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Method for detecting apple viruses by adopting reverse transcription loop-mediated isothermal amplification technology

A loop-mediated isothermal, apple virus technology, applied in the field of plant virology, can solve the problems of low accuracy, low sensitivity, cumbersome operation, etc., to reduce the detection cost and improve the detection efficiency.

Inactive Publication Date: 2012-08-15
NORTHWEST A & F UNIV
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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 apple viruses by adopting reverse transcription loop-mediated isothermal amplification technology
  • Method for detecting apple viruses by adopting reverse transcription loop-mediated isothermal amplification technology
  • Method for detecting apple viruses by adopting reverse transcription loop-mediated isothermal amplification technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] 1. Primer design

[0063] First, the inventors downloaded the whole genome sequences of the three major apple viruses from NCBI, used Primer4.0 (http: / / primerexplorer.jp / e / v4_manual / index.html) to design multiple sets of primers, and then based on the primers Comprehensive factors such as the conservation of the sequence region, the hairpin structure of the primer, the GC content of the dimer, and the Tm value have selected the primers for each virus. Finally, 4 primers were selected for each Apple virus, including 2 outer primers (F3 and B3) and 2 inner primers (FIP and BIP). The primers refer to SEQ ID NO. 1-12. For RT-PCR primers, the present invention uses the primers of Fan Xudong et al., see SEQ ID NO. 13-18. See Table 1 for information on all primers.

[0064] Table 1 Primer sequence SEQ ID NO.1-18 information:

[0065]

[0066] 2. RNA extraction

[0067] Use the polysaccharide polyphenol plant total RNA rapid extraction kit (centrifuge column) (Bioteke), according t...

Embodiment 2

[0080] Example 2 , Use agarose gel electrophoresis, SYBR Green I green fluorescence method to detect RT-LAMP amplification products

[0081] The RT-LAMP amplification products were detected by agarose gel electrophoresis and SYBR Green I green fluorescence method respectively.

[0082] RT-LAMP amplification products are positive in the gel electrophoresis pattern and are ladder-shaped ( figure 1 , A+).

[0083] In SYBR Green I fluorescence detection, dilute the stock solution of SYBR Green I 10 times and add 1μl to the reaction tube. The positive is emerald green ( figure 1 , B+), the negative is orange ( figure 1 , B-).

[0084] In this experiment, two methods were adopted to detect the results of RT-LAMP, one was to add SYBR Green I and observe directly with the naked eye, and the other was to analyze by 2% agarose gel electrophoresis. Under normal circumstances, the first method can be used to clearly distinguish between negative and positive results, and the second method can be ...

Embodiment 3

[0085] Example 3 , Comparison of sensitivity between RT-LAMP and RT-PCR:

[0086] Dilute the pMD18-T-ASGV plasmid by 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 Times is used as a template for RT-PCR and RT-LAMP. The result is diluted by 10 8 Times RT-PCR has not been detected ( figure 2 A), while RT-LAMP is diluted by 10 9 Times can also detect ( figure 2 B). This also shows that the sensitivity of RT-LAMP is more than 100 times higher than that of RT-PCR.

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Abstract

The invention relates to a method for detecting apple viruses by adopting a reverse transcription loop-mediated isothermal amplification technology, wherein the method comprises the following steps of total RNA (Ribose Nucleic Acid) extraction of apple viruses, RT-LAMP (reverse transcription loop-mediated isothermal amplification) reaction and electrophoresis detection and virus type determination, wherein the apple viruses are selected from an ASGV (Apple Stem Grooving Virus), an ACLSV (Apple Chlorotic Leaf Spot Virus) and an ASPV (Apple Stem Pitting Virus). According to the invention, an RT-LAMP primer is designed according to the gene conserved region of the apple viruses, and a rapid, flexible and highly specific method for detecting the apple viruses by adopting the one-step method reverse transcription loop-mediated isothermal amplification technology, and a new means for rapid detection of the apple viruses is provided.

Description

Technical field [0001] The invention belongs to the technical field of plant virology, and particularly relates to a method for detecting apple virus by adopting reverse transcription loop-mediated isothermal amplification technology. Background technique [0002] my country is the main producing area of ​​apples, and its planting area and output account for a quarter of the world. The apple production in Shaanxi Province ranks first in the country. Apples mainly reproduce through grafting, and the spread of viruses is also easy to cause during the grafting process. Investigation and research have found that the incidence of apple latent virus is very high, and the infection rate of apple stem groove virus (ASGV) can be as high as 90%, which brings serious harm to apple production. Once an apple tree is infected with the virus, it will carry the virus for life and cause long-lasting damage. The main manifestations are weakened growth, organ deformity, reduced resistance, and so...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/94
Inventor 郝兴安赵磊苏莹吴云锋
Owner NORTHWEST A & F UNIV
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