LAMP-CRISPR/Cas12b rapid detection platform for canine parvovirus detection

Abstract

Canine parvovirus (CPV) is one of the main pathogens causing toxic diarrhea in Chinese dogs, which is the cause of large-scale epidemic of dogs, and poses a great threat to the dog industry in China. Rapid, sensitive, and specific CPV testing facilitates the timely diagnosis and treatment of sick dogs. The aim of this study was to build a LAMP-CRISPR/Cas12b platform for CPV detection. The loop mediated isothermal amplification (LAMP) technique was combined with CRISPR-Cas12b analysis to establish a “two-step” and “one-tube” CRISPR/Cas12b rapid CPV method, respectively. The detection system was constructed with specific LAMP amplification primers and single guide RNA (sgRNA) for the highly conserved short fragment of CPV gene, which could be detected within 1h without cross-reaction with the other viruses causing canine diarrhea. The detection limits of both “two-step” and “one-tube” CRISPR/Cas12b reactions were 10-1 copies/μL, which was 100 times sensitive than qPCR and LAMP.In order to achieve point-of-care testing (POCT) of CPV, a one-tube LAMP-CRISPR/Cas12b nucleic acid extraction and detection platform based on magnetic nanoparticle enrichment technology was established to achieve "sample in-result out".The results of this method on simulated samples were compared with those of quantitative Real-time PCR,the results showed 100% consistency, and the time was shorter, which could detect the diseased dogs earlier and provide the basis for clinical diagnosis. The LAMP-CRISPR/Cas12b method established in this study provides a sensitive and specific method for rapid detection of CPV, and provides technical support for rapid diagnosis of CPV.