Use of chimeric influenza viruses as a novel internal control for diagnostic rRT-PCR assays

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• 作者：Xueliang Wang ; Fen Liu ; Lingli Jiang ; Yun Bao…
• 关键词：rRT ; PCR ; Internal control ; Influenza virus ; Hepatitis C virus ; Reverse genetics technology
• 刊名：Applied Microbiology and Biotechnology
• 出版年：2016
• 出版时间：February 2016
• 年：2016
• 卷：100
• 期：4
• 页码：1667-1676
• 全文大小：1,251 KB
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• 作者单位：Xueliang Wang (1)
  Fen Liu (2)
  Lingli Jiang (1)
  Yun Bao (1)
  Yanqun Xiao (1)
  Hualiang Wang (1)
  
  1. Shanghai Centre for Clinical Laboratory, 528 Hongshan Road, Shanghai, 200126, China
  2. Shanghai Institute of Biological Products, 1262 West Yanan Road, Shanghai, 200052, China
  
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Biotechnology
  Microbiology
  Microbial Genetics and Genomics
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1432-0614

文摘

Real-time quantitative reverse transcriptase polymerase chain reaction (rRT-PCR) is now widely used to detect viral pathogens in various human specimens. The application of internal controls to validate the entire process of these assays is necessary to prevent false-negative results caused by unexpected inhibition or inefficient extraction. In the present study, we describe a strategy to produce a stable internal control for rRT-PCR by packaging foreign RNA into influenza virions using plasmid-based reverse genetics technology. The envelope structure of influenza virus can effectively protect RNA segments from RNase digestion, which provides an advantage for its routine use as an internal control. Utilizing this approach, we successfully generated a recombinant influenza virus (rPR8-HCV) containing the 5′ untranslated region (5′ UTR) of the hepatitis C virus (HCV) RNA genome. After inactivation and purification, the rPR8-HCV particles were demonstrated to be RNase resistant and stable at 4 °C for at least 252 days in human plasma, with no degradation even after being frozen and thawed multiple times. These results were reproducible in the COBAS TaqMan HCV test for 164 days. Moreover, the chimeric influenza virus particles could be easily produced in embryonated eggs and were noninfectious after inactivation treatment. Additionally, this strategy could also be adapted for real-time clinical applications of other RNA targets, providing a universal approach with broad clinical applications in rRT-PCR assays. Keywords rRT-PCR Internal control Influenza virus Hepatitis C virus Reverse genetics technology