Application of Duplex Fluorescence Melting Curve Analysis (FMCA) to Identify Canine Parvovirus Type 2 Variants.

Zhicheng Liu,Junjie Shao,Chunhong Zhang,Jianfeng Zhang,Gali Bingga,Haiyan Shen,Junying Sun

doi:10.3389/fmicb.2019.00419

Abstract

Canine parvovirus (CPV-2) is an enteric virus causing morbidity and mortality in dogs worldwide. Since CPV-2 emerged as canine pathogen, the original CPV-2 strain has constantly evolved, and its primary variants (CPV-2a, CPV-2b, and CPV-2c) co-circulate to varying extents in canine populations worldwide. Thus, rapid and accurate laboratory diagnoses of CPV-2 variants are crucial to monitor CPV-2 evolution. Conventional methods for CPV-2 genotyping are laborious, time consuming, and determining the genotype of a CPV-2 variant often requires two or more reaction tubes. The present study developed a probe-based fluorescence melting curve analysis (FMCA) for genotyping six different CPV-2 variants (original CPV-2, CPV-2a, CPV-2b, CPV-2c, and vaccine strains of CPVpf and CPVint) in a single reaction tube using only two TaqMan probes. One of the TaqMan probes (FAM labeled) was designed to perfectly match with the target sequence of CPV-2a, this probe allows a 1-bp mismatched hybridization with the CPV-2b VP2 gene region (A4062G), and a 2-bp mismatched hybridization for CPV-2c (A4062G and T4064A); Another TaqMan probe (HEX labeled) was produced to perfectly match with the target sequence of original CPV-2, this probe enables 1-bp mismatched hybridization with the other CPV-2 variants (A3045T). Using the two TaqMan probes, all six CPV-2 variants were readily distinguished by their respective melting temperature values in a single reaction tube. The detection limits of this assay were 1–10 copies per reaction for six CPV-2 construction plasmids and no cross reactions were observed with several other common canine viruses. In this assay, co-infected samples were also directly identified via probe-based FMCA without using a mixing control; only a pure control is required. The clinical evaluation of this assay was demonstrated by analyzing 83 clinical fecal samples, among which 41 (49.39%), 8 (9.63%), and 14 (16.87%) samples were found to be positive for CPV-2a, CPV-2b, and CPV-2c, respectively. The concordance rate between probe-based FMCA and Sanger sequencing was 100%. Thus, the duplex FMCA is effective, rapid, simple, high-throughput, and straightforward for genotyping CPV-2 variants, and is useful to effectively diagnose and monitor CPV-2 epidemiology.

Highlights

Canine parvovirus type 2 (CPV-2), which was first identified in 1977–1978, causes acute gastroenteritis and leukopenia in dogs
Five plasmids of pCPV-2a, pCPV-2b, pCPV-2c, pCPVpf, and pCPVint were generated using the pMD18-T Vector Cloning Kit (Takara, Dalian, China), and the insert fragments of the plasmids were amplified from CPV-js1
The vaccine strain of CPVpf yielded a Tm value of 52.47 ± 0.33◦C, which was only 0.60◦C higher than that of CPV-2b, the Tm difference between them was 5.16◦C in the HEX channel

Summary

Introduction

Canine parvovirus type 2 (CPV-2), which was first identified in 1977–1978, causes acute gastroenteritis and leukopenia in dogs. A new antigenic variant (CPV-2a) completely replaced the originally identified CPV-2 virus between 1979 and 1980. Additional antigenic variants, CPV-2b and CPV-2c, were identified in 1984 (Parrish et al, 1991) and 2000 (Buonavoglia et al, 2001), respectively. Novel antigenic variants of CPV-2a/2b are still being identified, including new CPV-2a, new CPV-2b (Ohshima et al, 2008), Asp-300(2a), and Asp-300(2b) (Ikeda et al, 2000). The CPV-2 variant originally identified in 1977 can only be found in some vaccine strains. The primary determinants of the CPV-2 genotype are codons 84, 87, 101, 297, 300, 425, and 426 in the VP2 protein (Decaro et al, 2006a,b,c; Yoon et al, 2009; Decaro and Buonavoglia, 2012; Bingga et al, 2014)

Methods

Results

Conclusion