Testing of the influenza virus purification by CIEF.
In virological practice, the pre-concentration, purification and subsequent determination of the purity and concentration of the viruses from the cultural medium and/or from the real sample are required. The conventional techniques used today are equipment demanding, time-consuming and laborious. In this study, the CIEF of influenza viruses with UV detection has been developed and subsequently used to test the purification of the virus from the biological samples. The equine and swine influenza viruses present in infected allantoic fluid of specific pathogen free embryonated chicken eggs were precipitated by using PEG 6000 and sodium chloride. The precipitated viruses were centrifuged at 14 000 x g, and the impurities of different densities were removed by using the sucrose gradients. The efficiency of the virus purification technique was examined by the CIEF and compared to the results of real-time PCR. The pIs of both influenza viruses were determined. Simultaneously, the CIEF was found to be a suitable method for the rapid testing of the efficiency of the virus purification.
Horká
Marie
M
Institute of Analytical Chemistry of the ASCR, Brno, Czech Republic. horka@iach.cz
Kubícek
Oldrich
O
Kubesoví
Anna
A
Kubícková
Zuzana
Z
Rosenbergová
Katerina
K
Slais
Karel
K
D000818:Animals; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods*; D007526:Isoelectric Point; D009975:Orthomyxoviridae / Q000737:chemistry / Q000254:growth & development / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013395:Sucrose; D013552:Swine; D014776:Virus Cultivation / Q000379:methods
D013395:Sucrose