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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Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province, Ministry of Agriculture, Key Laboratory of Livestock Disease Prevention of Guangdong Province, Institute of Animal Health, Guangdong Academy of Agricultural Sciences</institution>,<addr-line>Guangzhou</addr-line>,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Bingga, Gali" sort="Bingga, Gali" uniqKey="Bingga G" first="Gali" last="Bingga">Gali Bingga</name><affiliation wicri:level="1"><nlm:aff id="aff2"><institution>Vocational and Technical College of Inner Mongolia Agricultural University</institution>,<addr-line>Baotou</addr-line>,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Zhang, Chunhong" sort="Zhang, Chunhong" uniqKey="Zhang C" first="Chunhong" last="Zhang">Chunhong Zhang</name><affiliation wicri:level="1"><nlm:aff id="aff1"><institution>Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province, Ministry of Agriculture, Key Laboratory of Livestock Disease Prevention of Guangdong Province, Institute of Animal Health, Guangdong Academy of Agricultural Sciences</institution>,<addr-line>Guangzhou</addr-line>,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Shao, Junjie" sort="Shao, Junjie" uniqKey="Shao J" first="Junjie" last="Shao">Junjie Shao</name><affiliation wicri:level="1"><nlm:aff id="aff3"><institution>Changzhou Wumu Animal Hospital</institution>,<addr-line>Changzhou</addr-line>,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Shen, Haiyan" sort="Shen, Haiyan" uniqKey="Shen H" first="Haiyan" last="Shen">Haiyan Shen</name><affiliation wicri:level="1"><nlm:aff id="aff1"><institution>Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province, Ministry of Agriculture, Key Laboratory of Livestock Disease Prevention of Guangdong Province, Institute of Animal Health, Guangdong Academy of Agricultural Sciences</institution>,<addr-line>Guangzhou</addr-line>,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Sun, Junying" sort="Sun, Junying" uniqKey="Sun J" first="Junying" last="Sun">Junying Sun</name><affiliation wicri:level="1"><nlm:aff id="aff1"><institution>Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province, Ministry of Agriculture, Key Laboratory of Livestock Disease Prevention of Guangdong Province, Institute of Animal Health, Guangdong Academy of Agricultural Sciences</institution>,<addr-line>Guangzhou</addr-line>,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Zhang, Jianfeng" sort="Zhang, Jianfeng" uniqKey="Zhang J" first="Jianfeng" last="Zhang">Jianfeng Zhang</name><affiliation wicri:level="1"><nlm:aff id="aff1"><institution>Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province, Ministry of Agriculture, Key Laboratory of Livestock Disease Prevention of Guangdong Province, Institute of Animal Health, Guangdong Academy of Agricultural Sciences</institution>,<addr-line>Guangzhou</addr-line>,<country>China</country></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author></analytic><series><title level="j">Frontiers in Microbiology</title><idno type="eISSN">1664-302X</idno><imprint><date when="2019">2019</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><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></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="An, D J" uniqKey="An D">D. J. An</name></author><author><name sortKey="Jeong, W" uniqKey="Jeong W">W. Jeong</name></author><author><name sortKey="Jeoung, H Y" uniqKey="Jeoung H">H. Y. Jeoung</name></author><author><name sortKey="Lee, M H" uniqKey="Lee M">M. H. Lee</name></author><author><name sortKey="Park, J Y" uniqKey="Park J">J. Y. 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Feng</name></author></analytic></biblStruct></listBibl></div1></back></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></noRegion><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></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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