Cargando…
Discriminating the eight genotypes of the porcine circovirus type 2 with TaqMan-based real-time PCR
BACKGROUND: The porcine circovirus type 2 (PCV2) is divided into eight genotypes including the previously described genotypes PCV2a to PCV2f and the two new genotypes PCV2g and PCV2h. PCV2 genotyping has become an important task in molecular epidemiology and to advance research on the prophylaxis an...
Autores principales: | , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2021
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8028161/ https://www.ncbi.nlm.nih.gov/pubmed/33827614 http://dx.doi.org/10.1186/s12985-021-01541-z |
_version_ | 1783675935114919936 |
---|---|
author | Link, Ellen Kathrin Eddicks, Matthias Nan, Liangliang Ritzmann, Mathias Sutter, Gerd Fux, Robert |
author_facet | Link, Ellen Kathrin Eddicks, Matthias Nan, Liangliang Ritzmann, Mathias Sutter, Gerd Fux, Robert |
author_sort | Link, Ellen Kathrin |
collection | PubMed |
description | BACKGROUND: The porcine circovirus type 2 (PCV2) is divided into eight genotypes including the previously described genotypes PCV2a to PCV2f and the two new genotypes PCV2g and PCV2h. PCV2 genotyping has become an important task in molecular epidemiology and to advance research on the prophylaxis and pathogenesis of PCV2 associated diseases. Standard genotyping of PCV2 is based on the sequencing of the viral genome or at least of the open reading frame 2. Although, the circovirus genome is small, classical sequencing is time consuming, expensive, less sensitive and less compatible with mass testing compared with modern real-time PCR assays. Here we report about a new PCV2 genotyping method using qPCR. METHODS: Based on the analysis of several hundred PCV2 full genome sequences, we identified PCV2 genotype specific sequences or single-nucleotide polymorphisms. We designed six TaqMan PCR assays that are specific for single genotypes PCV2a to PCV2f and two qPCRs targeting two genotypes simultaneously (PCV2g/PCV2d and PCV2h/PCV2c). To improve specific binding of oligonucleotide primers and TaqMan probes, we used locked nucleic acid technology. We evaluated amplification efficiency, diagnostic sensitivity and tested assay specificity for the respective genotypes. RESULTS: All eight PCV2 genotype specific qPCRs demonstrated appropriate amplification efficiencies between 91 and 97%. Testing samples from an epidemiological field study demonstrated a diagnostic sensitivity of the respective genotype specific qPCR that was comparable to a highly sensitive pan-PCV2 qPCR system. Genotype specificity of most qPCRs was excellent. Limited unspecific signals were obtained when a high viral load of PCV2b was tested with qPCRs targeting PCV2d or PCV2g. The same was true for the PCV2a specific qPCR when high copy numbers of PCV2d were tested. The qPCR targeting PCV2h/PCV2c showed some minor cross-reaction with PCV2d, PCV2f and PCV2g. CONCLUSION: Genotyping of PCV2 is important for routine diagnosis as well as for epidemiological studies. The introduced genotyping qPCR system is ideal for mass testing and should be a valuable complement to PCV2 sequencing, especially in the case of simultaneous infections with multiple PCV2 genotypes, subclinically infected animals or research studies that require large sample numbers. |
format | Online Article Text |
id | pubmed-8028161 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-80281612021-04-08 Discriminating the eight genotypes of the porcine circovirus type 2 with TaqMan-based real-time PCR Link, Ellen Kathrin Eddicks, Matthias Nan, Liangliang Ritzmann, Mathias Sutter, Gerd Fux, Robert Virol J Research BACKGROUND: The porcine circovirus type 2 (PCV2) is divided into eight genotypes including the previously described genotypes PCV2a to PCV2f and the two new genotypes PCV2g and PCV2h. PCV2 genotyping has become an important task in molecular epidemiology and to advance research on the prophylaxis and pathogenesis of PCV2 associated diseases. Standard genotyping of PCV2 is based on the sequencing of the viral genome or at least of the open reading frame 2. Although, the circovirus genome is small, classical sequencing is time consuming, expensive, less sensitive and less compatible with mass testing compared with modern real-time PCR assays. Here we report about a new PCV2 genotyping method using qPCR. METHODS: Based on the analysis of several hundred PCV2 full genome sequences, we identified PCV2 genotype specific sequences or single-nucleotide polymorphisms. We designed six TaqMan PCR assays that are specific for single genotypes PCV2a to PCV2f and two qPCRs targeting two genotypes simultaneously (PCV2g/PCV2d and PCV2h/PCV2c). To improve specific binding of oligonucleotide primers and TaqMan probes, we used locked nucleic acid technology. We evaluated amplification efficiency, diagnostic sensitivity and tested assay specificity for the respective genotypes. RESULTS: All eight PCV2 genotype specific qPCRs demonstrated appropriate amplification efficiencies between 91 and 97%. Testing samples from an epidemiological field study demonstrated a diagnostic sensitivity of the respective genotype specific qPCR that was comparable to a highly sensitive pan-PCV2 qPCR system. Genotype specificity of most qPCRs was excellent. Limited unspecific signals were obtained when a high viral load of PCV2b was tested with qPCRs targeting PCV2d or PCV2g. The same was true for the PCV2a specific qPCR when high copy numbers of PCV2d were tested. The qPCR targeting PCV2h/PCV2c showed some minor cross-reaction with PCV2d, PCV2f and PCV2g. CONCLUSION: Genotyping of PCV2 is important for routine diagnosis as well as for epidemiological studies. The introduced genotyping qPCR system is ideal for mass testing and should be a valuable complement to PCV2 sequencing, especially in the case of simultaneous infections with multiple PCV2 genotypes, subclinically infected animals or research studies that require large sample numbers. BioMed Central 2021-04-07 /pmc/articles/PMC8028161/ /pubmed/33827614 http://dx.doi.org/10.1186/s12985-021-01541-z Text en © The Author(s) 2021 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Research Link, Ellen Kathrin Eddicks, Matthias Nan, Liangliang Ritzmann, Mathias Sutter, Gerd Fux, Robert Discriminating the eight genotypes of the porcine circovirus type 2 with TaqMan-based real-time PCR |
title | Discriminating the eight genotypes of the porcine circovirus type 2 with TaqMan-based real-time PCR |
title_full | Discriminating the eight genotypes of the porcine circovirus type 2 with TaqMan-based real-time PCR |
title_fullStr | Discriminating the eight genotypes of the porcine circovirus type 2 with TaqMan-based real-time PCR |
title_full_unstemmed | Discriminating the eight genotypes of the porcine circovirus type 2 with TaqMan-based real-time PCR |
title_short | Discriminating the eight genotypes of the porcine circovirus type 2 with TaqMan-based real-time PCR |
title_sort | discriminating the eight genotypes of the porcine circovirus type 2 with taqman-based real-time pcr |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8028161/ https://www.ncbi.nlm.nih.gov/pubmed/33827614 http://dx.doi.org/10.1186/s12985-021-01541-z |
work_keys_str_mv | AT linkellenkathrin discriminatingtheeightgenotypesoftheporcinecircovirustype2withtaqmanbasedrealtimepcr AT eddicksmatthias discriminatingtheeightgenotypesoftheporcinecircovirustype2withtaqmanbasedrealtimepcr AT nanliangliang discriminatingtheeightgenotypesoftheporcinecircovirustype2withtaqmanbasedrealtimepcr AT ritzmannmathias discriminatingtheeightgenotypesoftheporcinecircovirustype2withtaqmanbasedrealtimepcr AT suttergerd discriminatingtheeightgenotypesoftheporcinecircovirustype2withtaqmanbasedrealtimepcr AT fuxrobert discriminatingtheeightgenotypesoftheporcinecircovirustype2withtaqmanbasedrealtimepcr |