Toward establishing a rapid constant temperature detection method for canine parvovirus based on endonuclease activities

Abstract

ABSTRACT Canine parvovirus (CPV) can cause high morbidity and mortality rates in puppies, posing a significant threat to both pet dogs and the breeding industry. Rapid, accurate, and convenient detection methods are important for the early intervention and treatment of canine parvovirus. In this study, we propose a visual CPV detection system called nucleic acid mismatch enzyme digestion (NMED). This system combines loop-mediated isothermal amplification (LAMP), endonuclease for gene mismatch detection, and colloidal gold lateral chromatography. We demonstrated that NMED can induce the binding of the amplicon from the sample to the specific labeling probe, which in turn triggers digestion by the endonuclease. The sensitivity and visual visibility of LAMP were increased by combining endonuclease and colloidal gold lateral chromatography assisted by a simple temperature-controlled device. The sensitivity of the NMED assay was 1 copy/μL, which was consistent with quantitative PCR (qPCR). The method was validated with 20 clinical samples that potentially had CPV infection; 15 positive samples and 5 negative samples were evaluated; and the detection accuracy was consistent with that of qPCR. As a rapid, accurate, and convenient molecular diagnostic method, NMED has great potential for application in the field of pathogenic microorganism detection.IMPORTANCEThe NMED method has been established in the laboratory and used for CPV detection. The method has several advantages, including simple sampling, high sensitivity, intuitive results, and no requirement for expensive equipment. The establishment of this method has commercial potential and offers a novel approach and concept for the future development of clinical detection of pathogenic microorganisms.

Keywords

• canine parvovirus
• endonuclease
• loop-mediated isothermal amplification
• specific labeling probe
• lateral chromatography