| Plant virus disease is a whole plant infection disease,which spreads quickly in the field and causes great harm,making it very difficult to control.The traditional plant virus disease control technology mainly includes three aspects:healthy seedling cultivation,cutting off the vector and later pesticide control,which has high requirements on plant germplasm research,virus infection mechanism research and pathogenesis research.In recent years,the promotion of the intensive production model of medicinal plants has intensified the prevalence and development of medicinal plant virus diseases.However,the above-mentioned techniques for controlling plant virus diseases are not suitable for small species such as medicinal plants,rapid and accurate virus detection technology is needed to control the spread of plant virus diseases.The traditional detection techniques of plant virus mainly include morphological detection methods,immunological detection methods,and molecular biological detection methods.They have disadvantages such as long detection time and high requirements for people and the environment.The new pathogen detection technology draws on materials such as paper-based and nano-magnetic beads to accelerate the extraction of the target to be tested.At the same time,it combines quantum dots and gold nano-materials to quickly characterize the detection results.Therefore,based on the development of new pathogen detection technology,a rapid and visible nucleic acid detection technology on plant virus was proposed,using Tobacco mosaic virus(TMV)as a model.The main test results are as follows:1.Rapid extraction of plant virus RNA based on functionalized magnetic beads.The test uses TMV specific capture probe(Amine-modified capture probe DNA sequence,ACPTMV)as a modifier to obtain TMV functionalized magnetic beads(CMBs-ACPTMV).Then,based on the base complementary pairing principle,the TMV in the test solution was extracted specifically.The results showed that the extraction capacity of functionalized magnetic beads was the best when the addition amount of ACPTMV(100 μmol/L)was 40 μL and the magnetic beads was 0.08 mg.In the subsequent optimization and evaluation process,it was found that the extraction temperature of CMBs-ACP TMV has a wide application range,and the extraction efficiency is the highest when the extraction time is 3 min.The extraction sensitivity of CMBs-ACPTMV is as low as 2.5 ng/μL.The stability of real sample detection is good,and compared with the traditional Trizol extraction method,it has significant advantages in sample consumption and detection speed.2.Establish a rapid visual detection technology for single-stranded nucleic acid based on gold nanoparticles(AuNPs).RdS random sequence was used to simulate single-stranded RNA,and a visual detection system was established based on the principle that the electrostatic adsorption of nucleic acid bases on the surface of AuNPs can improve their stability in high-salt environments.When the volume fraction of AuNPs is optimized,it is found that when the volume fraction is 35%,it has the two major advantages of good target response sensitivity and economic benefits.After screening,it is determined that the lowest NaCl concentration of this technology is 0.19 mol/L,and the optimum electrostatic coupling time is 5 min.Under these conditions,the detection limit of the visual detection technology is 1μM,the linear detection range is 0～5 μM,and the linear equation is y=0.0435x+0.0176(R2=0.957).Comprehensive performance evaluation shows that the technology has low sample consumption,short detection time,low detection cost,green and safe,and no strict requirements on detection environment.3.Integrated technology for rapid extraction of plant virus RNA and visual detection.The experiment conducted a fusion design and analysis of the above extraction and visual detection technology,and found that the integrated detection technology can effectively detect the TMV in the reaction system,however,no-load functional magnetic beads can influence the data characterization of the detection results to some extent,so the linear detection equation is modified to y=0.0071 x-0.0398(R2=0.957,ng/μL).Afterwards,the relevant performance of the integrated detection technology was evaluated,and it was found that the non-specific extraction volume of mala vein cleaning virus(MVCV)and broad bean wilt virus(BBWV)in the magnetic beads did not affect the judgment of the color result,but the non-specific extraction volume of soybean mosaic virus(SMV)had a certain impact on it,and its specificity should be optimized in follow-up research;The detection limit of naked eye observation is the same as that of UV-Vis absorption spectroscopy(UV-Vis),both are 25 ng/μL,which can meet the daily detection requirements;The detection of TMV in real sample is not affected by other substances,the results of the samples can be correctly determined by either UV-Vis or naked eye detection.The integrated detection technology has the advantages of low sample consumption,short detection time,simple and safe operation,and has high application potential. |
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