Development And Application Of Asymmetric RPA Amplification Combined With CRISPR-LbCas12a Technology For Visual Detection Of Pear Fire Blight

Pear Fire Blight is a bacterial disease caused by Erwinia amylovora which is devastating to fruit trees.It has a wide host range and spreads rapidly.So far,the control of E.amylovora is still an international problem.At present,the quarantine and identification methods of the disease mainly include symptom detection,molecular biological detection,and immunological detection.However,these techniques are complicated,time-consuming,and sensitive to certain limitations.Therefore,it is urgent to develop a scientific,effective and low-cost detection method that is suitable for the field production,so that people can timely control Pear Fire Blight.Recent studies have found that Cas12 a protein can indiscriminately cleave any single-stranded DNA in the system after recognizing its target.Based on this characteristic,this experiment took E.amylovora as the main research object.By combining asymmetric RPA amplification technology and CRISPR-LbCas12 a fluorescence detection technology,a field visual detection system was constructed,so as to achieve efficient and rapid detection of E.amylovora.The purpose of detection is to provide a scientific basis for the early prevention and control of pear fire blight.The specific research results are as follows:1.Based on the characteristics of CRISPR-LbCas12 a trans-cleavage activity,the effects of new PAM(YTV)and different types of target sequences on LbCas12 a were detected by fluorescent reporter groups.The results showed that new PAM(YTV)could assist LbCas12 a in identifying target sequences;among different types of targets,ssDNA activation of LbCas12 a does not require the assistance of the PAM sequence,while dsDNA activation of LbCas12 a must be carried out under the interaction of PAM and crRNA.2.We used symmetric RPA amplification and asymmetric RPA amplification of E.amylovora sequences as targets,and detected their effects on LbCas12 a cleavage activity by fluorescent reporter groups.The results show that the target sequence amplified by symmetric RPA in E.amylovora activates the cleavage activity of LbCas12 a only when the crRNA contains a PAM site(TTTV),while the target sequence amplified by asymmetric RPA in E.amylovora can be amplified in any type of crRNA activates the cleavage activity of LbCas12 a.It further indicated that this experiment established a visual detection system for E.amylovora by asymmetric RPA combined with CRISPR-LbCas12 a technology.3.The crude extract of bacterial DNA extracted by high temperature lysis was used to amplify the substrate without RPA.After 10 minutes for pre-amplification,it was combined with LbCas12 a,and it was detected by UV analyzer that green fluorescence was only displayed in the complete LbCas12 a system;therefore,a one-tube reaction system combining asymmetric RPA amplification and LbCas12 a fluorescence detection of E.amylovora were established,which effectively reduced the interference caused by opening the lid during the experiment.4.We used the crude extracts of microbial community DNA on different fruit surfaces as RPA amplification substrates.Through high-throughput sequencing and bioinformatics analysis and CRISPR-LbCas12 a fluorescence detection,we did not find the existence of E.amylovora.This result shows that CRISPR-LbCas12 a technology has high specificity.Under the optimal reaction conditions,the sensitivity of CRISPR-LbCas12 a to detect E.amylovora was 6.4×103 cfu·m L-1,which was consistent with the detection sensitivity of q PCR technology and was 10 times higher than that of conventional PCR.because.Therefore,it shows that the CRISPR-LbCas12 a technology established in this experiment has high specificity and sensitivity for E.amylovora visual detection system,and has great application potential in actual field detection.In this study,it was established that an asymmetric RPA amplification combined with CRISPR-LbCas12 a technology for visual detection of E.amylovora.Under constant temperature reaction conditions of 37 °C,the method greatly improved the reaction sensitivity through asymmetric RPA amplification,and broke through the PAM Due to the limitation of sequence(TTTV).Meanwhile the detection process is simple,and it required no large-scale detection equipment.The probe for bacterial DNA detection is versatile and can detect multiple samples within 50 minutes.The lower detection limit is 6.4×103 cfu·m L-1.It has good specificity and high sensitivity,low cost and high efficiency,and can identify field samples in actual production.