Establishment Of CRISPR-Cas12a Detection Method For Infectious Bursal Disease Virus And Its Application

Infectious Bursal disease(IBD)is a highly contagious disease of chickens caused by Infectious Bursal disease virus(IBDV).The disease is widely spread around the world,resulting in reduced immunosuppressive performance of chicken flocks and failure of immunization with other vaccines,which poses a serious threat and huge economic loss to the poultry industry.The simple,rapid and specific nucleic acid detection method is conducive to the early diagnosis of the disease,promoting early intervention and prevention and control,which is of a great significance for reducing the spread of the disease and reducing the economic loss of the poultry industry.In addition,the simple,rapid and specific nucleic acid detection method is also an important means of effective prevention and control of epidemic diseases in large-scale production and food quarantine,and it is also of great significance for food safety.So far,many detection methods have been used to detect IBDV.But,these existing detection methods cannot achieve effective detection of IBDV because of one or several shortcomings,such as complex operation steps,long reaction time,low sensitivity,false positive,poor detection specificity,and the need for expensive equipment and professionals.Recently,RNA-guided CRISPR-Cas nucleic acid detection method has shown great potential in rapid,efficient and specific nucleic acid detection and opening a new era of molecular diagnosis with the in-depth study of CRISPR-Cas gene editing system and the discovery of non-specific cleavage activity of some Cas proteins.Therefore,we established a visual fluorescence detection method for IBDV by adding ss DNA reporter groups modified by fluorescence groups and quenching groups into the detection system based on the non-specific single strand DNA cutting activity of CRISPR-Cas12 a system and combined with RPA in this study.The main content of this study is divided into the following three parts.(1)Expression,purification and activity identification of Cas12 a recombinant plasmid.The recombinant plasmid(p ET28-6×His-MBP-TEV-hu Lb Cas12a)was transformed into Escherichia coli Rosetta(DE3),BL21Star(DE3)and BL21(DE3),which were identified by SDSPAGE and Western blot after induction by IPTG.The results showed that the recombinant Cas12 a protein was highly expressed in Escherichia coli.We designed three cr RNAs at the conserved region of IBDV and the best cr RNA was screened out and a preliminary detection method of IBDV using CRISPR-Cas12 a was established.We used a recombinant Cas12 a protein with prokaryotic expression to replace the commercial Cas12 a protein in the CRISPR-Cas12 a assay system.The results showed that the recombinant Cas12 a protein with prokaryotic expression could specifically recognize IBDV sequences and non-specifically cut fluorescently labeled single-strand DNA reporter group(FQ-SSDNA),resulting in specificity fluorescent.(2)Establishment and evaluation of visual fluorescence method for detection of IBDV based on RPA-CRISPR-Cas12 a.First,we established an RPA detection method for IBDV.Then,we optimized the buffer system suitable for RPA amplification and CRISPR-Cas12 a experiment.RPA amplification and CRISPR-Cas12 a based visual fluorescence detection were successfully combined in a single detection system and a single reaction condition was used to detect IBDV.The detection process only needs 50 min reaction at 37 ℃ to complete,and the detection results can be observed by ultraviolet light or blue light.We named this method "RPA-Cas12 a DS".Finally,we used the method of RPA-Cas12 a DS to detect IBDV and other pathogens in poultry,and used the method of RT-PCR as a standard control to evaluate its specificity.The results showed that this method had good specificity for detecting IBDV,and the detection result was consistent with that of RT-PCR.The sensitivity of RPA-Cas12 a DS to detect IBDV was further evaluated,and real-time PCR(q PCR)was used as the standard control.The results showed that the lower limit of RPA-Cas12 a DS detection was 277 copies,while the lower limit of q PCR detection was 616 copies.Therefore,the sensitivity of RPA-Cas12 a DS is better than that of q PCR for IBDV,and its sensitivity is about two times of q PCR.(3)Application of RPA-Cas12aDS method in clinical and chicken detection.To evaluate the effect of the RPA-Cas12 a DS method in clinical and food detection,we applied the RPACas12 a DS method to the diagnosis of IBDV in clinical samples and chicken,and the commercial RT-PCR kit as a control.The results showed that the RPA-Cas12 a DS method was in good agreement with the commercial RT-PCR kit,indicating that the RPA-Cas12 a DS method established in this study can be used for the clinical detection of IBDV in poultry and chicken.In conclusion,we established a rapid,simple,specific and visual detection method for IBDV by combining recombinant enzyme polymerase amplification(RPA)with CRISPRCas12 a based nucleic acid detection in this study.We named it "RPA-Cas12 a DS".RPACas12 a DS method can detect IBDV in less than 50 minutes at 37 ℃.The detection process avoids contamination caused by lid opening and the detection results can be directly observed under blue or ultraviolet light.The whole detection process does not need complex instruments and equipment,and the method has the potential application for point-of-care testing.In addition,the application of RPA-Cas12 a DS method in the detection of infectious bursal disease virus in chicken will help to improve the biological safety of chicken food and strengthen the food production standards.