QUANTITATIVE ELISA POLYMERASE-CHAIN-REACTION AT SATURATION USING HOMOLOGOUS INTERNAL DNA STANDARDS AND CHEMILUMINESCENCE REVELATION

In this report, we describe the development and validation of a conven ient, versatile and high throughput quantitative polymerase chain reac tion (PCR) method. This assay is based on the use of only one concentr ation of an internal homologous standard (IS) easily obtained by repla cing an 18 nt specific sequence using recombinant PCR, Target and IS a mplicons are quantitated at the PCR plateau phase using ELISA which in cludes a hybridization step with either target or IS specific probes a nd luminometric revelation. Luminometry allows measurement of amplicon levels without the need for serial dilutions. Experimental values wer e obtained by comparing their target/IS signal ratios to those of an e xternal scale. A linear dynamic range over four orders of magnitude an d good reproducibility were obtained. We used this assay to investigat e variations of IL-13 mRNA expression in HIV-infected patients under h ighly active antiretroviral therapy, Furthermore, we also report a var iant of this method using Taqman(TM) assay in the ABI PRISM(TM) 7,700 apparatus.