title: High resolution melting curve analysis as a new tool for rapid identification of canine parvovirus type 2 strains.
abstract: A high resolution melting (HRM) curve method was developed to identify canine parvovirus type 2 (CPV-2) strains by nested PCR. Two sets of primers, CPV-426F/426R and CPV-87R/87F, were designed that amplified a 52 bp and 53 bp product from the viral VP2 capsid gene. The region amplified by CPV-426F/426R included the A4062G and T4064A mutations in CPV-2a, CPV-2b and CPV-2c. The region amplified by CPV-87F/87R included the A3045T mutation in the vaccine strains of CPV-2 and CPV-2a, CPV-2b and CPV-2c. Faecal samples were obtained from 30 dogs that were CPV antigen-positive. The DNA was isolated from the faecal samples and PCR-amplified using the two sets of primers, and genotyped by HRM curve analysis. The PCR-HRM assay was able to distinguish single nucleotide polymorphisms between CPV-2a, CPV-2b and CPV-2c using CPV-426F/426R. CPV-2a was distinguished from CPV-2b and CPV-2c by differences in the melting temperature. CPV-2b and CPV-2c could be distinguished based on the shape of the melting curve after generating heteroduplexes using a CPV-2b reference sample. The vaccine strains of CPV-2 were identified using CPV-87F/87R. Conventional methods for genotyping CPV strains are labor intensive, expensive or time consuming; the present PCR-based HRM assay might be an attractive alternative.
authors.affiliation: Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, China Agricultural University, Beijing, China; Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Guangdong, China.
authors.forename: Gali
authors.initials: G
authors.lastname: Bingga
authors.affiliation: Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Guangdong, China.
authors.forename: Zhicheng
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Guangdong, China.
authors.forename: Jianfeng
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Guangzhou Bortzi Biotech Ltd, Guangzhou, China.
authors.forename: Yujun
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Guangdong, China.
authors.forename: Lifeng
authors.initials: L
authors.lastname: Lin
authors.affiliation: Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, China Agricultural University, Beijing, China. Electronic address: dingsy@cau.edu.cn.
authors.forename: Shuangyang
authors.initials: S
authors.lastname: Ding
authors.affiliation: Guangdong Key Laboratory of Laboratory Animals, Guangdong Laboratory Animals Monitoring Institute, Guangzhou, China. Electronic address: 1518227522@qq.com.
authors.forename: Pengju
authors.initials: P
authors.lastname: Guo
mesh_terms: D000818:Animals; D001483:Base Sequence; D036022:Capsid Proteins / Q000235:genetics*; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004283:Dog Diseases / Q000821:virology; D004285:Dogs; D005243:Feces / Q000821:virology; D005838:Genotype; D008969:Molecular Sequence Data; D009154:Mutation; D009691:Nucleic Acid Denaturation; D010322:Parvoviridae Infections / Q000821:virology; D017993:Parvovirus, Canine / Q000145:classification / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity
chemical_list: D036022:Capsid Proteins; D004279:DNA, Viral; C095548:capsid protein VP2, parvovirus B19
keywords: Canine parvovirus; Heteroduplex; High resolution melting curve analysis; PCR; VP2 gene