Kit and method for detecting TYLCV (tomato yellow leaf curl virus) carried by tomato seedlings

A technology for tomato yellowing and curved leaves and tomato seedlings, applied in the biological field, can solve the problems of difficult promotion and application, complicated process, low detection efficiency, etc., and achieve the effect of easy promotion and application and overcoming time-consuming

Inactive Publication Date: 2012-11-14
CHINA AGRI UNIV +1
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AI Technical Summary

Problems solved by technology

[0003] At present, there are two common methods for detecting TYLCV. One is the molecular biology detection method, which mainly uses the PCR method, but PCR detection is time-consuming, the product is inconvenient to detect, the operation is cumbersome, and the reaction requires a PCR machine and special reagents, which is not suitable for production line use.
The second is the serological detection method, mainly ELISA method, but the detection sensitivity and specificity of ELISA are not high, so it is not suitable for rapid and accurate detection
The deficiencies of the prior art mainly include: the first is that the primers used are not suitable for the detection of TYLCV occurring in my country, resulting in low detection efficiency and high false negative rate; the second is that there is no suitable method for tomato seedling detection; Sampling method; Fourth, DNA needs to be prepared, the process is relatively complicated, and it is not easy to popularize and apply

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  • Kit and method for detecting TYLCV (tomato yellow leaf curl virus) carried by tomato seedlings
  • Kit and method for detecting TYLCV (tomato yellow leaf curl virus) carried by tomato seedlings
  • Kit and method for detecting TYLCV (tomato yellow leaf curl virus) carried by tomato seedlings

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Embodiment 1

[0036] Example 1. Preparation of kit for detecting tomato yellow leaf curl virus carried by tomato seedlings

[0037] 1. Design of primers

[0038] Primers were designed according to the nucleic acid sequences of the dominant strains of tomato yellow leaf curl virus that occurred in my country, and the primer sequences were as follows:

[0039] TYLCV-C-F3:5'-AACGCCATTCTCTGCTTGA-3' (SEQ ID NO: 1)

[0040] TYLCV-C-B3:5'-GAGCCACTGTTCGCAAGT-3' (SEQ ID NO: 2)

[0041] TYLCV-C-FIP:5'-ACTACCTCCACATCAACTGCAAGGAGCAGTGATGAGTTCCC-3' (SEQ ID NO: 3)

[0042] TYLCV-C-BIP: 5'-ATGAGCAGCCACAGTCTAGGTGGTTCAACACAAGATAGCCA-3' (SEQ ID NO: 4)

[0043] 2. Preparation of reaction solution and kit for detection of tomato yellow leaf curl virus

[0044] The reaction solution is 24 μL, and the primers added and the final concentration are 0.2 μmol / L for TYLCV-C-F3 and TYLCV-C-B3 respectively; 1.6 μmol / L for TYLCV-C-FIP and TYLCV-C-BIP; Other components and final concentrations were betaine 5mol / L; d...

Embodiment 2

[0046] Example 2, Application of the kit for detecting tomato yellow leaf curl virus carried by tomato seedlings

[0047] 1. LAMP reaction of the kit for detecting tomato yellow leaf curl virus carried by tomato seedlings

[0048] 1. Sampling and LAMP reaction

[0049] In the seedling tray, 7-week-old (3-4 leaves) number 1-3 virus-infected tomato seedlings (with yellow leaves, curly plants, dwarfing) and number 4 tomato seedlings without virus infection (without yellow leaves) were randomly selected in the seedling tray. Symptoms such as wilting, curling, plant dwarfing, etc.), use a toothpick (or a suction head) to directly pierce the second leaf from the top of the tomato seedling (or the petiole of the second leaf from the top), and then put the toothpick into the container. Gently stir 3 times in the small tube containing the reaction solution of 1 and 2 above. After taking out the toothpick, add 1 μL of Bst DNA polymerase (8U / μL) to the tube respectively, for a total of 25...

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Abstract

The invention discloses a kit and a method for detecting TYLCV (tomato yellow leaf curl virus) carried by tomato seedlings. A primer group provided by the invention comprises a primer 1, a primer 2, a primer 3 and a primer 4; and nucleotide sequences of the primer 1, the primer 2, the primer 3 and the primer 4 respectively and sequentially are a sequence 1, a sequence 2, a sequence 3 and a sequence 4 in a sequence list. The experiment provided by the invention proves that aiming at whether the tomato seedlings are infected by the TYLCV, LAMP (loop mediated isothermal amplification) primer is designed according to the gene group sequence of TYLCV superior strain generated in China, the experiment puts forward that a tomato leaf or a leaf stem is punctured directly by utilizing a toothpick or a sucker, DNA (deoxyribonucleic acid) is not required to be extracted, the LAMP reaction is carried out directly, and the kit and the method for detecting TYLCV carried by the tomato seedlings have the advantages of simpleness and convenience, speediness, flexibility and the like, and overcome the defects that in the traditional technology, time is consumed, requirements for extracting nucleic acid and instruments are high, the steps are complex and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a kit and method for detecting tomato yellow leaf curl virus carried by tomato seedlings. Background technique [0002] Tomato yellow leaf curl virus (Tomato yellow leaf curl virus, TYLCV) is an important source of virus that causes a devastating disease in tomato production, "Tomato yellow leaf curl virus disease", which has caused serious economic damage to tomato production in my country. loss. In production, the poisonous tomato seedlings not only cause the loss of production, but also are an important source of the rapid spread of the disease. The use of non-toxic tomato seedlings and timely and rapid detection of tomato seedlings are the key measures for comprehensive control of the disease. However, tomato seedlings are There is an incubation period of 14-30 days after virus infection until symptoms appear. This feature causes difficulties and problems in the diagnosis and con...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 周涛梁彦师迎春郑建秋范在丰
Owner CHINA AGRI UNIV
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