Patent ID: 6955901

Claim:
Method for detecting in a sample, comprising a plurality of sample nucleic acids of different sequence, the presence of at least one specific single stranded target nucleic acid sequence comprising a first and a second segment, and optionally a third segment being located between the first and second segments, the segments located essentially adjacent to one another, comprising, in a reaction mixture, the steps of: contacting the sample nucleic acids with a plurality of different probe sets, each probe set comprising a first nucleic acid probe having a first target specific region complementary to the first segment of said target nucleic acid sequence and a first non-complementary region, 3′ from the first target specfic region, being essentially non-complementary to said target nucleic acid sequence, comprising a first tag sequence, a second nucleic acid probe having a second target specific region complementary to the second segment of said target nucleic acid sequence and a second non-complementary region, 5′ from the second target specific region, being essentially non-complementary to said target nucleic acid sequence, comprising a second tag sequence, and, optionally, a third nucleic acid probe having a third target specific region, complementary to the third segment, incubating the plurality of sample nucleic acids with the probes allowing hybridisation of complementary nucleic acids, connecting to one another the first, second and optionally the third nucleic acid probes, hybridised to the first, second and, if present, third segment of the same target nucleic acid sequence, respectively, the hybridised probes being located essentially adjacent to one another, forming a connected probe assembly, amplifying the connected probe assemblies, wherein amplification is initiated by binding of a first nucleic acid primer specific for the first tag sequence followed by elongation thereof, detecting an amplicon, wherein the amount of at least the first nucleic acid probe of at least one probe set in the mixture is less than 40 femtomoles, and molar ratio between the first nucleic acid primer and the first nucleic acid probe being at least 200.