Development Of Rapid Visual Detection Technology For Nosema Bombycis Based On CRISPR/Cas12a System

Pebrine disease of silkworm is an infectious protozoan disease caused by Nosema bombycis.It can cause a large number of deaths of silkworm within 5-7 days through oral and traumatic infection,and can be vertically transmitted to the next generation through silkworm eggs,seriously endangering the high-quality development of the sericulture industry.At present,the main diagnostic technology of pebrine disease of silkworm is the microscopic examination method of female moth,which is time-consuming and labor-consuming,and lacks in timeliness,sensitivity and accuracy.Therefore,it is extremely necessary to develop a new detection technology with rapid,high sensitivity and high specificity for Nosema bombycis,so that it can be applied to grassroots silkworm seed stations and quarantine departments,and become a routine detection method.In recent years,CRISPR/Cas system has not only been widely used in gene editing of humans and other organisms,but also played an important role in pathogen detection,food safety quarantine,environmental ecological detection and other fields.Since the development of CRISPR/Cas12a nucleic acid detection system,combined with PCR amplification technology or LAMP,RPA and other constant temperature amplification technologies,not only has shown ultra-high sensitivity and specificity in improving the reliability of detection results,but also rapidly stood out from many nucleic acid detection technologies due to its extremely short detection time,portable design and low detection cost,by the majority of researchers and grassroots medical institutions high attention and favor.At present,this technology has been successfully applied to the visual rapid diagnosis of a variety of malignant viruses and serious diseases,such as Severe Acute Respiratory Syndrome Coronavirus 2（SARS-Co V-2）,human immunodeficiencyvirus（HIV）,human papilloma virus（HPV）,Zika virus（ZIKV）,dengue virus（DENV）,influenza A and B virus,malignant malaria,etc.,but it has not been reported in the detection of insect pathogens.In order to break through the application limitations of the existing detection technology of Nosema bombycis,this study independently expressed in large quantities and purified As Cas12a protein,combined with RPA technology and the use of microplate reader and immunochromatographic strips,successfully established a new rapid visual detection technology for Nosema bombycis.The main research results are as follows:1.Mass expression and purification of As Cas12a proteinen As Cas12a-HF1proteinwasexpressedby p ET-28b-T7-hen As Cas12a-HF1-NLS-6×His expression vector.It was found that when the induction temperature was 16℃and 20℃,the optimal concentration of IPTG to induce the expression of As Cas12a protein was 0.25 m M and 0.75 m M respectively.When the induction conditions were 0.25 m M IPTG at 16℃and 0.75 m M IPTG at 20℃,As Cas12a protein was mainly expressed in the form of inclusion body protein and soluble protein,respectively.In this study,soluble As Cas12a protein was expressed in large quantities under the induction condition of 0.75 m M IPTG at 20℃,and relatively pure As Cas12a protein was purified by affinity chromatography.The purified As Cas12a protein concentration was up to 1.254 mg/m L,and its biological activity was confirmed.2.Establishment and optimization of rapid visual detection technology of Nosema bombycis based on CRISPR/Cas12a systemBased on the nucleotide sequence of Nbssu gene and the PAM sequence recognized by As Cas12a protein,the detection sites of Nbssu-61 and Nbssu-915 were selected and successfully amplified by RPA technology.Through in vitro transcription experiments,cr RNA was successfully obtained to recognize different detection sites.Different ss DNA reporter molecules were designed according to different detection tools.Through the optimization of different reaction components or reaction time,it was found that based on the detection site of Nbssu-61,the optimal reaction system of CRISPR/Cas12a fluorescence detection technology is:5μL NEBuffer ^TMr2.1,15 n M As Cas12a protein,5 n M cr RNA,5n M ss DNA reporter molecule,2μL RPA product,dd H₂O supplemented to 50μL;The optimal reaction system of CRISPR/Cas12a immunochromatographic detection technology is:5μL NEBuffer ^TMr2.1,50 n M As Cas12a protein,25 n M cr RNA,300 n M ss DNA reporter molecule,2μL RPA product,dd H₂O supplemented to 50μL.For CRISPR/Cas12a immunochromatographic detection technology,the optimal time of CRISPR/Cas12a reaction was 25 min,the optimal incubation buffer was 5%PEG,and the optimal incubation time was 5 min.3.Evaluation of rapid visual detection technology of Nosema bombycis based on CRISPR/Cas12a systemThe rapid visual detection technology of Nosema bombycis based on CRISPR/Cas12a system was used to detect the standard of positive plasmid,the standard of Nosema bombycis genome and the standard of simulated infected silkworm eggs.It was found that the detection sensitivity of the technology to the above standards was 2 copies/μL,2 fg/μL and 10²spores/100 eggs,respectively,which was 100-1000 times more sensitive than the PCR assay and showed high sensitivity.In addition,CRISPR/Cas12a detection technology was used to detect the midgut tissues of silkworm infected with Bombyx mori nucleopolyhedrovirus（Bm NPV）,Bombyx mori cytoplasmic polyhedrosis virus（Bm CPV）,Bombyx mori bidensovirus（Bm BDV）and Beauveria bassiana.It was found that the detection results of the technology were negative except for positive control samples,indicating that the technology has good specificity.Finally,actual samples of silkworm larvae,pupae,moth,mixed silkworm egg and newly hatched larvae infected with Nosema bombycis were detected with CRISPR/Cas12a,and the study confirmed that the technology has high accuracy.By adding different compounds to the optimal reaction system of CRISPR/Cas12a fluorescence detection technology,it was found that the fluorescence response of CRISPR/As Cas12a was significantly enhanced when 0.1 M L-Proline was added with 1%～10%Glycerol.In summary,this study established a new rapid visual detection technology of Nosema bombycis,which has the advantage of rapidness,accuracy,visualization,low cost,low equipment requirements and simple operation,and is suitable for the on-site detection of Nosema bombycis.It can be popularized and demonstrated in grassroots silkworm seed stations and quarantine departments at all levels,so as to control the occurrence and spread of pebrine disease of silkworm in the early stage,and provide guarantee for the safe production and high-quality development of sericulture industry.