A quadruplex real-time PCR assay combined with a conventional PCR for the differential detection of Marek’s disease virus vaccines and field strains
To evaluate the effect of the vaccine and differentiate vaccine from virulent MDV, a new quadruplex real-time PCR assay based on TaqMan probes was developed to differentiate and accurately quantify HVT, CVI988 and virulent MDV-1. The results showed that the limit of detection (LOD) of the new assay...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2023-05-01
|
| Series: | Frontiers in Veterinary Science |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fvets.2023.1161441/full |
| _version_ | 1827949668044111872 |
|---|---|
| author | Shaopeng Wu Shaopeng Wu Shaopeng Wu Tian Ding Tian Ding Tian Ding Hongxia Shao Hongxia Shao Hongxia Shao Kun Qian Kun Qian Kun Qian Jianqiang Ye Jianqiang Ye Jianqiang Ye Aijian Qin Aijian Qin Aijian Qin |
| author_facet | Shaopeng Wu Shaopeng Wu Shaopeng Wu Tian Ding Tian Ding Tian Ding Hongxia Shao Hongxia Shao Hongxia Shao Kun Qian Kun Qian Kun Qian Jianqiang Ye Jianqiang Ye Jianqiang Ye Aijian Qin Aijian Qin Aijian Qin |
| author_sort | Shaopeng Wu |
| collection | DOAJ |
| description | To evaluate the effect of the vaccine and differentiate vaccine from virulent MDV, a new quadruplex real-time PCR assay based on TaqMan probes was developed to differentiate and accurately quantify HVT, CVI988 and virulent MDV-1. The results showed that the limit of detection (LOD) of the new assay was 10 copies with correlation coefficients >0.994 of CVI988, HVT and virulent MDV DNA molecules without cross-reactivity with other avian disease viruses. The intra-assay and inter-assay coefficients of variation (CVs) of Ct values for the new assay were less than 3%. Analysis of replication kinetics of CVI988 and virulent MDV of collected feathers between 7 and 60 days post-infection (dpi) showed MD5 had no significant effect on the genomic load of CVI988 (p > 0.05), while vaccination with CVI988 could significantly reduce the viral load of MD5 (p < 0.05). Combined with meq gene PCR, this method can effectively identify virulent MDV infections in immunized chickens. These results demonstrated that this assay could distinguish between the vaccine and virulent MDV strains and had the advantages of being reliable, sensitive and specific to confirm the immunization status and monitor the circulation of virulent MDV strains. |
| first_indexed | 2024-04-09T13:13:34Z |
| format | Article |
| id | doaj.art-83eae00202634a5da89167355a27c8f1 |
| institution | Directory Open Access Journal |
| issn | 2297-1769 |
| language | English |
| last_indexed | 2024-04-09T13:13:34Z |
| publishDate | 2023-05-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Veterinary Science |
| spelling | doaj.art-83eae00202634a5da89167355a27c8f12023-05-12T05:31:55ZengFrontiers Media S.A.Frontiers in Veterinary Science2297-17692023-05-011010.3389/fvets.2023.11614411161441A quadruplex real-time PCR assay combined with a conventional PCR for the differential detection of Marek’s disease virus vaccines and field strainsShaopeng Wu0Shaopeng Wu1Shaopeng Wu2Tian Ding3Tian Ding4Tian Ding5Hongxia Shao6Hongxia Shao7Hongxia Shao8Kun Qian9Kun Qian10Kun Qian11Jianqiang Ye12Jianqiang Ye13Jianqiang Ye14Aijian Qin15Aijian Qin16Aijian Qin17Ministry of Education Key Laboratory for Avian Preventive Medicine, Yangzhou University, Yangzhou, Jiangsu, ChinaJiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, Jiangsu, ChinaJoint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu, ChinaMinistry of Education Key Laboratory for Avian Preventive Medicine, Yangzhou University, Yangzhou, Jiangsu, ChinaJiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, Jiangsu, ChinaJoint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu, ChinaMinistry of Education Key Laboratory for Avian Preventive Medicine, Yangzhou University, Yangzhou, Jiangsu, ChinaJiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, Jiangsu, ChinaJoint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu, ChinaMinistry of Education Key Laboratory for Avian Preventive Medicine, Yangzhou University, Yangzhou, Jiangsu, ChinaJiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, Jiangsu, ChinaJoint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu, ChinaMinistry of Education Key Laboratory for Avian Preventive Medicine, Yangzhou University, Yangzhou, Jiangsu, ChinaJiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, Jiangsu, ChinaJoint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu, ChinaMinistry of Education Key Laboratory for Avian Preventive Medicine, Yangzhou University, Yangzhou, Jiangsu, ChinaJiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, Jiangsu, ChinaJoint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu, ChinaTo evaluate the effect of the vaccine and differentiate vaccine from virulent MDV, a new quadruplex real-time PCR assay based on TaqMan probes was developed to differentiate and accurately quantify HVT, CVI988 and virulent MDV-1. The results showed that the limit of detection (LOD) of the new assay was 10 copies with correlation coefficients >0.994 of CVI988, HVT and virulent MDV DNA molecules without cross-reactivity with other avian disease viruses. The intra-assay and inter-assay coefficients of variation (CVs) of Ct values for the new assay were less than 3%. Analysis of replication kinetics of CVI988 and virulent MDV of collected feathers between 7 and 60 days post-infection (dpi) showed MD5 had no significant effect on the genomic load of CVI988 (p > 0.05), while vaccination with CVI988 could significantly reduce the viral load of MD5 (p < 0.05). Combined with meq gene PCR, this method can effectively identify virulent MDV infections in immunized chickens. These results demonstrated that this assay could distinguish between the vaccine and virulent MDV strains and had the advantages of being reliable, sensitive and specific to confirm the immunization status and monitor the circulation of virulent MDV strains.https://www.frontiersin.org/articles/10.3389/fvets.2023.1161441/fullMarek’s disease virusvaccinationCVI988/Rispensreal-time PCRdetection |
| spellingShingle | Shaopeng Wu Shaopeng Wu Shaopeng Wu Tian Ding Tian Ding Tian Ding Hongxia Shao Hongxia Shao Hongxia Shao Kun Qian Kun Qian Kun Qian Jianqiang Ye Jianqiang Ye Jianqiang Ye Aijian Qin Aijian Qin Aijian Qin A quadruplex real-time PCR assay combined with a conventional PCR for the differential detection of Marek’s disease virus vaccines and field strains Frontiers in Veterinary Science Marek’s disease virus vaccination CVI988/Rispens real-time PCR detection |
| title | A quadruplex real-time PCR assay combined with a conventional PCR for the differential detection of Marek’s disease virus vaccines and field strains |
| title_full | A quadruplex real-time PCR assay combined with a conventional PCR for the differential detection of Marek’s disease virus vaccines and field strains |
| title_fullStr | A quadruplex real-time PCR assay combined with a conventional PCR for the differential detection of Marek’s disease virus vaccines and field strains |
| title_full_unstemmed | A quadruplex real-time PCR assay combined with a conventional PCR for the differential detection of Marek’s disease virus vaccines and field strains |
| title_short | A quadruplex real-time PCR assay combined with a conventional PCR for the differential detection of Marek’s disease virus vaccines and field strains |
| title_sort | quadruplex real time pcr assay combined with a conventional pcr for the differential detection of marek s disease virus vaccines and field strains |
| topic | Marek’s disease virus vaccination CVI988/Rispens real-time PCR detection |
| url | https://www.frontiersin.org/articles/10.3389/fvets.2023.1161441/full |
| work_keys_str_mv | AT shaopengwu aquadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT shaopengwu aquadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT shaopengwu aquadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT tianding aquadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT tianding aquadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT tianding aquadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT hongxiashao aquadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT hongxiashao aquadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT hongxiashao aquadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT kunqian aquadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT kunqian aquadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT kunqian aquadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT jianqiangye aquadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT jianqiangye aquadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT jianqiangye aquadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT aijianqin aquadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT aijianqin aquadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT aijianqin aquadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT shaopengwu quadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT shaopengwu quadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT shaopengwu quadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT tianding quadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT tianding quadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT tianding quadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT hongxiashao quadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT hongxiashao quadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT hongxiashao quadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT kunqian quadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT kunqian quadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT kunqian quadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT jianqiangye quadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT jianqiangye quadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT jianqiangye quadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT aijianqin quadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT aijianqin quadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains AT aijianqin quadruplexrealtimepcrassaycombinedwithaconventionalpcrforthedifferentialdetectionofmareksdiseasevirusvaccinesandfieldstrains |