Isolation And Identification Of Canine Parvovirus And Development Of A Loop Mediated Isothermal Amplification Method

Canine parvovirus is one of the major pathogens that cause bleeding enteritis and myocarditis in dogs.CPV was highly contagious.Its infectious ratio is 20%～100%,and its mortality is 10%～50%.The wide prevalence and spreading of the disease brought out serious threat to dog industry and huge loss in many nations and regions in the world. The study comprised two parts:Part1. To study the variations and prevalence of Canine parvovirus infected dogs in Shanghai of China.The result of studies indicated: Virus has been isolated from fecal samples of suspected canine parvovirus infected cases from Shanghai Pet Hospital, by morphology, physical chemistry and molecular virology we detected a canine parvovirus.Cell culture of canine parvovirus shows cell swelling, and there appears small particles in the cytoplasm.The virus particles are approximately 20～24 nm in diameter and have not cyst membrane at the electron microscope. Animal experiments showed that the test group suffered from clinical symptoms of canine parvovirus.Primers were designed on the basis of the complete genomic sequence of three Canine parvovirus serotypes in the GenBank, and the genomic sequence was amplified by PCR. The PCR products were cloned to T vector and sequenced, phylogenetic tree was constructed by the Neighbor-joining method with Mega 4 software. The complete genomic sequence gene of CPV-SH was 4 758bp in length, and compared with the VP2 gene of those three Canine parvovirus serotypes in GenBank.The result revealed that the highest homology is with genotype 2a (99.7 %) ,with 2 genotypes,2b genotypes and 2c genotypes of homology were 98.8%,99.5%,99.5%. We performed the phylogenetic analysis based on amino acid sequences of capsid protein of CPV-SH and 20 other referenced representative strains from different regions. Results confirmed that CPV-SH belonged to genotype 2a and closely clustered with a strain of china (CPV/BJ082).Part2. Study on a loop-mediated isothermal amplification (LAMP) method to detect canine parvovirus (CPV).We developed a loop-mediated isothermal amplification (LAMP) method to detect canine parvovirus (CPV) genomic DNA. A set of primers, which composed of outer primers, inner primers and loop primers was designed from a published CPV sequence data. The experiments results showed that the optimal reaction time and temperature for LAMP were determined to be 60 min at 63℃. LAMP turned out to be more sensitive than conventional PCR, with a detection limit of 5.7 copies of CPV genomic DNA per reaction. No cross reactivity was observed from the samples of other related viruses including Canine distemper virus (CDV), parvovirus B19, porcine parvovirus. The result indicated that as a simple, rapid procedure for the detection of CPV in a clinical setting, the technique had potential usefulness.