Response surface methodology-based optimisation of agarose gel electrophoresis for screening and electropherotyping of rotavirus.
Management of rotavirus diarrhoea cases and prevention of nosocomial infection require rapid diagnostic method at the patient care level. Diagnostic tests currently available are not routinely used due to economic or sensitivity/specificity constraints. Agarose-based sieving media and running conditions were modulated by using central composite design and response surface methodology for screening and electropherotyping of rotaviruses. The electrophoretic resolution of rotavirus genome was calculated from input parameters characterising the gel matrix structure and running conditions. Resolution of rotavirus genome was calculated by densitometric analysis of the gel. The parameters at critical values were able to resolve 11 segmented rotavirus genome. Better resolution and electropherotypic variation in 11 segmented double-stranded RNA genome of rotavirus was detected at 1.96% (w/v) agarose concentration, 0.073 mol l(-1) ionic strength of Tris base-boric acid-ethylenediamine tetraacetic acid buffer (1.4x) and 4.31 h of electrophoresis at 4.6 V cm(-1) electric field strength. Modified agarose gel electrophoresis can replace other methods as a simplified alternative for routine detection of rotavirus where it is not in practice.
Mishra
Vikas
V
Department of Paediatrics, Chhatrapati Shahuji Maharaj Medical University, Lucknow, Uttar Pradesh, 226003, India.
Nag
Vijaya Lakshmi
VL
Tandon
Ritu
R
Awasthi
Shally
S
D002648:Child; D003428:Cross Infection / Q000175:diagnosis / Q000821:virology; D003967:Diarrhea / Q000209:etiology / Q000821:virology; D004587:Electrophoresis, Agar Gel / Q000295:instrumentation / Q000379:methods*; D016679:Genome, Viral; D006801:Humans; D008962:Models, Theoretical; D012367:RNA, Viral / Q000032:analysis; D012401:Rotavirus / Q000145:classification / Q000235:genetics; D012400:Rotavirus Infections / Q000150:complications / Q000175:diagnosis*; D014773:Virology / Q000379:methods*
D012367:RNA, Viral