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Nucleic acid probe and kit for simultaneously detecting multiple tomato viroids and application of nucleic acid probe and kit

A nucleic acid probe and viroid technology, applied in the field of nucleic acid probes for simultaneous detection of multiple tomato viroids, can solve the problems of cumbersome process, time-consuming, cumbersome preparation process and the like

Pending Publication Date: 2021-12-10
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This not only requires careful design and selection of suitable enzyme cutting sites, but also the preparation process is cumbersome and time-consuming
Taking the detection of six types of viruses (PSTVd, PCFVd, TCDVd, TPMVd, TASVd, and CLVd) that infect tomato as an example, five times of subcloning are required to prepare polyprobes that can simultaneously detect these six types of viruses. cumbersome

Method used

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  • Nucleic acid probe and kit for simultaneously detecting multiple tomato viroids and application of nucleic acid probe and kit
  • Nucleic acid probe and kit for simultaneously detecting multiple tomato viroids and application of nucleic acid probe and kit
  • Nucleic acid probe and kit for simultaneously detecting multiple tomato viroids and application of nucleic acid probe and kit

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preparation example Construction

[0065] In yet another specific embodiment of the present invention, a method for preparing the above-mentioned universal nucleic acid probe is provided, comprising the steps of:

[0066] (1) Through sequence comparison, find out the common sequence between the above-mentioned 6 kinds of tomato viroids, design specific primers Pospi6-probe-F1 and Pospi6-probe-R1, as shown in sequence listing sequence 1,2, to contain The recombinant plasmid of the PSTVd ​​genome sequence (GenBank: U23058.1) was used as a template, amplified by PCR, purified and recovered, and cloned into the vector pGEM-T to obtain a plasmid for probe preparation;

[0067] (2) digest the plasmid obtained in the step (1) with restriction endonuclease Spe I, use the PCR product purification kit to purify the digested product;

[0068] (3) to the purified linearized plasmid that described step (2) obtains, synthesize universal nucleic acid probe by the method for in vitro transcription.

[0069] In yet another speci...

Embodiment 1

[0104] 1 material

[0105] 1.1 Plasmids to be tested:

[0106] Recombinant plasmids containing the genomic sequences of PSTVd, PCFVd, TASVd, TCDVd, TPMVd and CLVd.

[0107] 1.2 Main reagents and instruments:

[0108]Main reagents: Restriction endonuclease and T7 / T3 / SP6 RNA polymerase were purchased from TaKaRa, reagents required for PCR amplification and PCR product purification kit were purchased from Beijing Quanshijin Biotechnology Co., Ltd. (TransGen Biotech), Sangon Bioengineering (Shanghai) Co., Ltd., DNA gel recovery kit and plasmid DNA mini-prep kit (produced by Corning Life Sciences Co., Ltd.), pGEM-T vector produced by Beijing Puluo Promega Biotechnology Co., Ltd. (Promega) and reagents related to probe preparation (ROCHE) were purchased from Beijing Zhongyuan Heju Biotechnology Co., Ltd.

[0109] Main instruments: Bio-Rad PCR instrument, KCBIO-2800 gel imaging system, DYY-6B stabilized voltage electrophoresis instrument, constant temperature water bath, constant ...

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Abstract

The invention provides a nucleic acid probe and kit for simultaneously detecting multiple tomato viroids and application of the nucleic acid probe and kit, and belongs to the technical field of bioengineering and molecular detection. The sequence of each viroid in a potato spindle tuber viroid family is deeply analyzed to find a section of common sequence which is about 60bp and exists in six viroid genomes, PSTVd plasmids are used as templates, specific primers are designed, the sequence is obtained through PCR amplification, pGEM-T vectors are inserted, recombinant plasmids are digested with restriction enzymes, and then the universal nucleic acid probe is obtained through an in-vitro transcription method. By using the nucleic acid probe, six different tomato viroids can be simultaneously detected at a time. The cost for preparing the nucleic acid probe is reduced, and the detection efficiency is greatly improved. Therefore, the nucleic acid probe can be used for large-scale detection of field tomato samples, disease diagnosis can be rapidly and accurately carried out, and good practical application value is achieved.

Description

technical field [0001] The invention belongs to the technical field of bioengineering and molecular detection, and in particular relates to a nucleic acid probe, a kit and an application thereof for simultaneously detecting multiple tomato-like viruses. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily to be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] Viroid is a kind of circular, non-coding, single-stranded, low-molecular-weight RNA, which is the smallest plant pathogen and can cause serious economic losses after infecting plants (Flores R, Hernández C, Alba AEMD, Daròs JA , DiSerio F.Viroids and viroid-host interactions.[J].Annual Review of Phytopathology,2005,43(1):117-139.). Various viroids of the family Pota...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/70C12Q1/6841
CPCC12Q1/701C12Q1/6841C12Q2600/16C12Q2537/143C12Q2543/10
Inventor 张志想张煜泓李世访
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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