Crispr-Cas13a Based Detection Of Canine Parvovirus Type 2

Canine parvovirus type 2(CPV-2)infection is a lethal disease of dogs with higher mortality in puppies worldwide.It is crucial to find out a simple,accurate,portable,and economic CPV-2 detection method,with suitability for on-site detection.CRISPR-Cas13 a is an RNAactivated ribonuclease(RNase)capable of cr RNA processing and foreign RNA degradation upon binding.In this study,we expressed Lw Cas13 a protein in a prokaryotic expression system and purified it by nickel column.CRISPR-Cas13 a was then combined with recombinase polymerase amplification(RPA)to establish a molecular system for the nucleic acid detection,termed as specific high sensitivity enzymatic reporter unlocking(SHERLOCK)that can be employed for rapid and analytically sensitive nucleic acid detection.Harnessing SHERLOCK,we established a system for the on-site detection of CPV-2 DNA.The main results are listed below:1.The IPTG induced Cas13 a protein,expressed by E.coli was collected and confirmed by SDS-PAGE with clear protein bands.2.For checking the activity and specificity of Cas13 a we made four groups,two negative controls and two experimental groups.The results showed that the fluorescence values of the experimental groups were significantly higher than the negative control groups,validating that Cas13 a protein has RNase activity,and possess excellent specificity for the target.3.To evaluate the minimum detection limit and time of the detection system,we used known sample concentrations with different time intervals.To improve the detection sensitivity of the system,RPA and T7 in vitro transcription were harnessed,enabling the detection of CPV-2 DNA at concentration as low as 100 a M within 30 minutes.In short,we established a system for the detection of CPV-2 virus with significant sensitivity and specificity.In future,our system can be used for the rapid on-site detection of CPV-2during the disease outbreak.