Titration of human-bovine rotavirus reassortants using a tetrazolium-based colorimetric end-point dilution assay.
A colorimetric end-point dilution assay was developed for the titration of rotavirus-containing samples that uses commercially available tetrazolium dyes as an indicator of virus infection. This assay offers several advantages over both plaque assays and traditional end-point dilution methods. The latter assays require manual counting of plaques or the scoring of wells for the presence of virus based on observed cytopathic effects. The colorimetric end-point dilution assay enables the scoring of wells based upon absorbance readings alone, thereby eliminating time-consuming and subjective manual screenings. This method also has the potential for automating the analysis of large numbers of samples. Virus titers of human-bovine rotavirus reassortants obtained using this method are comparable to those determined by plaque assay. The scoring of wells based on absorbance readings was also found to agree with manual scoring of cytopathic effects and with the production of viral antigen.
DiStefano
D J
DJ
Department of Cellular and Molecular Biology, Merck Research Laboratories, Rahway, NJ 07065, USA.
Gould
S L
SL
Munshi
S
S
Robinson
D K
DK
D000818:Animals; D000956:Antigens, Viral; D002213:Capsid / Q000235:genetics; D036022:Capsid Proteins; D002417:Cattle; D002460:Cell Line; D002522:Chlorocebus aethiops; D003124:Colorimetry / Q000379:methods*; D004396:Coloring Agents; D003588:Cytopathogenic Effect, Viral; D006801:Humans; D008253:Macaca mulatta; D016865:Reassortant Viruses / Q000254:growth & development / Q000302:isolation & purification*; D012401:Rotavirus / Q000254:growth & development / Q000302:isolation & purification*; D013778:Tetrazolium Salts; D013844:Thiazoles; D017186:Titrimetry; D014709:Vero Cells
D000956:Antigens, Viral; D036022:Capsid Proteins; D004396:Coloring Agents; D013778:Tetrazolium Salts; D013844:Thiazoles; C060339:VP4 protein, Rotavirus; C050779:VP7 protein, Rotavirus; C070380:3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; C022616:thiazolyl blue