REAL-TIME FLUORESCENCE DETECTION OF RNA AMPLIFIED BY Q-BETA REPLICASE

Amplification of RNA probes by Q beta replicase can be used to detect a wide range of analytes with a potential sensitivity of a single mole cule. A system has been developed in which Q beta amplification of mid ivariant-(MDV)-based RNA is measured in real time by fluorescence. Thi s was accomplished by including a fluorescent intercalating dye, propi dium iodide, in the reactions and monitoring the fluorescence change u sing a custom fluorometer. The time at which fluorescence is detectabl e above background is referred to as the ''response time'' and is calc ulated using curve-fitting algorithms. A response time is inversely an d linearly proportional to the logarithm of the number of template RNA molecules which initiated the reaction. Therefore, this system permit s an unknown amount of input RNA probe to be quantified through 11 ord ers of magnitude when compared to a standard curve. Under the describe d conditions with MDV RNA, the response time occurs when about 3 X 10( 11) RNA molecules are synthesized and occurs within the exponential ph ase of the reaction, before the number of active enzyme molecules are saturated with RNA templates. This system has been used to determine t he replication properties of MDV RNA reporter molecules bearing specif ic probe sequences and to develop hybridization assays for the clinica l diagnostic field. (C) 1995 Academic Press, Inc.