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Primers, TaqMan probe and kit used for detecting tomato yellow leaf curl virus

A technology of tomato yellowing curved leaves and a kit, applied in the quantitative field, can solve the problems of false positive reaction, long time required, and difficulty in detecting virus infection, etc.

Active Publication Date: 2012-04-18
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The biological detection method is to judge whether the infection occurs through the tomato phenotype symptoms. This method has certain shortcomings due to the subjectivity of observation and the uncertainty of symptoms.
At present, enzyme-linked immunosorbent assay (ELISA) is the most widely used serological detection method, which takes a long time to prepare antiserum, often has false positive reactions, and is difficult to detect early viral infection

Method used

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  • Primers, TaqMan probe and kit used for detecting tomato yellow leaf curl virus
  • Primers, TaqMan probe and kit used for detecting tomato yellow leaf curl virus
  • Primers, TaqMan probe and kit used for detecting tomato yellow leaf curl virus

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1. (Design of primers and TaqMan probes for qualitative and quantitative detection of TYLCV)

[0031] According to the conserved sequence of TYLCV in GenBank, degenerate primers TY1 / F and TY2 / R were designed to amplify the coat protein (CP) sequence, and the amplified length was 356bp. Design specific primers and probes. The specific primer and probe sequences are as follows:

[0032] Upstream primer: 5'-CACCAATAACTGTAGCATGAAAT-3' (SEQ ID NO: 1 in the sequence listing), the annealing temperature is 54.5°C;

[0033] Downstream primer: 5'-GCCCAATGGATTTTGGAC-3' (SEQ ID NO: 2 in the sequence listing), the annealing temperature is 54.3°C;

[0034] Probe sequence: 5’- TCCTCATCACTTGAAACCTATCMCGCAAATC -3’ (SEQ ID NO: 3 in the sequence listing), the annealing temperature is 65.3°C.

[0035]SEQ ID NO: 1 in the sequence listing consists of 23 bases, which is the reverse complement of bases 856-878 at the 5' end of the tomato yellow leaf curl virus (TYLCV-CH; JF4142...

Embodiment 2

[0038] Embodiment 2, (use the primer of the present invention to carry out routine PCR detection to TYLCV)

[0039] Tomato ingredients described in this example:

[0040] Tomato plants infected with TYLCV in Hangzhou and Haining.

[0041] The detection method is carried out according to the following steps:

[0042] 1. Extraction of genomic DNA: Take 0.1 g of the leaves of the tomato susceptible plants, add liquid nitrogen and grind them into powder, extract the DNA according to the conventional CTAB method, and store them separately for later use;

[0043] 2. PCR amplification: Add 20 ng of DNA from each sample extracted in step (1) to each PCR tube, then add 0.2 μM each of the upstream and downstream primers, dNTP 0.25 mM, MgCl 2 2.0 mM, 1X PCR buffer, 1 unit of Taq DNA polymerase, add ddH 2 After 0 to 20 μL, follow the PCR reaction program: pre-denaturation at 94°C for 3 minutes, 30 seconds at 94°C, annealing at 56-59°C for 30 seconds, 30 seconds at 72°C, 40 cycles of a...

Embodiment 3

[0046] Embodiment 3, (use the primer of the present invention and TaqMan probe to carry out the real-time fluorescent quantitative PCR detection of TYLCV)

[0047] Tomato ingredients described in this example:

[0048] Tomato lines 07-020, 07-025, 07-026, 07-027, 07-040 and 07-029 were inoculated with TYLCV for 40 days.

[0049] Follow these steps:

[0050] 1. Genomic DNA extraction: Take 0.1 g of tomato plant leaves inoculated with TYLCV for 40 days, add liquid nitrogen and grind it into powder, extract DNA according to the conventional CTAB method, and store them separately for future use.

[0051] 2. Preparation of real-time fluorescent quantitative PCR standard products

[0052] (1) Recovery of PCR amplification products of TYLCV

[0053] Take 100 μL of the PCR amplification product of the detection sample obtained in Example 2, perform 1.5% agarose gel electrophoresis on it, cut out a 123bp target fragment under ultraviolet light, and then recover and purify the fragme...

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Abstract

The invention relates to primers and a probe used for carrying out qualitative and quantitative detections upon a virus through a biological method, and especially relates to primers, a TaqMan probe and a kit used for carrying out qualitative and quantitative detections upon tomato yellow leaf curl virus (TYLCV) through a real-time fluorescence quantitative PCR technology. The base sequence of an upstream primer of the primers is represented by SEQ ID NO: 1, and the base sequence of a downstream primer of the primers is represented by SEQ ID NO: 2. The DNA sequence of the probe is represented by SEQ ID NO: 3. A 5' terminal of the probe is labeled with reporting fluorophores, and a 3' terminal of the probe is labeled with quenching fluorophores. The kit comprises the primers and the probe. The primers and the probe provided by the invention can be used in quantitative and qualitative analysis of TYLCV. The primers and the probe have important significances in research fields such as the determinations of a permanent planting regularity and a replication level of the virus in a plant, and the determinations of whether the virus is removed after medication. The primers, the probe and the kit can play important roles in large-scale quarantine, epidemiological study and early diagnosis of TYLCV.

Description

technical field [0001] The present invention relates to primers and probes for qualitative and quantitative detection of viruses by molecular biological methods, in particular to real-time fluorescence quantitative PCR (Real-time fluorescence quantitative PCR, FQ-PCR) technology for detection of tomato yellow leaf curl virus. Primers, TaqMan probes and kits for qualitative and quantitative detection. Background technique [0002] tomato( Lycopersicon esculentum Mill.) is an important vegetable crop in the world, with many varieties, rich nutrition and wide application. Disease prevalence and adverse conditions can limit the development of tomato production. Tomato yellow leaf curl virus disease is one of the important diseases restricting tomato production. The disease causes symptoms such as slow growth or stagnation of tomato plants, difficulty in flowering and fruiting, and small fruit size, resulting in a serious reduction in tomato yield and quality. The disease is ca...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/94
Inventor 叶青静杨悦俭王荣青周国治阮美颖姚祝平李志邈万红建
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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