Compositions, methods, kits and apparatus for determining the presence or absence of target molecules

A method of determining the presence or absence of a target molecule, said method comprising the following steps:

• a.) providing a first RNA molecule and a second RNA molecule, said first RNA molecule capable of binding to a target molecule and having the following formula:
• 5′—A—B—C—3′;
• wherein A is a section of said RNA molecule having 10 to 100,000 nucleotides which section is capable of being received by an RNA replicase and with another RNA sequence, F, being replicated and the letter “B” denotes a section of the RNA molecule having approximately 10 to 50,000 nucleotides which section is capable of binding to the target molecule and the letter “C” denotes a section of the RNA molecule having approximately 1 to 10,000 nucleotides which section is capable of being ligated to another RNA sequence, “D”;
• said second RNA molecule capable of binding to said target molecule and having the following formula:
• 5′—D—E—F—3′;
• wherein D is a section of the RNA molecule having approximately 1 to 10,000 nucleotides which is capable being ligated to another RNA sequence, “C”, and the letter “E” denotes a section of the RNA molecule having approximately 10 to 50,000 nucleotides which section is capable of binding to the target molecule, and the letter “F” denotes a section of the RNA molecule capable of being received by an RNA replicase and with another sequence, “A”, being replicated;
• said first and the second RNA molecules are capable of forming a third RNA molecule having the following formula:
• 5′—A—B—C—D—E—F—3′;
• said third RNA molecule formed by ligating the C and D sections, as the E and the B sections are bound to target, said third RNA molecule capable of being received by an RNA replicase and being replicated by such enzyme as a indication of the presence or absence of the target molecule;
• b.) combining a sample potentially containing the target molecule with said first and said second RNA molecules and imposing conditions which allow said first and said second RNA molecules and said target molecule to form a first second RNA molecule target complex;
• c.) imposing RNA ligase conditions on said sample to form said third RNA molecule in the presence of the target molecule;
• d.) imposing amplification conditions on said sample to form an amplification product in the presence of target;
• and
• e.) monitoring the sample for the presence or absence of the third RNA molecule as indicative of the presence or absence of the target molecule.