Develop quantitative NASBA assay to detect E. coli.

The nucleic acid sequence-based amplification (NASBA) is a very sensitive and rapid technique for amplifying RNA and for detecting bacteria. NASBA is a specific, isothermal method of nucleic acid amplification which is highly suited for amplifying RNA. However, when using the NASBA, it is not possible to quantify the production yield of the tested target sequence.

Competitive assays have 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 of these assays entails the considerable effort that is required to construct an appropriate RNA or DNA internal standard sequence using genetic engineering. Much effort has to be put into designing a sequence that can equally compete with the target sequence for amplification in the same reaction tube.

So, 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, Cornell University scientists were able to show that the careful design of NASBA-deletion primers can lead to a competitor molecule within only two NASBA reactions.

A quantitative competitive NASBA assay that determines the initial concentration of E. coli RNA can be achieved within 3 hours with the use of an electrochemiluminesence (ECL) detection method and a simple lateral-flow assay. The ECL technique is extremely sensitive. Its dynamic range spans over several orders of magnitude.

Cornell researchers integrated a lateral-flow biosensor into the internal standards onto the same assay. This made it possible to detect and quantify the target RNA in just one simple assay. Internal standards constructed by this method competed equally with the RNA of the target E. coli.

Further information. Antje Baeumner, Department of Biological and Environmental Engineering, Cornell University, 318 Riley-Robb Hall, Ithaca, NY 14853; phone: [phone]; fax: [phone]; email: [e-mail].