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.
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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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