato yellow leaf curl virus positive plasmid, tomato spotted wilt virus positive plasmid and tomato brown wrinkle fruit virus positive plasmid; The papaya virus E positive plasmid is obtained by connecting PpVE target amplified fragments shown in SEQ ID NO.13 into a pCB301 vector, the tomato yellow leaf curl virus positive plasmid is obtained by connecting TYLCV target amplified fragments shown in SEQ ID NO.14 into the pCB301 vector, the tomato spotted wilt virus positive plasmid is obtained by connecting TSWV target amplified fragments shown in SEQ ID NO.15 into the pCB301 vector, and the tomato brown leaf curl virus positive plasmid is obtained by connecting ToBRFV target amplified fragments shown in SEQ ID NO.16 into the pCB301 vector.

Description

Primer combination and kit for simultaneously detecting various plant viruses infecting tomatoes and application of primer combination and kit Technical Field The invention relates to the technical field of plant virus detection, in particular to a primer combination and a kit for simultaneously detecting a plurality of tomato-infected plant viruses and application thereof. Background The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art. The virus seriously damages important economic crops such as tomatoes, which often cause crop yield reduction, quality reduction and even harvest failure, and causes huge economic loss for agricultural production. New tomato plants showing similar virus symptoms are found in vegetable greenhouses in the texas market in Shandong province in 2025, the leaves of the plants show obvious yellowing phenomenon, dark green spots with different sizes can be seen on the surfaces of immature green fruits, green and yellow spots appear on mature red fruits and are accompanied with discoloration phenomenon, and the incidence rate reaches 10-60 percent, so that the yield and quality of tomatoes are seriously affected. Identified as being caused by a new papaya virus E (papaya virusE, ppVE), there is still a lack of effective detection and control means for it. Tomato Yellow Leaf Curl Virus (TYLCV) is an important gemini virus threatening tomato production and can systematically infect tomatoes, causing typical yellow leaf curl symptoms. PpVE (RNA virus) and TYLCV (DNA virus) have common transmission medium bemisia tabaci, and are extremely easy to generate complex infection. The method is used for accurately evaluating the poisoning condition of the field bemisia tabaci population, and is important to the early warning of virus diseases, the analysis of a virus transmission mechanism and the formulation of a prevention and control strategy. However, in practical operation, two kinds of viruses with different nucleic acid types are efficiently extracted from a single-head or micro-mediator sample and accurately detected in a combined way, and technical bottlenecks such as low nucleic acid abundance, complicated extraction process and the like exist. More importantly, the traditional direct detection of DNA viruses such as TYLCV cannot effectively distinguish the substantial infection from the inactive DNA pollution of the body surface. Tomato Spotted Wilt Virus (TSWV), tomato brown wrinkle virus (ToBRFV) and PpVE all cause significant fruit symptoms, but the symptoms are not easily distinguishable. Therefore, the four plant viruses are detected timely, accurately and efficiently, and the method is a key premise for developing prevention and control of virus diseases, preventing virus transmission and diffusion and guaranteeing agricultural production safety. Currently, detection methods for plant viruses mainly comprise an immunodetection method (such as ELISA (enzyme-linked immunosorbent assay) and a molecular detection method (such as PCR (polymerase chain reaction), RT-PCR (reverse transcription-polymerase chain reaction), qPCR (quantitative polymerase chain reaction) and the like), wherein the molecular detection method has become a mainstream technology for detecting plant viruses due to high sensitivity and high specificity. However, the genomic structure, the distribution of conserved regions, and the variation characteristics of the four viruses PpVE, TYLCV, TSWV and ToBRFV differ significantly. TYLCV is DNA virus, genome is small (about 2.7 kb), conserved region is concentrated but recombination variation is easy to occur, TSWV is segmented negative sense RNA virus, variation rate is high, conserved region sequence is limited, toBRFV is positive sense single-stranded RNA virus, homology with tobacco mosaic virus, tomato mosaic virus and other closely related virus sequences is high, specific discrimination is difficult to realize, ppVE is new plant virus, and related researches are rarely reported. The above characteristics result in great difficulty in designing primers for detecting four viruses, and multiple requirements of high specificity, no cross reaction, tm value matching, no primer dimer formation, no competition for templates and the like need to be satisfied at the same time, the screening workload of primers is huge, and genomic variation (particularly primer binding region variation) of any virus may cause deviation in detection results. Disclosure of Invention In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a primer combination, a kit and applications thereof for simultaneously detecting a plurality of tomato-infected plant viruses. Specifically, the invention rela