Research On Rapid Detection Of Genetically Modified Crops Based On LAMP-CRISPR/Cas12a

Recently,more and more genetically modified organisms(GMOs)have been created.Correspondingly,the regulation of GM products in various countries is becoming more and more stringent,thus the rapid and accurate detection of GMOs is of great significance for their supervision.At present,the methods for detecting GMOs mainly include PCR technology based on DNA identification and isothermal amplification technology,but these technologies rely on expensive and complex instruments and equipment,which is time-consuming and labor-intensive.The in-depth study of the CRISPR/Cas system provides conditions for real-time field detection of GMOs.Among them,CRISPR/Cas12 a,from the type V-A CRISPR system,has been harnessed for molecular diagnostics on the basis of its trans activity on non-specific cleavage of ss DNA.The ss DNA reporter molecule with fluorophores and quencher groups(FAM-TTATT-BHQ1)is added to the reaction system to achieve visual detection of GMOs.Loop-mediated isothermal amplification(LAMP)is a method of amplifying target DNA at constant temperature(60-65 °C)using strand-displacement DNA polymerase(Bst DNA polymerase).This method does not require complex variable temperature instruments and is a rational choice for field testing.In this study,which combines LAMP with CRISPR/Cas12 a,a novel nucleic acid molecular diagnostic method developed can simply and quickly monitor GMOs by detecting p Ca MV35 S promoters,NOS terminators,NPTII or HPT.The main findings are as follows:(1)Firstly,in terms of isothermal amplification,we successfully screened specific LAMP primers that can amplify four target genes within 30 min.An alkaline PEG lysis buffer that can quickly extract plant leaf DNA was successfully improved,the buffer is compatible with LAMP buffer,and the target DNA fragment can be amplified from the crude genomic DNA extract without purification.The crude DNA extract was obtained by grinding the leaves with PEG lysis buffer,and it was found that the concentration of the DNA crude extract after dilution 500 times could also meet the amplification requirements of LAMP.The limit of detection(LOD)for LAMP amplification was determined.The LOD of p Ca MV35 S and HPT can reach 25 copies,the LOD of t NOS and NPTII can also reach 100 copies.(2)Secondly,in terms of CRISPR/Cas system detection,optimizing Cas digestion buffer-SF buffer,this buffer can significantly increase trans-cleavage activity.In addition,a hairpin structure reporter was designed,and the fluorescence signal value of this reporter gene was significantly increased compared with that of traditional ss DNA reporter.In this study,we successfully screened the cr RNA that targeted the target gene and was suitable for Lb-Cas12a-RR,the cr RNA of p Ca MV35 S,t NOS and HPT could complete the recognition of the target gene and be cleaved by Cas nuclease within 5 minutes,while the cr RNA of NPTII could complete the recognition and cleavage of the target gene within 10 minutes when the PAM was TCCC.In addition,cr RNAs exhibit good specificity and no cross-reactivity between different targets.The sensitivity of LAMP-CRISPR/Cas12 a was detected by flow strips,and it was found that the method could detect 0.01% of the GMOs of the DNA extracted using a CTAB extraction method.In the mixed detection of positive and negative samples,the ratio of p Ca MV35 S detected by this method reaches 1:29,and the ratio of t NOS,NPTII and HPT also reaches 1:9.In addition,this method can achieve dual determination of p Ca MV35 S and HPT.(3)Finally,to prevent amplicon contamination and improve the portability of detection,an integrated reaction device was designed to realize the reaction of LAMP and Cas digestion in the same closed tube.Flow strips or blue LED lights(470 nm)were used to visualize the test results.In order to determine the reliability of the new method based on LAMP-CRISPR/Cas12 a,a blind field examination was performed and the results of this method were found to be highly consistent with the traditional PCR results.In summary,a new method for molecular diagnosis of GMOs based on LAMPCRISPR/Cas12 a was established,which simplified the detection process of GMOs in the field,and could detect p Ca MV35 S,t NOS,NPTII and HPT under constant temperature conditions without large and complex instruments and equipment,realizing the characteristics of high sensitivity,good specificity,time-saving and labor-saving,and providing a new options for the detection of GMOs.