Quantification of eubacteria in competitive polymerase chain reaction

摘要

Quantitative determination of eubacteria comprises performing a competitive, quantitative polymerase chain reaction (PCR) on a test sample, using forward and reverse primers (FP and RP) that bind to eubacterial 16S rRNA genes, in presence of a known amount of standard DNA (I). (I) is produced in a preliminary PCR using FP or RP plus a hybrid primer (HP) which: (a) binds to a sequence within the amplicon produced by FP and RP; and (b) has at its 5'-end the sequence for FP or RP. Both products (from sample and from (I)) are then quantified by: (a) separation on agarose electrophoretic gel, staining with ethidium bromide and video-densitometry; or (b) when using one of RP and FP in 5'-biotinylated form, by binding to immobilised streptavidin, reaction with two digoxigenin (dig)-labelled probes, binding to different amplicons, and enzyme-linked oligonucleotide sorbent assay (ELOSA) using enzyme-coupled anti-dig antibodies. The content of eubacteria in the sample is then calculated from the ratio of product concentrations and the known amount of (I).