In various fields such as a clinical field, a pathologic field, etc., a polymerase chain reaction (PCR) method is used widely for the purpose of a gene expression analysis, a gene function analysis, genetic diagnoses, and the like. The PCR method generally includes three steps:
1) denaturing DNA (dissociation from double-stranded DNA into single-stranded DNA) by a heat treatment;
2) annealing a primer to single-stranded DNA template; and
3) extending the primer by DNA polymerase as a cycle and, by repeating the cycle, amplifies DNA complementary to a target nucleic acid in a sample.
However, when a biological sample, especially a whole blood sample, is subjected to PCR, because of the heat treatment for the DNA denaturation, saccharide and protein contained in the sample are denatured and an insoluble precipitate, turbidity, or the like, is generated. The generation of such precipitate, or the like does not become a big problem when the presence or absence of the amplification product is confirmed by electrophoresis, for example. However, when the presence or absence of the amplification product is confirmed by an optical unit, because the precipitate or the turbidity blocks an irradiated light, there is a problem that accurate measurements cannot be obtained. Especially, with a method of monitoring a preparation process of the amplification product with time in PCR (what is called, a real time PCR method), for example, quantitative analysis of the amplification product per cycle and a count of the number of the cycles when the amplification product reaches a specified quantity (threshold value) can be performed. Further, on the basis of those information, quantitative analysis of the target nucleic acid in the biological sample also can be performed. Therefore, a high emphasis is placed on a realization of an accurate measurement by the optical unit.
In order to solve such problem, conventionally, methods are taken in which the biological sample is purified preliminarily in advance of the use as a PCR sample (pretreatment) and in which a PCR reaction solution is purified after a reaction (aftertreatment). Examples of the pretreatment of the biological sample include methods: in which the heat treatment is preliminarily applied to the biological sample and the generated precipitate or the like is removed and then an obtained supernatant is used as the PCR sample; and in which a causative substance of the precipitate, the turbidity, or the like is removed preliminarily from the biological sample. Further, an example of the aftertreatment of the PCR reaction solution includes a method in which the precipitate or the like generated after the PCR reaction is removed in advance of a detection of the amplification product.
On the other hand, with the PCR using the biological sample, from the perspective of ease and swiftness of an operation, it is desired that the biological sample is used as it is without applying the pretreatment such as the purification. In this state, it is reported that the ratio of the whole blood sample in the PCR reaction solution is in a range of about 1 to 10% by volume (Patent Document 1, Non-Patent Documents 1 and 2).
However, as described above, when the detection of the amplification product is carried out by the optical unit with a former method, the pretreatment (a purification treatment) of the biological sample is required. Further, with a latter method, although the biological sample can be used as it is, because a removal of the precipitate after completion of the reaction is required, the operation becomes as complicated as the former method. Moreover, there is a problem that the analysis can be performed only after the completion of the reaction. In other words, although the amplification product finally obtained can be analyzed, it is difficult to monitor the preparation process of the amplification product with time by the optical unit. Further, with respect to the biological sample such as the whole blood sample, without applying the pretreatment, there is a problem that the PCR reaction may be hindered due to components contained therein (Non-Patent Documents 3 and 4).    [Patent Document 1] JP3727667B    [Non-Patent Document 1] Nucleic Acids Research, Vol. 18, No. 19, 5908 (1990)    [Non-Patent Document 2] Nucleic Acids Research, Vol. 19, No. 5, 1151 (1991)    [Non-Patent Document 3] Nucleic Acids Research, Vol. 16, No. 20, 9775-9787 (1998)    [Non-Patent Document 4] Journal of Clinical Microbiology, Vol. 39, No. 2. p 485-493 (2001)