Method for detecting tobacco virus using reverse transcription loop-mediated isothermal amplification technique

A loop-mediated isothermal, tobacco virus technology, applied in the field of plant virology, can solve the problems of low sensitivity, low accuracy, long test period, etc., and achieve the effect of improving detection efficiency and reducing detection cost.

Inactive Publication Date: 2011-12-21
SHAANXI TOBACCO RES INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above methods have different disadvantages, for example, the required test period is long, the operation is cumbersome, the sensitivity is not high, the specificity is not strong, the accuracy is low, and it is easy to be limited by the detection materials, etc.

Method used

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  • Method for detecting tobacco virus using reverse transcription loop-mediated isothermal amplification technique
  • Method for detecting tobacco virus using reverse transcription loop-mediated isothermal amplification technique
  • Method for detecting tobacco virus using reverse transcription loop-mediated isothermal amplification technique

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] 1. Primer Design

[0087] At first the inventor downloaded the sequences of the CP genes of the five main viruses of tobacco from NCBI, (CMV, EF424775) (PVY, AM236802) (TEV, AY787757) (TMV, AF318213) (TVBMV, AM236820) using Primer4.0 ( http: / / primerexplorer.jp / e / v4_manual / index.html) designed multiple sets of primers, and then selected according to comprehensive factors such as the conservation of the sequence region where the primers are located, the hairpin structure of the primers, the GC content of the dimer, and the Tm value primers for each virus. Finally, 4 primers were selected for each tobacco virus, including 2 outer primers (F3 and B3) and 2 inner primers (FIP and BIP). The primers refer to SEQ ID NO.1-20. The primers for RT-PCR of TEV, the present invention uses the primers of Zheng Xuan et al., refer to SEQ ID NO.21-22. See Table 1 for information on all primers.

[0088] The information of the primer sequence SEQ ID NO.1-22 in table 1:

[0089]

[...

Embodiment 2

[0105] Embodiment 2, adopt agarose gel electrophoresis method, SYBR Green I green fluorescence method to detect RT-LAMP amplification product

[0106] The amplified products of RT-LAMP were detected by agarose gel electrophoresis and SYBR GreenI green fluorescence method, respectively.

[0107] The RT-LAMP amplification product was positive in the gel electrophoresis pattern and was ladder-like ( figure 1 ,1).

[0108] In SYBR Green I fluorescence detection, dilute the stock solution of SYBR Green I 10 times, take 1 μl and add it to the reaction tube, the positive is emerald green ( figure 2 , left), the negative is orange ( figure 2 ,right).

[0109] In this experiment, two methods were adopted to detect the results of RT-LAMP, one was directly observed by adding SYBR GreenI, and the other was analyzed by 2% agarose gel electrophoresis. When SYBR GreenI was added and directly observed with the naked eye, the inventors found that the orange color of the negative control ...

Embodiment 3

[0110] Embodiment 3, the impact of different temperatures on the RT-LAMP reaction:

[0111] In the present invention, different temperatures are set for each group of primers, and TEV is taken as an example to illustrate as follows. Under the condition of other conditions unchanged, TEV was tested at four temperatures of 65°C, 63°C, 61°C, and 59°C, and the results were better at 63°C and 61°C ( image 3 ), with the same method, the present invention finds that the optimal temperature of CMV, TMV, TVBMV is 63°C, and PVY is 65°C. The results of five kinds of virus RT-LAMP gel electrophoresis under optimal conditions are as follows: Figure 4 , adding SYBR Green I fluorescent dye to directly observe the results as Figure 5 .

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Abstract

The invention relates to a method for detecting tobacco viruses by a reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) technology, which comprises the steps of tobacco virus total ribonucleic acid (RNA) extraction, RT-LAMP reaction, electrophoresis detection and virus type determination, wherein tobacco viruses are selected from two or more than two kinds of viruses from cucumber mosaic viruses (CMV), potato viruses Y (PVY), tobacco etch viruses (TEV), tobacco mosaic viruses (TMV) and tobacco vein banding mosaic viruses (TVBMV). LAMP primers are designed according to a case protein gene conserved region of the tobacco viruses, and a fast, sensitive and high-specificity method is built for detecting various main tobacco viruses by the one-step RT-LAMP technology. A newmeasure is provided for the fast detection of the tobacco viruses.

Description

technical field [0001] The invention belongs to the technical field of plant virology, in particular to a method for detecting tobacco virus by adopting reverse transcription loop-mediated isothermal amplification technology. Background technique [0002] Tobacco is one of the most important economic crops in the world. China's planting area ranks first in the world, and it increases the country's fiscal revenue by 200-300 billion yuan every year. However, with the development of the industry and the continuous expansion of the planting area, after the 1970s, the harm of tobacco virus disease has increased year by year. In the tobacco field, it is often manifested as a mixed infection of various viruses, which causes heavy losses to tobacco production. The output of severely diseased fields is reduced by 20%-50%, and the output of summer tobacco is reduced by more than 70%. The trend of continuous expansion and gradual aggravation. [0003] Tobacco viruses that endanger Ch...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 成巨龙赵磊胡翠华郝兴安满建云吴云锋
Owner SHAANXI TOBACCO RES INST
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