Application of Duplex Fluorescence Melting Curve Analysis (FMCA) to Identify Canine Parvovirus Type 2 Variants
Identifieur interne : 000852 ( Ncbi/Checkpoint ); précédent : 000851; suivant : 000853Application of Duplex Fluorescence Melting Curve Analysis (FMCA) to Identify Canine Parvovirus Type 2 Variants
Auteurs : Zhicheng Liu [République populaire de Chine] ; Gali Bingga [République populaire de Chine] ; Chunhong Zhang [République populaire de Chine] ; Junjie Shao [République populaire de Chine] ; Haiyan Shen [République populaire de Chine] ; Junying Sun [République populaire de Chine] ; Jianfeng Zhang [République populaire de Chine]Source :
- Frontiers in Microbiology [ 1664-302X ] ; 2019.
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.
Url:
DOI: 10.3389/fmicb.2019.00419
PubMed: 30891024
PubMed Central: 6411689
Affiliations:
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<front><div type="abstract" xml:lang="en"><p>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.</p>
</div>
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</TEI>
<affiliations><list><country><li>République populaire de Chine</li>
</country>
</list>
<tree><country name="République populaire de Chine"><noRegion><name sortKey="Liu, Zhicheng" sort="Liu, Zhicheng" uniqKey="Liu Z" first="Zhicheng" last="Liu">Zhicheng Liu</name>
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<name sortKey="Bingga, Gali" sort="Bingga, Gali" uniqKey="Bingga G" first="Gali" last="Bingga">Gali Bingga</name>
<name sortKey="Shao, Junjie" sort="Shao, Junjie" uniqKey="Shao J" first="Junjie" last="Shao">Junjie Shao</name>
<name sortKey="Shen, Haiyan" sort="Shen, Haiyan" uniqKey="Shen H" first="Haiyan" last="Shen">Haiyan Shen</name>
<name sortKey="Sun, Junying" sort="Sun, Junying" uniqKey="Sun J" first="Junying" last="Sun">Junying Sun</name>
<name sortKey="Zhang, Chunhong" sort="Zhang, Chunhong" uniqKey="Zhang C" first="Chunhong" last="Zhang">Chunhong Zhang</name>
<name sortKey="Zhang, Jianfeng" sort="Zhang, Jianfeng" uniqKey="Zhang J" first="Jianfeng" last="Zhang">Jianfeng Zhang</name>
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