36B-29 |
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W.-Y. LO, Food Science, Cornell Univ., 1018 Bradfield Hall, Ithaca, NY 14850 and A. J. Baeumner, Dept. of Biological & Environmental Engineering, Cornell Univ., 318 Riley-Robb Hall, Ithaca, NY 14853-5701. Nucleic Acid Sequence Based Amplification (NASBA) is a very sensitive and rapid method for RNA amplification and subsequent detection. However, similar to PCR (polymerase chain reaction) or RT-PCR (reverse transcriptase polymerase chain reaction), using NASBA it is not possible to quantify the production yield of the tested target sequence. Competitive assays have therefore been developed to quantify the target sample. These assays use internal standards that compete with the target sequence for multiplication during the amplification reaction. One current drawback is the considerable effort that is required to construct an appropriate RNA or DNA internal standard sequence by genetic engineering. Considerable effort has to be put into the design of a sequence which can equally compete with the target sequence for amplification in the same reaction tube. Therefore, the development of a simple procedure that produces a competitor molecule is of great interest. Instead of cloning sequences, carrying out deletion-PCR reactions and sequencing the successful construct, we were able to prove that the careful design of NASBA-deletion primers resulted in a competitor molecule within only two NASBA reactions. A quantitative competitive NASBA assay to determine the initial concentration of E. coli RNA can subsequently be achieved within 3 h by the use of eletrochemiluminesence (ECL) detection method and a simple lateral-flow assay. ECL provides a great sensitivity and its dynamic range spans over several orders of magnitude in concentration. A lateral flow biosensor was integrated the internal standards onto the same assay and thus allowed the detection and quantification of the target RNA in just one simple assay. Internal standards constructed by our novel method were shown to equally compete with target E.coli RNA and the detection limit in this competitive assay of target RNA is 14 fg for both methods.
Session 36B, Biotechnology: General
2005 IFT Annual Meeting, July 15-20 - New Orleans, Louisiana |