Screening of an enterovirus specific RT-PCR ELISA method for the quantification of enterovirus genomes in human body fluids by means of a three-level experimental design.
In order to obtain a detection limit as low as possible for a quantitative enterovirus specific RT-PCR ELISA assay, optimal reaction conditions, which give rise to the highest response, need to be determined. This was done by investigating the influence of 13 factors, selected from RT and PCR, in a multivariate approach by means of a well-balanced three-level screening design, derived from a three-level Plackett--Burman design. Optimal reaction conditions could be determined by calculation and evaluation of the effects of the different factors on the response, i.e. the measured absorbance of the ELISA detection. The method will be used to study a possible longitudinal relationship between enteroviruses and the development of multiple sclerosis and juvenile diabetes.
Lauwers
S
S
Department of Microbiology and Hygiene, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium.
Vander Heyden
Y
Y
Rombaut
B
B
D001826:Body Fluids / Q000821:virology*; D017931:DNA Primers / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D004770:Enterovirus / Q000235:genetics / Q000502:physiology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D016679:Genome, Viral; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D010926:Placental Hormones / Q000032:analysis; D011786:Quality Control; D012367:RNA, Viral / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D012107:Research Design; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013696:Temperature; D013997:Time Factors; D019562:Viral Load
D017931:DNA Primers; D003838:Deoxyadenine Nucleotides; D010926:Placental Hormones; D012367:RNA, Viral; C022052:placental ribonuclease inhibitor; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D008274:Magnesium; C026600:2'-deoxyadenosine triphosphate