Target nucleic acid amplification/detection systems and methods for the use thereof

A method for determining whether at least one specific RNA target sequence is present in a nucleic acid containing sample, said method comprising priming a replicatable extension product and detecting the presence or absence of said product wherein detection of said replicatable extension prodect is indicative of the presence of said specific RNA target sequence in the sample. said extension product being an extension from a second nucleic acid primer hybridized with a strand separated from a first extension product that contains a sequence of a first nucleic acid primer hybridizable with a target RNA sequence, said replicatable extension product functioning as a reporter molecule for said target,

• wherein said first nucleic acid primer comprises:
• (a) at its 5'-end, a first portion of an autocatalytically replicatable RNA, wherein the complement of said first portion is a segment of an autocatalytically replicatable RNA which comprises the 3'-end of an autocatalytically replicatable RNA which is capable of RNA-dependent RNA polymerase-directed autocatalytic replication, and wherein said first portion is a segment of a (+) strand or a (-) strand of an autocatalytically replicatable RNA, including the 5'-end;
• and
• (b) at its 3'-end, a first probe sequence which is capable of hybridizing to a segment of a target RNA sequence, and priming an extension reaction, using the target RNA sequence as template, to produce a first primer extension product which includes an RNA sequence complementary to the target RNA sequence;
• and
• wherein said second nucleic acid primer comprises:
• (a) at its 5'-end, a second portion derived from the opposite strand of said autocatalytically replicatable RNA, wherein the complement of said second portion comprises the 3'-end of an autocatalytically replicatable RNA which is capable of RNA-dependent RNA polymerase-directed autocatalytic replication, and wherein said second portion is a segment of a (+) strand or a (-) strand of said autocatalytically replicatable RNA, including the 5'-end;
• and
• (b) at its 3'-end, a second probe sequence which is capable of hybridizing to a segment of said RNA sequence complementary to the target RNA sequence and priming an extension reaction, using the first extension product as template, to produce a second primer extension product which is autocatalytically replicatable RNA having incorporated therein a target RNA sequence;
• provided that either the first or second portion of said autocatalytically replicatable RNA is derived from the (+) strand and the other portion of said autocatalytically replicatable RNA is derived from the (-) strand of the same autocatalytically replicatable RNA, and further provided that one of said cofunctioning primers includes a replicase binding site.

A method for determining whether at least one specific RNA target sequence is present in a nucleic acid containing sample, said method comprising:

• hybridizing, under suitable conditions, said RNA target sequence with a first nucleic acid primer comprising:
• (a) at its 5'-end, a first portion of an autocatalytically replicatable RNA, wherein the complement of said first portion is a segment of an autocatalytically replicatable RNA which comprises the 3'-end of an autocatalytically replicatable RNA which is capable of RNA-dependent RNA polymerase-directed autocatalytic replication, and wherein said first portion is a segment of a (+) strand or a (-) strand of an autocatalytically replicatable RNA, including the 5'-end;
• and
• (b) at its 3'-end, a first probe sequence which is capable of hybridizing to a segment of a target RNA sequence, and priming an extension reaction, using the target RNA sequence as template, to produce a first primer extension product which includes an RNA sequence complementary to the target RNA sequence;
• chain extending said hybridized nucleic acid primer,
• strand separating the resulting extension product,
• hybridizing the strand separated in the previous step containing the sequence that is the complement of one susceptible to replication with a second nucleic acid primer, said second nucleic acid primer comprising:
• (a) at its 5'-end, a second portion derived from the opposite strand of said autocatalytically replicatable RNA, wherein the complement of said second portion comprises the 3'-end of an autocatalytically replicatable RNA which is capable of RNA-dependent RNA polymerase-directed autocatalytic replication, and wherein said second portion is a segment of a (+) strand or a (-) strand of said autocatalytically replicatable RNA, including the 5'-end;
• and
• (b) at its 3'-end, a second probe sequence which is capable of hybridizing to a segment of said RNA sequence complementary to the target RNA sequence and priming an extension reaction, using the first extension product as template, to produce a second primer extension product which is autocatalytically replicatable RNA having incorporated therein a target RNA sequence;
• provided that either the first or second portion of said autocatalytically replicatable RNA is derived from the (+) strand and the other portion of said autocatalytically replicatable RNA sequence is derived from the (-) strand of the same autocatalytically replicatable RNA, and further provided that one of said cofunctioning primers includes a replicase binding site,
• chain extending said hybridized second nucleic acid primer,
• permitting the second extension product of the previous step, optionally after strand separation, to undergo replication by contacting with an appropriate RNA-dependent RNA polymerase, and
• detecting the presence or absence of replication product, wherein detection of said replication product is indicative of the presence or said specific RNA target sequence in the sample.

A kit useful for the detection of at least one specific RNA target sequence in a sample containing nucleic acid, said kit comprising:

• (1) a first nucleic acid primer comprising:
• (a) at its 5'-end, a first portion of an autocatalytically replicatable RNA, wherein the complement of said first portion is a segment of an autocatalytically replicatable RNA which comprises the 3'-end of an autocatalytically replicatable RNA which is capable of RNA-dependent RNA polymerase-directed autocatalytic replication, and wherein said first portion is a segment of a (+) strand or a (-) strand of an autocatalytically replicatable RNA, including the 5'-end;
• and
• (b) at its 3'-end, a first probe sequence which is capable of hybridizing to a segment of a target RNA sequence, and priming an extension reaction, using the target RNA sequence as template, to produce a first primer extension product which includes an RNA sequence complementary to the target RNA sequence;
• and
• (2) a second nucleic acid primer comprising:
• (a) at its 5'-end, a second portion derived from the opposite strand of said autocatalytically replicatable RNA, wherein the complement of said second portion comprises the 3'-end of an autocatalytically replicatable RNA which is capable of RNA-dependent RNA polymerase-directed autocatalytic replication, and wherein said second portion is a segment of a (+) strand or a (-) strand of said autocatalytically replicatable RNA, including the 5'-end;
• and
• (b) at its 3'-end, a second probe sequence which is capable of hybridizing to a segment of said RNA sequence complementary to the target RNA sequence and priming an extension reaction, using the first extension product as template, to produce a second primer extension product which is autocatalytically replicatable RNA having incorporated therein a target RNA sequence;
• provided that either the first or second portion of said autocatalytically replicatable RNA is derived from the (+) strand and the other portion of said autocatalytically replicatable RNA is derived from the (31 ) strand of the same autocatalytically replicatable RNA, and further provided that one of said cofunctioning primers includes a replicase binding site;
• and
• (3) RNA-dependent RNA polymerase.