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Mol Cell Probes. 2014 Oct-Dec;28(5-6):271-8. doi: 10.1016/j.mcp.2014.08.001. Epub 2014 Aug 23.

High resolution melting curve analysis as a new tool for rapid identification of canine parvovirus type 2 strains.

Author information

1
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.
2
Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Guangdong, China.
3
Guangzhou Bortzi Biotech Ltd, Guangzhou, China.
4
Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, China Agricultural University, Beijing, China. Electronic address: dingsy@cau.edu.cn.
5
Guangdong Key Laboratory of Laboratory Animals, Guangdong Laboratory Animals Monitoring Institute, Guangzhou, China. Electronic address: 1518227522@qq.com.

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.

KEYWORDS:

Canine parvovirus; Heteroduplex; High resolution melting curve analysis; PCR; VP2 gene

PMID:
25159576
DOI:
10.1016/j.mcp.2014.08.001
[Indexed for MEDLINE]

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