ABSTRACT
The Qiagen QIAsymphony SP is a high-throughput (up to 96 samples per run), fully-automated nucleic acid isolation system. It was implemented in the authors' laboratory to cope with the high demand for pandemic H1N1 influenza testing in 2009. This study evaluated the QIAsymphony SP for viral nucleic acid isolation from quality control materials, pure cultures and various clinical specimens. The effect of varying sample volume on detection sensitivity was investigated using serial 10-fold dilutions of pure viral specimens and target nucleic acids were detected by real-time polymerase chain reaction (PCR) assays. Little variability in detection sensitivity was observed for all the viral targets tested, although variation in cycle threshold values was apparent in some cases. Importantly, pathogens were detectable over a broad concentration range and from diverse clinical specimens. Removal of PCR inhibitors was generally effective, as demonstrated by detection of viral nucleic acids and/or internal controls. The results demonstrate that the QIAsymphony SP is suitable for use in routine virology molecular diagnostics, and provides a high-throughput capacity, which is needed in peak seasons of infection or in centralised laboratories.
Subject(s)
DNA, Viral/isolation & purification , Nucleic Acids/isolation & purification , Pathology, Molecular/methods , RNA, Viral/isolation & purification , Reagent Kits, Diagnostic , Animals , Automation, Laboratory , Cells, Cultured , Chick Embryo , DNA, Viral/analysis , DNA, Viral/genetics , Dogs , Humans , Influenza A Virus, H1N1 Subtype/genetics , Nucleic Acids/analysis , Pathology, Molecular/instrumentation , Polymerase Chain Reaction/methods , Quality Control , RNA, Viral/analysis , RNA, Viral/genetics , Reagent Kits, Diagnostic/standards , Real-Time Polymerase Chain ReactionABSTRACT
Coxsackievirus B3 (CVB3), along with other enteroviruses, is involved in about 50% of myocarditis cases and in the pathogenesis of dilated cardiomyopathy. Prevention of CVB3 infection is therefore highly desirable. Virus-like particles (VLPs) are structurally similar to native virus particles and therefore are far better immunogens than any other subunit vaccines. Recombinant baculoviruses carrying either the intact, entire coding region of CVB3 or the four individual coding regions for virus proteins 1-4 (VP1-4) were constructed. Expression of CVB3 capsid proteins in insect cells infected with recombinant baculovirus was detected by immunofluorescence and Western blot analysis. Sucrose gradient ultracentrifugation fractions of the infected cell lysates contained peaks of CVB3 antigen with an approximate density of 1.14g/ml. Electron microscopy demonstrated the presence of VLP in these sucrose fractions. The CVB3 VLP was non-infectious in tissue culture. SWR (H-2(q)) mice vaccinated with CVB3 VLP developed antibodies to CVB3 capsid proteins after the first boost. Antibody titre was comparable to the level induced by an attenuated CVB3 vaccine. Vaccinated animals were protected from myocarditis when subsequently challenged with cardiovirulent CVB3 (chimera-2). Vaccination with VLP produced from the complete CVB3 coding region gave a greater immune response and afforded better protection than with VLP from the quadruple expression vector. These results demonstrate that CVB3 capsid proteins expressed in insect cells have the intrinsic capacity to assemble into non-infectious VLP, which afforded protection from CVB3 infection to mice when used as a vaccine.