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Liquid phase chip detection primer of tomato spotted wilt virus, and detection method thereof

A liquid-phase chip detection technology for tomato spotted wilt virus, which is applied in biochemical equipment and methods, microbial measurement/testing, DNA/RNA fragments, etc., to achieve high throughput, good specificity, fast and simple detection methods

Active Publication Date: 2014-03-26
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This technology has been used in the detection of plant viruses such as Plum pox virus (PPV), and some have obtained patents; so far, there has been no report on the application of this technology in the detection of tomato ringspot virus at home and abroad

Method used

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  • Liquid phase chip detection primer of tomato spotted wilt virus, and detection method thereof
  • Liquid phase chip detection primer of tomato spotted wilt virus, and detection method thereof
  • Liquid phase chip detection primer of tomato spotted wilt virus, and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1, Preparation of Tomato Spotted Wilt Virus Liquid Chip Detection Primers, Probes and Kit

[0042]According to the nucleotide sequence of tomato spotted wilt virus reported by NCBI, primers and probes were designed using Primer Premier 5.0 software, and finally a pair of primers and probes with high specificity were screened out and artificially synthesized. The sequences are shown in Table 1 shown.

[0043] Table 1 Designed primers and expected target fragment length

[0044]

[0045] The 5' end of the primer shown in Sequence 2 above was labeled with Biotin.

[0046] After each of the above primers and probes is packaged independently, it can be used as a component of a kit for detecting tomato spotted wilt virus liquid chip.

Embodiment 2

[0047] Example 2, Application of Tomato Spotted Wilt Virus Liquid Chip Detection Primers, Probes and Kits

[0048] 1. Establishment of liquid chip detection method for tomato spotted wilt virus

[0049] 1. Detection of cDNA acquisition of samples

[0050] 1) Extraction of detection sample RNA

[0051] Use Invitrogen's Trizol Reagent (product number: 15596-026) to extract RNA from the sample to be tested, and follow the steps in the instructions.

[0052] 2) cDNA synthesis: Add 1 μL total RNA, 2 μL (10 μmol / L) downstream primer TSWV-S-R (no biotin labeling required), 1 μL dNTP Mix (10 mmol / L) and 10 μL DEPC-H to a 0.2 mL reaction tube 2 O; after mixing, heat at 70°C for 10 min, quickly transfer to ice for 5 min, and centrifuge briefly; then add 5 μL of 5× reverse transcription buffer and 0.5 μL RNase inhibitor (40 U / μL) to the reaction tube, 0.5 μL reverse transcriptase (200 U / μL); gently mix, centrifuge briefly; react at 42°C for 1 hour, quench on ice for 5 minutes, and obt...

Embodiment 3

[0091] Example 3, Primers and detection method for detection of tomato spotted wilt virus liquid chip detection actual sample to be tested

[0092] In order to better verify the practical application effect of the primer probe of the present invention, the present invention uses tomato (Lycopersicon.esculentum) leaves (sample 1) infected with tomato spotted wilt virus and detects with RT-PCR method to determine that it is not infected by tomato spot. Four samples (sample 2, sample 3, sample 4, sample 5) infected with wilt virus were tested at the same time.

[0093] The method is the same as Step 1 in Example 2.

[0094] The results show (Table 4), the test result of the infected sample is positive, and the sample that has not been infected by tomato spotted wilt virus is negative, thereby proving that the method of the present invention can effectively detect whether there is tomato in the sample to be tested. Spotted wilt virus.

[0095] Table 4 liquid chip method detects ...

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Abstract

The invention discloses a liquid phase chip detection primer of a tomato spotted wilt virus, and a detection method thereof. The invention provides a kit used for the liquid phase chip detection or the assisted detection of the tomato spotted wilt virus. The kit comprises a primer group and a probe, and the primer group is composed of a primer 1 and a primer 2. Experiments prove that a sample can be rapidly detected through the detection method, and a process from sample treatment to result obtaining only costs about 4h; and there is no cross reaction of the primers designed in the invention and common plant viruses on tomatoes, so the false negative result caused by the non-specific amplification is reduced.

Description

technical field [0001] The invention belongs to the field of plant virus detection, and in particular relates to a liquid phase chip detection primer and a detection method for tomato spotted wilt virus. Background technique [0002] Tomato spotted wilt virus is a representative member of the genus Tospovirus in the family Bunyaviridae. Since tomato spotted wilt was first reported in Australia in 1915, and its pathogen was determined to be a virus in 1930, many countries have reported the occurrence and damage of the virus one after another. Tomato spotted wilt virus has a wide range of hosts, including more than 80 families and nearly a thousand dicotyledonous and monocotyledonous plants in temperate, subtropical and tropical regions. It can cause serious diseases of many important horticultural plants and crops, causing extremely serious economic losses. . In the United States, the virus was first found on peanuts grown in southern Texas in 1971, and its damage worsened ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6816C12Q1/70C12Q2563/149
Inventor 张永江刘洪义陈林辛言言张洪祥李桂芬刘忠梅朱水芳
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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