Development of Strand-Specific Real-Time RT-PCR to Distinguish Viral RNAs during Newcastle Disease Virus Infection
Newcastle disease virus (NDV) causes large losses in the global fowl industry. To better understand NDV replication and transcription cycle, quantitative detection methods for distinguishing NDV genomic RNA (gRNA), antigenomic RNA (cRNA), and messenger RNA (mRNA) in NDV-infected cells are indispensi...
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| Format: | Article |
| Language: | English |
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Wiley
2014-01-01
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| Series: | The Scientific World Journal |
| Online Access: | http://dx.doi.org/10.1155/2014/934851 |
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| author | Xusheng Qiu Yang Yu Shengqing Yu Yuan Zhan Nana Wei Cuiping Song Yingjie Sun Lei Tan Chan Ding |
| author_facet | Xusheng Qiu Yang Yu Shengqing Yu Yuan Zhan Nana Wei Cuiping Song Yingjie Sun Lei Tan Chan Ding |
| author_sort | Xusheng Qiu |
| collection | DOAJ |
| description | Newcastle disease virus (NDV) causes large losses in the global fowl industry. To better understand NDV replication and transcription cycle, quantitative detection methods for distinguishing NDV genomic RNA (gRNA), antigenomic RNA (cRNA), and messenger RNA (mRNA) in NDV-infected cells are indispensible. Three reverse transcription primers were designed to specifically target the nucleoprotein (NP) region of gRNA, cRNA, and NP mRNA, and a corresponding real-time RT-PCR assay was developed to simultaneously quantify the three types of RNAs in NDV-infected cells. This method showed very good specificity, sensitivity, and reproducibility. The detection range of the assay was between 5.5×102 and 1.1×109 copies/μL of the target gene. These methods were applied to investigate the dynamics of the gRNA, cRNA, and mRNA synthesis in NDV La Sota infected DF-1 cells. The results showed that the copy numbers of viral gRNA, cRNA, and NP mRNA all exponentially increased in the beginning. The viral RNA copy number then plateaued at 10’h postinfection and gradually decreased from 16 h postinfection. No synthesis priority was observed between replication (gRNA and cRNA amounts) and transcription (mRNA amounts) during NDV infection. However, the cRNA accumulated more rapidly than gRNA, as the cRNA copy number was three- to tenfold higher than gRNA starting from 2 h postinfection. Conclusion. A real-time RT-PCR for absolute quantitation of specific viral RNA fragments in NDV-infected cells was developed for the first time. The development of this assay will be helpful for further studies on the pathogenesis and control strategies of NDV. |
| format | Article |
| id | doaj-art-4010729ccbe44cbe800e53184b863ef1 |
| institution | Kabale University |
| issn | 2356-6140 1537-744X |
| language | English |
| publishDate | 2014-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | The Scientific World Journal |
| spelling | doaj-art-4010729ccbe44cbe800e53184b863ef12025-08-20T03:55:36ZengWileyThe Scientific World Journal2356-61401537-744X2014-01-01201410.1155/2014/934851934851Development of Strand-Specific Real-Time RT-PCR to Distinguish Viral RNAs during Newcastle Disease Virus InfectionXusheng Qiu0Yang Yu1Shengqing Yu2Yuan Zhan3Nana Wei4Cuiping Song5Yingjie Sun6Lei Tan7Chan Ding8Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, ChinaShanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, ChinaShanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, ChinaShanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, ChinaShanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, ChinaShanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, ChinaShanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, ChinaShanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, ChinaShanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, ChinaNewcastle disease virus (NDV) causes large losses in the global fowl industry. To better understand NDV replication and transcription cycle, quantitative detection methods for distinguishing NDV genomic RNA (gRNA), antigenomic RNA (cRNA), and messenger RNA (mRNA) in NDV-infected cells are indispensible. Three reverse transcription primers were designed to specifically target the nucleoprotein (NP) region of gRNA, cRNA, and NP mRNA, and a corresponding real-time RT-PCR assay was developed to simultaneously quantify the three types of RNAs in NDV-infected cells. This method showed very good specificity, sensitivity, and reproducibility. The detection range of the assay was between 5.5×102 and 1.1×109 copies/μL of the target gene. These methods were applied to investigate the dynamics of the gRNA, cRNA, and mRNA synthesis in NDV La Sota infected DF-1 cells. The results showed that the copy numbers of viral gRNA, cRNA, and NP mRNA all exponentially increased in the beginning. The viral RNA copy number then plateaued at 10’h postinfection and gradually decreased from 16 h postinfection. No synthesis priority was observed between replication (gRNA and cRNA amounts) and transcription (mRNA amounts) during NDV infection. However, the cRNA accumulated more rapidly than gRNA, as the cRNA copy number was three- to tenfold higher than gRNA starting from 2 h postinfection. Conclusion. A real-time RT-PCR for absolute quantitation of specific viral RNA fragments in NDV-infected cells was developed for the first time. The development of this assay will be helpful for further studies on the pathogenesis and control strategies of NDV.http://dx.doi.org/10.1155/2014/934851 |
| spellingShingle | Xusheng Qiu Yang Yu Shengqing Yu Yuan Zhan Nana Wei Cuiping Song Yingjie Sun Lei Tan Chan Ding Development of Strand-Specific Real-Time RT-PCR to Distinguish Viral RNAs during Newcastle Disease Virus Infection The Scientific World Journal |
| title | Development of Strand-Specific Real-Time RT-PCR to Distinguish Viral RNAs during Newcastle Disease Virus Infection |
| title_full | Development of Strand-Specific Real-Time RT-PCR to Distinguish Viral RNAs during Newcastle Disease Virus Infection |
| title_fullStr | Development of Strand-Specific Real-Time RT-PCR to Distinguish Viral RNAs during Newcastle Disease Virus Infection |
| title_full_unstemmed | Development of Strand-Specific Real-Time RT-PCR to Distinguish Viral RNAs during Newcastle Disease Virus Infection |
| title_short | Development of Strand-Specific Real-Time RT-PCR to Distinguish Viral RNAs during Newcastle Disease Virus Infection |
| title_sort | development of strand specific real time rt pcr to distinguish viral rnas during newcastle disease virus infection |
| url | http://dx.doi.org/10.1155/2014/934851 |
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