Evaluation of internal standards in a competitive nucleic acid sequence-based amplification assay

An end-point quantitative nucleic acid sequence-based amplification (NASBA) reaction with two exogenous internal standards for the detection of the model analyte E. coli clpB mRNA was developed and statistically analyzed. Electrochemiluminescence was chosen as a highly sensitive detection means allowing careful evaluation of the internal standards used. The two internal standards examined had been designed previously using a novel and rapid NASBA-based method. Initially, each standard was used separately in a NASBA reaction; subsequently, two internal standards were added into one reaction at different concentrations. The accuracy and precision of the data obtained were analyzed using linear and multiple regression analysis. In the case of single-standard reactions, the accuracy was > 95% and the precision > 98.5%. In the case of double-standard reactions, the accuracy increased to > 97%. With a single internal standard, 3 orders of magnitude of target sequence could be quantified; using three different concentrations of one internal standard, the dynamic range increased to 5 orders of magnitude. In both cases, a detection limit as low as 0.14 pg of target sequence was obtained. In the case of double-internal standard reactions, a dynamic range with 5 orders of magnitude and a detection limit of 1.76 pg was determined. The high-performance quality of the internal standards was assumed to be in part due to the unique synthesis process using two NASBA reactions rather than traditional cloning techniques.