title: Use of chimeric influenza viruses as a novel internal control  for diagnostic rRT-PCR assays.
abstract: 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.
authors.affiliation: Shanghai Centre for Clinical Laboratory, 528 Hongshan Road, Shanghai, 200126, China.
authors.forename: Xueliang
authors.initials: X
authors.lastname: Wang
authors.affiliation: Shanghai Institute of Biological Products, 1262 West Yanan Road, Shanghai, 200052, China.
authors.forename: Fen
authors.initials: F
authors.lastname: Liu
authors.affiliation: Shanghai Centre for Clinical Laboratory, 528 Hongshan Road, Shanghai, 200126, China.
authors.forename: Lingli
authors.initials: L
authors.lastname: Jiang
authors.affiliation: Shanghai Centre for Clinical Laboratory, 528 Hongshan Road, Shanghai, 200126, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Bao
authors.affiliation: Shanghai Centre for Clinical Laboratory, 528 Hongshan Road, Shanghai, 200126, China.
authors.forename: Yanqun
authors.initials: Y
authors.lastname: Xiao
authors.affiliation: Shanghai Centre for Clinical Laboratory, 528 Hongshan Road, Shanghai, 200126, China. wanghualiang@sccl.org.cn.
authors.forename: Hualiang
authors.initials: H
authors.lastname: Wang
mesh_terms: D016174:Hepacivirus / Q000235:genetics; D006526:Hepatitis C / Q000175:diagnosis*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods / Q000592:standards*; D009975:Orthomyxoviridae / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D011995:Recombination, Genetic; D012015:Reference Standards; D059386:Reverse Genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards*
chemical_list: D012367:RNA, Viral