| Citrus Huanglongbing is a widespread citrus disease with ACP,caused by phloem-restricted bacterial Candidatus Liberibacter asiaticu infection.At present,it has been raging in the main citrus producing areas around the world,lacking effective control and treat methods.Therefore,the rapid and accurate detection of citrus HLB is of great significance for controlling the disease in orchards.However,the distribution of HLB pathogen in citrus plants is extremely uneven and the content is extremely low.Conventional molecular diagnostic techniques based on PCR can no longer meet the needs of highly sensitive detection for early diagnosis of infection.Based on nano material CRISPR detection technology is widely used in the fields of nucleic acid detection,disease diagnosis,and point-of-care detection due to its high sensitivity,high specificity,and signal transduction.For the above problem,this paper designed two DNA-modified CRISPR responsive nanomaterials-sensors,established new methods for the highly sensitive detection of HLB,and realized the visual detection of HLB.The main research contents are as follows:1.Preparation of CRISPR/Cas12a-responsive mesoporous silicon sensors andfluorescence detection of 16 S rDNA of citrus huanglongbingAt present,the detection of HLB is still mainly based on PCR technology,which has high false positive,and difficulty in early diagnosis.Therefore,the apply to CRISPR/Cas12 a detection technology developed in recent years is of great significance to solve the problem of difficult early diagnosis of HLB.In this section,based on nanotechnology and CRISPR/Cas12 a nucleic acid recognition and transduction capabilities,a responsive MSN gated material was constructed for the highly sensitive fluorescence detection of HLB.First,MSN loaded with BHQ and FITC were prepared,and forming a gated fluorescent probe FITC-BHQ@MSN-ss DNA,due to adsorbed to the surface ss DNA to close the surface pores.Then,amplify the HLB16 S rDNA in a way of inducing amplification reaction to generate H3 that can bind to cr RNA.Finally overcome the HLB specific detection,resulting in H3 binds cr RNA to activate Cas12 a trans-cleavage activity,cleavage sensor surface adsorbed ss DNA to release fluorescent signals.After the amplification enzyme content,the probe/Cas12 a incubation time was optimized,and finally the purpose of specific detection of HLB16 S rDNA was achieved.The detection range was 10-1000 pmol/L,the detection limit was as low as 2.5 pmol/L,and the recovery rate 95%-102%.2.Construction of CRISPR/Cas12a-responsive DNA gel sensors and visualdetection of 16 S rDNA of citrus huanglongbingIn order to reduce the incubation time of the probe and achieve the purpose of rapid and simple detection of HLB bacteria.On the basis of the first chapter,this section designs DNA hydrogel nanoparticles that can respond to DNA trans-cleavage activity for the visual and sensitive detection of HLB,relied on DNA hydrogel enzyme responsive activity and huge cargo loaded capacity.First,HRP@DNA gel NP were prepared by freezing-radical polymerization technique and molecular entrapment method as visualization probes.Then,the HLB16 S rDNA is amplified in a manner of inducing amplification reaction,and produce the H3 that can bind to the cr RNA.Finally,H3 binds cr RNA to activate Cas12 a trans-cleaving activity,probe HRP@DNA gel NP responds to the change of Cas12 a activity,and gel cleavage releases HRP.H2O2 and TMB in the internal HRP catalyzed solution reaction,to achieve the purpose of visual detection of HLB.For the amplification enzyme content and Cas12 a enzyme content,the specific visual detection of HLB16 S rDNA was finally realized,the detection range was 20-1000pmol/L,and the detection limit was 13.6 pmol/L.Compared with the first system,the detection time is faster for the DNA gel nanosensor,and the presence of HLB can be observed with the naked eye in only 35 min. |
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