| In recent years,with the rapid development of China’s agricultural economy,planting high value-added crops has been bringing huge economic benefits to farmers.However,after years of overloading cultivation,lots of soil pathogens involving bacteria,viruses and fungi have accumulated in the soil.Under such circumstances,it is difficult to avoid the outbreak of large-scale soil-borne diseases within a certain period of time,which will bring huge economic losses to farmers.In particular,fungi,as one of the deadly pathogenic microorganisms,are abundant in soil and are not easy to be killed.In order to successfully remediate soil,reduce the deterioration of soil disease,build a rapid and portable diagnostic method for soil-borne diseases,and return the soil to a natural and healthy state is imperative.At present,although there are many methods for detecting pathogenic bacteria,most of them are cumbersome in steps,time-consuming and highly dependent on instruments,which cannot meet the needs of rapid on-site detection.To this end,we first combined the recombinase polymerase amplification（RPA）technology with the CRISPR/Cas system to construct a rapid and sensitive detection system,which named RPA/CRISPR/Cas12a.We also developed a portable Slip Chip that combines the RPA/CRISPR/Cas12a detection system can realize the digital detection of pathogenic bacteria on the chip,reduce the risk of contamination.The developed RPA/CRISPR/Cas12a/Slip Chip detection stragedy does not require complex instruments and equipment,and can meet the needs of on-site point-of-care detection with high sensitivity and specificity.1.The target fungi included Phytophthora hibernalis and 11 similar species involving Phytophthora hibernalis,Phytophthora syringae,Phytophthora capsici,Phytophthora cinnamomi,Phytophthora palmivora,Phytophthora niederhauserii,Phytophthora cactorum,Pythium oligandrum,Phytophthora citrophthora,Pythium ultimum,Pythium aphanidermatum,and Phytophthora ramorum.The RPA primers and probes were designed according to the ITS sequences using DNAMAN 8.0.For optimal conditions,the optimal Mg OAc concentration was 14 m M,and the optimal primer concentration was 420 n M,and the amplification was performed at 39°C for 30 minutes.Compared with the PCR amplification(detection of Phytophthora hibernalis was 1×10-1 ng/μL),the sensitivity of RPA detection of Phytophthora hibernalis was 1×10-2 ng/μL,which can improve detection sensitivity by an order of magnitude.2.Integtating RPA and CRISPR/Cas12a,i.e.RPA/CRISPR/Cas12a,could meet the requirements of rapid on-site detection of Phytophthora hibernalis with the detection limit of1×10-2 ng/μL,which was the same as that of the RPA method,but the RPA/CRISPR/Cas12a method showed a higher fluorescence signal when the concentration of 1×10-2 ng/μL genomic DNA was detected..3.In the practical application of RPA/CRISPR/Cas12a,a portable incubator pad or box was used to provide the required temperature（37～40℃）for the RPA pre-amplification and Cas12a/cr RNA reaction.Thus,this RPA/CRISPR/Cas12a could be applied in remote field or at port for detecting Phytophthora hibernalis on suspected plant,monitoring the disease occurrence,thereby timely management would be used to prevent the spread of the aggressive pathogen. |
