Research On The Development Of A Rapid Detection Method For Ralstonia Solanacearum Based On The CRISPR-Cas12a System

Bacterial wilt（BW）,caused by the aerobic gram-negative pathogen Ralstonia solanacearum（RS）,is a major disease affecting global commercial agriculture and is one of the top ten bacterial plant pathogens in the world.The Asian branch phylotype I RS is the pathogen of tomato bacterial wilt,which has caused serious economic losses in southern China for many years.Once the disease occurs,the condition progresses rapidly and ultimately leads to wilting and death of the plant.Traditional RS detection methods have the disadvantages of complex operation and low timeliness,so developing a fast,sensitive,and effective RS detection method is the top priority for controlling bacterial wilt.In recent years,with the rise of a series of isothermal amplification technologies and the gradual popularization of nucleic acid rapid detection methods,there is a problem of decreased detection specificity behind the rapid and efficient amplification.The discovery of the trans-cleavege activity to single-stranded DNA of the CRISPR/Cas12a system makes this system applicable to the field of rapid detection from a gene editing tool.Providing single-stranded DNA with two-terminal modifications in the system can detect specific DNA targets.This technology combined with isothermal amplification technology can greatly improve the detection sensitivity.Based on this,this study established a method for rapid visual detection of RS using loop-mediated isothermal amplification technology（LAMP）and CRISPR/Cas12a system,and the main results are as follows:1.Multiple strains of RS were identified for phylotypes,and strains of phylotype I were screened and used as targets.Through multiple sequence alignment of the conserved genes,the 215bp fragment anchored on the hrpb gene was finally selected as the target sequence for subsequent experiments.2.Successfully constructed p UC57-hrpb detection target plasmid and obtained p ET30a-Lba Cas12a expression plasmid.Cas12a protein was expressed and purified in E.coli expression system,and finally obtained high-quality Lba Cas12a protein.3.LAMP primers were successfully designed to extend the 215 bp specific fragment,and the reaction process of LAMP was optimized.A relatively mature LAMP reaction system was obtained by adding an outer to inner primer ratio of 1:8（2μM:16μM）,8 m M Mg²⁺,1.4 m M d NTP,and 0.64 U/μL Bst polymerase.4.The cr RNA sequence was designed and synthesized for the target sequence,and cr RNA sequences with both cis and trans cleavage activity were screened and optimized to establish a CRISPR/Cas12a fluorescence detection system.A CRISPR/Cas12a detection system was established by adding 33 n M of Lba Cas12a,180n M of cr RNA,800 n M of FQ-reporter,and 3μL of LAMP reaction product under fluorescence detection conditions.For test strip detection conditions,the FQ-reporter was replaced with a 250 n M concentration of FB-reporter.5.The LAMP/Cas12a fluorescence detection system was constructed,and key factors such as Cas12a concentration,cr RNA concentration,reporter group concentration,and LAMP reaction time were optimized to obtain a highly specific and sensitive visual fluorescence detection method.The target DNA detection was completed within 2 hours,and the detection accuracy reached the level of single copy number.6.The LAMP/Cas12a combination immunochromatographic test strip method was constructed,and the T-line intensity in the negative control group was weakened to the maximum by controlling the FB-reporter concentration and reaction buffer system.Compared with the fluorescence detection method,this method also showed good specificity and sensitivity.7.Soil and plant samples suspected of suffering from bacterial wilt were collected from tomato fields in Changyang,Hubei and Tianyang,Guangxi,and the rapid detection methods based on the LAMP/Cas12a system were used for rapid detection,and the results were verified by PCR.The results showed that the two methods constructed in this study obtained accurate detection results in soil and plant samples suspected of suffering from bacterial wilt,proving that the rapid detection method based on the LAMP/Cas12a system has the feasibility of on-site rapid detection in agricultural production,and has good development and application prospects in agricultural disease warning and control.