In recent years, a variety of gene amplification methods for amplifying a gene aiming at the detection of a trace amount of a target gene have been developed. Of these methods, a Polymerase Chain Reaction method utilizing a thermostable nucleic acid polymerase (U.S. Pat. Nos. 4,683,195, 4,683,202, hereinafter referred to as “PCR”), a Ligase Chain Reaction method utilizing a thermostable nucleic acid ligase (U.S. Pat. No. 5,792,607, hereinafter referred to as “LCR”), a Strand Displacement Amplification method utilizing a nucleic acid polymerase having strand displacement activity (JP 2076096, hereinafter referred to as a “SDA method”), and an Isothermal and Chimeric primer-initiated Amplification of Nucleic acids method (WO 00/56877, hereinafter referred to as an “ICAN method”) are gene amplification methods which have been developed taking advantage of characteristics of enzymes synthesizing nucleic acids, respectively.
These gene amplification methods make use of the reaction in which only a specific region of a gene is replicated repetitively, thereby a gene fragment that consists of the specific region is amplified. Accordingly, it is difficult to detect the products amplified by the gene amplification methods in a simple and easy way, because they are linear gene fragments. In the gene detection methods employed in kits for gene diagnosis on the market, a target gene is mainly detected in combination with EIA (enzyme immunoassay) or by previously labeling a gene with a fluorescent substance.
EIA and the measurement in which a gene is labeled with a fluorescent substance, however, need special instruments and reagents, and are complicated in their procedures, requiring more than one hour before judgment. Thus, a simple and inexpensive method for detection of genes amplified by conventional gene amplification methods has been awaited.
On the other hand, the present applicants have already proposed a novel isothermal amplification method of nucleic acid without using an enzyme (a method for forming a self-assembly substance of probes) (U.S. Pat. No. 6,261,846, JP 3267576 and EP 1,002,877A). This method makes use of a pair of probes comprising 3 regions (HoneyComb Probe, hereinafter referred to as an “HCP”), in which a first probe and a second probe are designed such that the 3 regions in each of the probes has base sequences complementary to each other, and when both probes are allowed to react, only one region of the first probe hybridizes to one region of the second probe. This design makes it possible for a plurality of pairs of the probes to hybridize with each other upon reaction and form a self-assembly substance of the probes (Probe alternation link self-assembly reaction, hereinafter referred to as a “PALSAR method”).