The Isolation Of 5 CPV-2 Strains And The Detection Of CPV-2 Based On CRISPR Cas13a Techonology

The canine parvovirus disease is caused by canine parvovirus type 2(CPV-2).The disease is highly contagious infectious,which has brought great damage to the canine industry.Recent evidence suggests that more and more CPV-2 virulent strains have presented,while few studies have investigated the systematic epidemiological investigation of canine parvovirus disease in China.In this study,five CPV-2 strains were isolated from canine specimens collected in Wuhan through continuous passage in vitro.Further genotypic analysis showed that four of the five strains belong to CPV-2c subtype and the other one was CPV-2a subtype.Given the uneven subtype distribution,the full-length sequences of CPV-2 VP2 gene from China published in NCBI were collected and the relevant information was summarized.The epidemiological findings show that among the strains isolated in this study,one strain is closely related to the vaccine strain NL-35-D,while the other four strains cluster separately,suggesting local prevail trending characteristics.Further analysis revealed a significant subtype switching of CPV-2 in China,with the dominant CPV-2 strain converting from subtype 2a to subtype 2c during the past 20 years.The SHERLOCK,an emerging detection tool,is based on the CRISPR/Cas type VI system.With multiple advantages concluding high sensitivity and specificity as well as simple reaction conditions,this method is potential to be a new generation of pathogen detection assay.In this study,we constructed the CPV-2-detection SHERLOCK system based on the conserved sequence on VP2 gene.Through progressively optimizing the reaction conditions of CPV-2 Recombinase polymerase amplification(RPA)and CPV-2CRISPR/Cas13 a,the component of CPV-2 RPA-CRISPR system was established.The data show that the CPV-2 RPA-CRISPR system established in this study can be completed at 37°C for 1 h;the system has both good sensitivity and specificity,no crossreactivity with other canine viral diarrhoea pathogens(CCo V,CDV,CAV,Ca HV,CCV),and is able to detect viral sequences at 1 copy level;the system can work in a clinical setting,and for the 50 clinical samples tested by CPV-2 RPA-CRISPR system,there was100% overlap with the PCR method.In conclusion,a rapid,specific and sensitive method for CPV-2 detection has been established in this study,which has shown good potentiality for virus-detecting application.In addition,the data in this study provides theoretical support for the prevention of canine parvovirus disease.