Establishment And Application Of An Accurate And Rapid PRRSV Detection Method Based On Cas13a

Porcine reproductive and respiratory syndrome(PRRS)is a viral infection of porcine dysreproductive disorder.PRRS often lead to miscarriage of pregnant sows and large number of piglets dying.Its characteristics of persistent infection and difficult purification greatly increase its threat to the pig breeding industry in China.At present,the prevention and control methods of PRRS mainly include timely detection and diagnosis and vaccination.However,Porcine reproductive and respiratory syndrome virus(PRRSV)is prone to mutate and often recombines with vaccine strains to further increase difficulty of prevention and control.Therefore,the timely and accurate detection of PRRSV is very important.The CRISPR/Cas system is an acquired immune system widely found in bacteria and archaea.Because of its specific targeting function and cutting function,it is often used in gene editing and virus strain identification.With the continuous development of CRISPR/Cas system,CRISPR/Cas detection system has been widely used in clinical detection of a variety of viruses.Compared with RT-PCR,RT-q PCR and other methods widely used in PRRSV clinical detection,the nucleic acid detection method based on CRISPR/Cas system has the advantages of rapid and simple,visualization of detection results and low equipment dependence,and is easier to be popularized in the grassroots.In the CRISPR/Lw Cas13a detection system,CRISPR/Lw Cas13a protein was guided by cr RNA to recognize and bind the target RNA,and generated non-specific RNA enzymatic activity after binding,which carried out indiscriminate cutting of RNA in the detection system.The presence of target RNA can be determined by detecting the fluorescence produced by cutting the RNA pribe in the system.RT-RPA is a nucleic acid isothermal amplification of DNA template under the joint action of single-stranded DNA binding protein(SSB),strand-displacement DNA polymerase and recombinase.In this study,we combined RT-RPA and CRISPR/Lw Cas13a detection system to establish a two-step detection method of RT-RPA-Lw Cas13a to realize accurate and rapid visual detection of PRRSV.The main results of this study are as follows:(1)A CRISPR/Lw Cas13a detection system targeting PRRSV M gene was established by screening cr RNA with high activity,low background and high conserved in multiple strains of PRRSV.The concentration of each component and treatment conditions of the detection system were optimized.It was found that the combination of high activity and low background cr RNA could improve the detection speed and sensitivity of CRISPR/Lw Cas13a detection system targeting PRRSV M gene.(2)A cr RNA combination CRISPR/Lw Cas13a detection system targeting PRRSV M gene was optimized.The sensitivity of the optimized detection system reached6.0×10~7 copies/μL after incubation at 37°C for 45 min.(3)RT-RPA was combined with T7 in vitro transcription to achieve a one-tube reaction,and the reaction product was added into the optimized cr RNA combination CRISPR/Lw Cas13a detection system as a target for detection,and a two-step detection method of RT-RPA-Lw Cas13A targeting PRRSV M gene was constructed.The lowest detection limit(LOD)was 6.0 copies/μL,which was similar to the detection limit of RT-q PCR.(4)RT-RPA-Lw Cas13a two-step detection method and RT-q PCR were used to detect 20 unknown negative and positive samples,and the detection result coincidence rate reached 100%,with no false positive and false negative,suggesting that this detection method shows good specificity.(5)The two-step method of RT-RPA-Lw Cas13a was used to detect PRRSV M gene in pig serum.The positive and negative coincidence rate of the detection results and RT-q PCR results reached 100%,and the fluorescence value was completely consistent with the trend of PRRSV M gene copy number in samples.It provides a great possibility for the application of RT-RPA-Lw Cas13a two-step method in the clinical detection of PRRSV.To sum up,this study established a precise and rapid visual two-step detection method targeting PRRSV M gene RT-RPA-Lw Cas13a,achieving the lowest detection limit of 6.0copies/μL within 75 min,with good specificity,and the cr RNA used in the detection system was highly conserved in multiple strains of PRRSV.In addition,this two-step method can accurately detect PRRSV M gene in pig serum,which lays a foundation for its popularization in clinical detection.