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 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 × 10(2) and 1.1 × 10(9) 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.
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, China.
Xusheng
X
Qiu
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, China.
Yang
Y
Yu
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, China.
Shengqing
S
Yu
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, China.
Yuan
Y
Zhan
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, China.
Nana
N
Wei
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, China.
Cuiping
C
Song
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, China.
Yingjie
Y
Sun
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, China.
Lei
L
Tan
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, China.
Chan
C
Ding
D000818:Animals; D045463:Avulavirus Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D017931:DNA Primers / Q000138:chemical synthesis; D057230:Limit of Detection; D009522:Newcastle disease virus / Q000235:genetics* / Q000302:isolation & purification; D009698:Nucleoproteins / Q000235:genetics / Q000378:metabolism; D011200:Poultry; D011201:Poultry Diseases / Q000175:diagnosis* / Q000821:virology; D012333:RNA, Messenger / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014158:Transcription, Genetic; D019562:Viral Load; D014779:Virus Replication
D017931:DNA Primers; D009698:Nucleoproteins; D012333:RNA, Messenger; D012367:RNA, Viral