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Primer and method for quantitatively detecting turnip mosaic virus (TuMV) in limonium sinuatum

A technology for quantitative detection of turnip mosaic virus, which is applied in biochemical equipment and methods, microorganism-based methods, and microbial determination/inspection, etc., can solve the problems of cumbersome operation, low sensitivity, complicated operation, etc. Sexual, high-sensitivity effects

Pending Publication Date: 2022-01-07
KUNMING INST OF BOTANY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Biological methods have the problems of long cycle and cumbersome operation, and irreparable damage has been caused by the time plants show corresponding symptoms
The method of microscopic observation can visually see the virion, but the operation is relatively complicated and time-consuming
Immunological methods such as ELISA have the characteristics of low sensitivity and poor specificity
Ordinary PCR can only conduct qualitative or semi-quantitative analysis of the virus, and the occurrence of false positive results cannot be ruled out
However, there is no relevant technical research and application report on TuMV real-time fluorescent quantitative PCR detection method

Method used

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  • Primer and method for quantitatively detecting turnip mosaic virus (TuMV) in limonium sinuatum
  • Primer and method for quantitatively detecting turnip mosaic virus (TuMV) in limonium sinuatum
  • Primer and method for quantitatively detecting turnip mosaic virus (TuMV) in limonium sinuatum

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Embodiment 1

[0039] In 2019, more than 50 samples of forget-me-not leaves infected with turnip mosaic virus (TuMV) were collected in the main planting area of ​​forget-me-nots in Yunnan. Deformation, dwarfing, difficulty in blooming flower stems, etc. (see figure 2 ). 15 samples were randomly selected for testing.

[0040] Before the sample inspection, the TuMV CP gene sequences AB701704, KC119189 and AP017831 were screened in GenBank, and primers were designed in the conserved region of the gene. Design multiple pairs of primers on Premier 5.0, and select a pair of primers with the best effect and the most stability according to the results of multiple PCR experiments, namely: upstream primer TuMV-F:5'-CGACCATACATGCCACGACA-3', downstream primer TuMV -R: 5'-TCCATCCAAGCCGAACGAA-3', the size of the product amplified by this primer is 180bp.

[0041] Then, the general plant total RNA extraction kit was used to extract the RNA of the forget-me-not infected leaves, and then the RNA was reve...

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Abstract

The invention discloses a primer and a method for quantitatively detecting turnip mosaic virus (TuMV) in limonium sinuatum, and belongs to the technical field of plant disease detection. The primer used in the invention is designed based on conserved regions of a TuMV CP gene sequence AB701704, KC119189 and AP017831; and the primer is as follows: an upstream primer TuMV-F:5'-CGACCATACATGCCACGACA-3', and a downstream primer TuMV-R:5'-TCCATCCAAGCCGAACGAA-3', and the primer can be used for both RT-PCR amplification and RT-qPCR amplification. The method for quantitatively detecting the TuMV in the limonium sinuatum comprises the following steps of: extracting total RNA of infected leaves of the limonium sinuatum; performing reverse transcription on the total RNA to form cDNA, and performing common PCR and real-time fluorescent quantitative PCR by taking the cDNA as a template once. The primer designed by the invention is high in sensitivity, strong in specificity and good in repeatability; a set of method capable of rapidly and sensitively detecting the TuMV is established in the invention; and a technical support can be provided for early monitoring and early warning of the TuMV of the limonium sinuatum.

Description

technical field [0001] The invention belongs to the technical field of plant disease detection, in particular to a primer and a method for rapid quantitative detection of forget-me-not turnip mosaic virus. Background technique [0002] Forget-me-not is also known as Limonium sinuatum. This species has rich flower colors, such as purple, pink, white, blue, yellow, etc. It is highly ornamental, and its petals are waxy. It can be used as fresh cut flowers and dried flowers, and is widely planted all over the world. Since forget-me-nots were introduced to Kunming for cultivation in 1991, the forget-me-not industry has developed rapidly and has become one of the main forces in the flower cultivation industry in Yunnan Province. At present, the mass-produced forget-me-nots mainly come from tissue culture, and the virus continues to accumulate in multiple subcultures, which greatly restricts the development of the forget-me-not industry. The frequent occurrence of viral diseases ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/94
CPCC12Q1/701C12Q1/6851C12Q2531/113C12Q2563/107C12Q2545/114
Inventor 姚林伶秦姣张石宝胡虹王华
Owner KUNMING INST OF BOTANY - CHINESE ACAD OF SCI
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