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

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 world...

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Autores principales: Liu, Zhicheng, Bingga, Gali, Zhang, Chunhong, Shao, Junjie, Shen, Haiyan, Sun, Junying, Zhang, Jianfeng
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411689/
https://www.ncbi.nlm.nih.gov/pubmed/30891024
http://dx.doi.org/10.3389/fmicb.2019.00419
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author Liu, Zhicheng
Bingga, Gali
Zhang, Chunhong
Shao, Junjie
Shen, Haiyan
Sun, Junying
Zhang, Jianfeng
author_facet Liu, Zhicheng
Bingga, Gali
Zhang, Chunhong
Shao, Junjie
Shen, Haiyan
Sun, Junying
Zhang, Jianfeng
author_sort Liu, Zhicheng
collection PubMed
description 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.
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spelling pubmed-64116892019-03-19 Application of Duplex Fluorescence Melting Curve Analysis (FMCA) to Identify Canine Parvovirus Type 2 Variants Liu, Zhicheng Bingga, Gali Zhang, Chunhong Shao, Junjie Shen, Haiyan Sun, Junying Zhang, Jianfeng Front Microbiol Microbiology 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. Frontiers Media S.A. 2019-03-05 /pmc/articles/PMC6411689/ /pubmed/30891024 http://dx.doi.org/10.3389/fmicb.2019.00419 Text en Copyright © 2019 Liu, Bingga, Zhang, Shao, Shen, Sun and Zhang. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Liu, Zhicheng
Bingga, Gali
Zhang, Chunhong
Shao, Junjie
Shen, Haiyan
Sun, Junying
Zhang, Jianfeng
Application of Duplex Fluorescence Melting Curve Analysis (FMCA) to Identify Canine Parvovirus Type 2 Variants
title Application of Duplex Fluorescence Melting Curve Analysis (FMCA) to Identify Canine Parvovirus Type 2 Variants
title_full Application of Duplex Fluorescence Melting Curve Analysis (FMCA) to Identify Canine Parvovirus Type 2 Variants
title_fullStr Application of Duplex Fluorescence Melting Curve Analysis (FMCA) to Identify Canine Parvovirus Type 2 Variants
title_full_unstemmed Application of Duplex Fluorescence Melting Curve Analysis (FMCA) to Identify Canine Parvovirus Type 2 Variants
title_short Application of Duplex Fluorescence Melting Curve Analysis (FMCA) to Identify Canine Parvovirus Type 2 Variants
title_sort application of duplex fluorescence melting curve analysis (fmca) to identify canine parvovirus type 2 variants
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411689/
https://www.ncbi.nlm.nih.gov/pubmed/30891024
http://dx.doi.org/10.3389/fmicb.2019.00419
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