Two-step hybridization and capture of a polynucleotide

A method for determining the presence of a target polynucleotide in a sample, consisting essentially of the steps of:

• a. providing a capture probe capable of hybridizing to a target polynucleotide present in a sample;
• b. mixing the capture probe with a sample suspected of containing the target polynucleotide at a first hybridization condition that favors hybridization of the capture probe and the target polynucleotide, thereby producing a capture probe:target polynucleotide complex formed by hybridization of the capture probe to the target polynucleotide, wherein the first hybridization condition disfavors hybridization of an immobilized probe and the capture probe;
• c. providing an immobilized probe capable of hybridizing to the capture probe;
• d. incubating a mixture containing the capture probe:target polynucleotide complex and the immobilized probe at a second hybridization condition that favors hybridization of the immobilized probe and the capture probe, thereby producing a captured target polynucleotide consisting essentially of an immobilized probe:capture probe:target polynucleotide complex formed by hybridization of the immobilized probe to the capture probe in the capture probe:target polynucleotide complex;
• e. purifying the captured target polynucleotide from other sample components by washing the captured target polynucleotide, thereby producing a purified target polynucleotide retained in the immobilized probe:capture probe:target polynucleotide complex;
• f. amplifying the purified target polynucleotide by using an amplification oligonucleotide which hybridizes specifically to and initiates polymerization from a position in the target polynucleotide, thereby producing an amplified nucleic acid;
• and
• g. detecting the amplified nucleic acid which indicates the presence of the target polynucleotide in the sample.