期刊
FOOD AND ENVIRONMENTAL VIROLOGY
卷 5, 期 4, 页码 231-235出版社
SPRINGER
DOI: 10.1007/s12560-013-9122-4
关键词
Size-exclusion chromatography; Rotavirus; Purification; Characterisation; Quantification
资金
- Royal Society of New Zealand [ESR-1001]
This article describes a rapid method for purifying infectious rotavirus particles from cell culture for environmental research. The method is based on size-exclusion chromatography using TOSOH TSKgel(A (R)) G5000PWXL-CP with a TSKgel(A (R)) Size Exclusion G2500PWxl guard column, set up on an AKTA Explorer10. Four peaks were identified from the chromatogram and the corresponding fractions were collected and analysed by electron microscopy, 1-step quantitative reverse transcription polymerase chain reaction (RT-PCR) and qNano measurement. Infectivity potential of the recovered virus particles was determined using cell culture. Our analysis reveals that the first fraction contains majority of the intact triple-layered infectious virions while the other three fractions contain mixtures of empty capsids and intact infectious virions. Our results also indicate that there is a gross overestimation of rotaviruses in crude extracts due to encapsidated RNA in the order of 2.3 x 10(11) particles and we note that estimates by qNano are similarly skewed (1.36 x 10(13) particle) possibly due to empty capsids and cellular debris. In summary we present a method for purification (similar to 12 h) of rotaviruses for a more robust and accurate quantification of virus size, surface charge and particle concentration in environmental contexts.
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