Screening The DNAzyme Of Pathogenic Bacteria And Constructing The DNAzyme Sensor Based On CRISPR-Cas12a

As a large agricultural country,the stable development of grain production in China is of great significance to guaranteeing national food security.Bacterial plant diseases are common diseases in our country’s agricultural production.Due to the diverse transmission methods and the lack of control agents,the difficulty of prevention and control is increasing,and the safety of food crops is seriously threatened.Rice is the main food crop in our country,and its bacterial disease Bacterial blight occurs in all major rice regions,causing serious rice yield reduction and economic losses.Therefore,developing a sensitive,accurate and convenient pathogen detection method to realize the early diagnosis of bacterial diseases is of great significance for reducing the amount of pesticides and improving the control effect.Deoxyribozymes(DNAzymes),as a type of functional nucleic acid,can specifically recognize targets and have specific catalytic activity.It has the characteristics of convenient synthesis and easy modification,and has been widely used in the design of various biosensors.The systematic evolution technology of exponential enrichment of ligands(SELEX)is the main way to obtain specific DNAzyme for the target.At the same time,the gene editing technology CRISPR-Cas12a system has trans-cleavage activity for single-stranded DNA molecules.By designing in conjunction with sensors,it can achieve signal amplification at room temperature.In this study,the first attempt was made to screen DNAzymes that specifically recognize Xoo using SELEX.At the same time,using the target recognition element DNAzyme and the CRISPR-Cas12a system to design a nucleic acid biosensor capable of signal amplification at room temperature.This article mainly conducts the following two aspects of research:1.Bacterial growth will secrete a variety of substances.In order to avoid tedious separation and purification,we extract the extracellular secretion(CEM)of Xoo as the screening target directly.By screening the ssDNA library,designing with RNA-cleaving fluorescent DNAzyme(RFD),and screening by SELEX,we have carried out a total of fourteen rounds of positive screening and reverse screening(CEM for culture medium,buffer and other bacteria).In the screening process,the screening efficiency is increased by reducing the amount of screening library round by round and shortening the reaction time.In the end,we found that the screening library was enriched by monitoring the screening process and high-throughput sequencing.By testing the specific cleavage ability of the first three DNA sequences with the highest enrichment,it was found that no specific DNAzyme against the target bacterial blight bacteria was obtained.The other enriched sequences have certain homology and have further analysis value.2.Escherichia coli(E.coli)is a kind of gram-negative bacilli pathogenic microorganism,which exists widely in the environment.We used this bacterium as a model,based on the DNAzyme sequence reported in the literature,combined with magnetic bead separation technology and the CRISPR-Cas 12a system to design a new type of nucleic acid biosensor.Using DNAzyme’s recognition function for the target E.coli to generate an activator DNA,which activates the cutting of the signal molecule by the Cas12a system to generate a fluorescent signal.After the optimization of this system,the target and magnetic DNAzyme complex are incubated for 2 h,the detection sensitivity can reach 102 CFU/mL.And the sensor can realize the specific detection of the target,and still has a certain detection ability in complex samples.By designing the sensor combined with invertase,the added sucrose is converted into glucose,and the real-time outdoor detection of pathogenic bacteria is initially realized by the blood glucose meter.,and the design needs to be further optimized.In summary,we extracted the CEM of Xoo,and used SELEX technology to screen the DNAzyme of Xoo.In addition,we also designed a type of DNAzyme-regulated CRISPR-Cas12a sensor,which can achieve signal amplification at room temperature and outdoor detection capabilities.It has a certain reference value for the later optimization of screening conditions to obtain the DNAzyme of bacterial blight,and the design of this type of sensor to achieve isothermal signal amplification,so as to realize the early diagnosis of rice bacterial blight in the field.