JP2018157799A - Gene examination method, gene examination chip, and examination system - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a gene examination method which performs type decision for each of a plurality of genes in a short time.SOLUTION: The gene examination method includes: a heating step for mixing a detection reagent comprising a plurality of fluorescent dyes and a biological specimen comprising a plurality of target genes and then heating analytes obtained by modifying each of the plurality of target genes by fluorescent dyes each different from the other among the plurality of fluorescent dyes, the heating being performed in such a manner that a periodical change is repeated within a predetermined temperature range; a fluorescence detection step for projecting a plurality of excitation light beams each with a different wavelength onto the analytes while heating the analytes at a constant rate of temperature rise and detecting a fluorescent change of the analytes; and a decision step for performing a type decision for the plurality of target genes based on the change of fluorescence intensity obtained in the fluorescence detection step. The heating step includes: a first heating step (S101) for heating the analytes by repeating the periodical change by a first number of repetitions in a first period; and a second heating step (S102) for heating the analytes by repeating the periodical change by a second number of repetitions in a second period different from the first period.SELECTED DRAWING: Figure 1A

Description

  The present disclosure relates to a genetic testing method, a genetic testing chip, and a genetic testing system.

  A gene polymorphism is an individual difference in the base sequence of DNA constituting a gene, and is generally defined to occur at a frequency of 1% or more of a population. The gene polymorphism includes a single nucleotide polymorphism (SNP: single nucleotide polymorphism) in which only one base of a DNA base sequence is mutated, and a microsatellite polymorphism that is a difference in the number of repeats of one unit of about 2 to 4 bases ( microsatellite polymorphism), base deletion and insertion. It is known that some gene polymorphisms affect the susceptibility to illness and drug metabolism. The base of the SNP site is used for diagnosing the morbidity of disease, the effect of administered drugs, and predicting side effects. Judgment is made.

  Patent Document 1 discloses a technique for determining three types of genes using three probes modified with fluorescent dyes having different wavelengths. Non-Patent Document 1 discloses a touch-down PCR method that improves amplification performance by lowering the annealing temperature in a PCR cycle step by step for each number of times.

Japanese Patent No. 5279492

Touchdown 'PCR to Circumferential spur priming dur ing gene ampli? Cation; H. Don et al. Nucleic Acid Research, Vol. 19, no. 14 (1991) 4008

  However, in the prior art disclosed in Patent Document 1, when the PCR cycle is shortened in order to perform the type determination of a plurality of types of genes in a short time, the difference in amplification characteristics between genes gradually becomes remarkable, and erroneous determination is made. There is a problem that arises. Moreover, although the conventional technique disclosed in Non-Patent Document 1 can determine the genotype for a single SNP, there is a problem that it is difficult to determine the genotype for a plurality of SNPs.

  Therefore, an object of the present disclosure is to provide a genetic testing method, a genetic testing chip, and a genetic testing system that can realize type determination of a plurality of types of genes in a short time.

  In order to achieve the above object, a genetic testing method according to an embodiment of the present disclosure includes mixing a detection reagent containing a plurality of types of fluorescent dyes and a biological sample containing a plurality of types of target genes, A heating step in which samples obtained by modifying different fluorescent dyes among each of a plurality of types of fluorescent dyes are repeatedly heated within a predetermined temperature range and heated at a constant temperature increase rate. A fluorescence detection step for irradiating a sample with a plurality of excitation lights having different wavelengths and detecting a fluorescence change of the sample, and a determination step for performing type determination of a plurality of types of target genes based on a change in fluorescence intensity obtained in the fluorescence detection step And the heating step includes a second cycle different from the first cycle, and a first heating step of heating the specimen by repeating the periodic change a first number of times in the first cycle. Comprising a second heating step of heating the sample a periodic change is repeated a second number, the.

  In order to achieve the above object, a genetic test chip according to one embodiment of the present disclosure heats a specimen by repeating periodic changes in a predetermined temperature range, and amplifies a plurality of types of target genes contained in the specimen. A sample holding unit for holding the sample, and a medium for holding an identifier associated with the heating cycle and the number of times of heating for amplifying a plurality of types of target genes.

  Furthermore, in order to achieve the above object, a genetic test system according to one embodiment of the present disclosure mixes a detection reagent containing a plurality of types of fluorescent dyes and a biological sample, and a plurality of types of target genes are mixed with each of a plurality of types of target genes. Specimens obtained by modifying different fluorescent dyes among the fluorescent dyes are heated in a predetermined temperature range by repeating periodic changes and a heating unit that heats the specimen at a constant temperature increase rate while the wavelength of the specimen is adjusted. A fluorescence detection unit that irradiates a plurality of different excitation lights and detects a fluorescence change of the specimen, and a determination unit that performs type determination of a plurality of types of target genes based on a change in fluorescence intensity obtained in the fluorescence detection unit, The fluorescence detection unit heats the specimen by repeating a periodic change a first number of times in the first cycle by the heating unit, and then at least some of the target genes of the plurality of types of target genes by the fluorescence detection unit 1st fluorescence detection part which detects the change of the fluorescence intensity of 1 target gene, and after a detection by the 1st fluorescence detection part, it is different from the 1st period by a heating part, and a periodical change is carried out by the 2nd number of times. A second fluorescence detection unit that detects a change in fluorescence intensity of a second target gene that is at least a part of a plurality of types of target genes included in the sample by the fluorescence detection unit after repeatedly heating the sample; Is provided.

  According to the present disclosure, it is possible to provide a genetic testing method, a genetic testing chip, and a genetic testing system that can realize type determination of a plurality of types of genes in a short time.

The flowchart which shows the genetic test method which concerns on the 1st aspect in 1st Embodiment. The graph which shows the fluorescence change of one of the multiple types of target genes (SNP3) which shows the timing chart of the genetic testing method which concerns on the 1st aspect in 1st Embodiment. The flowchart which shows the genetic test method which concerns on the 2nd aspect in 1st Embodiment. Timing chart of the genetic testing method according to the second aspect of the first embodiment Flowchart showing a genetic testing method according to the second embodiment Graph for detecting a change point of fluorescence intensity of one of a plurality of types of target genes (SNP1) in the genetic testing method according to the second embodiment Graph for detecting a change point of fluorescence intensity of one of a plurality of types of target genes (SNP2) in the genetic testing method according to the second embodiment Graph for detecting a change point of fluorescence intensity of one of a plurality of types of target genes (SNP3) in the genetic testing method according to the second embodiment The graph which shows the fluorescence change of one of several types of target genes (SNP3) in the 1st heating step (PCR1) which concerns on the 1st aspect in 1st Embodiment. The graph which shows the fluorescence change of one of several types of target genes (SNP3) in the 2nd heating step (PCR2) which concerns on the 1st aspect in 1st Embodiment. The perspective view which shows an example of the genetic test chip | tip concerning 3rd Embodiment. The flowchart which shows an example of the genetic testing method using the genetic testing chip concerning 3rd Embodiment Block diagram showing a genetic test system according to a fourth embodiment An example of a timing chart of a genetic testing method according to another embodiment Another example of a timing chart of a genetic testing method according to another embodiment

  Hereinafter, embodiments of the present disclosure will be described with reference to the drawings. Note that each of the embodiments described below shows a preferred specific example of the present disclosure. Therefore, the numerical values, shapes, materials, components, arrangement positions and connection forms of components, and steps and the order of steps shown in the following embodiments are merely examples, and are not intended to limit the present disclosure. Absent. Therefore, among the constituent elements in the following embodiments, constituent elements that are not described in the independent claims indicating the highest concept of the present disclosure are described as arbitrary constituent elements.

  Each figure is a schematic diagram and is not necessarily illustrated strictly. Moreover, in each figure, the same code | symbol is attached | subjected to the substantially same structure, The overlapping description is abbreviate | omitted or simplified.

(Embodiment)
[A. Genetic testing method]
Hereinafter, the genetic testing method according to the present disclosure will be described with reference to FIGS. 1A to 5C.

  In the genetic testing method according to the present disclosure, a detection reagent containing a plurality of types of fluorescent dyes and a biological sample containing a plurality of types of target genes are mixed, and each of the plurality of types of target genes is different from each other among the plurality of types of fluorescent dyes. A heating step (so-called PCR: Polymerase chain reaction) for heating a specimen obtained by modifying a fluorescent dye by repeating periodic changes in a predetermined temperature range, while heating the specimen at a constant heating rate A fluorescence detection step of irradiating the specimen with a plurality of excitation lights having different wavelengths and detecting a fluorescence change of the specimen.

  In the heating step (PCR), a sample prepared by modifying a plurality of types of target genes with different fluorescent dyes in advance is used to amplify a plurality of types of target genes including the target SNP site. A PCR amplification product is obtained.

  In the subsequent fluorescence detection step, the genotype is determined based on the difference in dissociation temperature at which the probe labeled with the fluorescent dye is separated from the PCR amplification product of the target gene while heating the specimen after the heating step at a constant heating rate.

  Here, the genetic test method of the present disclosure includes a genetic test method (first embodiment) in which analysis conditions for a plurality of types of target genes are determined in advance, and analysis conditions ( Specifically, it can be classified as a genetic test method (second embodiment) for determining the temperature range of heating in PCR, the number of heating cycles in PCR, the number of PCR executions, and the like.

  Hereinafter, the genetic testing method according to the first embodiment and the second embodiment will be described in detail.

[A-1. First Embodiment]
As described above, the genetic testing method according to the present embodiment is a genetic testing method in which analysis conditions for a plurality of types of target genes are determined in advance.

  In the present embodiment, the heating step includes a first heating step in which the specimen is heated by repeating the periodic change in the first cycle a first number of times, and a cycle in a second cycle different from the first cycle. And a second heating step of heating the specimen by repeating the mechanical change a second number of times.

  In the present embodiment, the second period may be shorter than the first period.

  In the present embodiment, the heating step may further include a third heating step of heating the specimen by repeating the periodic change a third number of times in a third period shorter than the second period.

  Hereinafter, the genetic testing method according to the present embodiment will be described by dividing it into two modes using examples. These modes include the number of times of heating by repeating a periodic change in a predetermined temperature range in the heating step (PCR) (number of heating cycles), the period of heating, the number of times of performing the heating step (PCR), and the fluorescence detection step. They are classified based on the target gene to be subjected to fluorescence change detection and the timing of genotype determination. In the following example, three types of target genes, SNP1, SNP2, and SNP3, are detection targets. In addition, the genetic testing method according to each aspect is configured to include the first, second, and third fluorescence detection circuits that can perform each of the above steps and detect SNP1, SNP2, and SNP3, respectively. Implemented using a genetic testing system. Details of this system will be described later as a fourth embodiment.

[A-1-1. First aspect]
The genetic testing method according to the first aspect will be described with reference to FIGS. 1A and 1B. FIG. 1A is a flowchart showing an example of the genetic testing method according to the first aspect, and FIG. 1B is a timing chart of an example of the genetic testing method according to the first aspect. FIG. 2A is a graph showing changes in fluorescence of one of a plurality of types of target genes (SNP3) in the first heating step (PCR1) according to the first aspect of the first embodiment. FIG. 2B is a graph showing changes in fluorescence of one of a plurality of types of target genes (SNP3) in the second heating step (PCR2) according to the first aspect of the first embodiment.

  As shown in FIG. 1A, in the first embodiment, first, a genetic test chip into which a specimen has been injected is set in the genetic test system (S100).

  Next, in order to amplify the target gene, the first heating step (PCR1) that is the first heating step is performed (S101), and then the second heating step (PCR2) that is the second heating step is performed. (S102).

  Next, the fluorescence intensity of the target genes (SNP1, SNP2, and SNP3) is measured in the fluorescence detection step (S103). In the fluorescence detection step S103, the fluorescence detection circuit provided in the genetic test system is used. Each fluorescence detection circuit will be described later.

  Next, Tm values (Tm1, Tm2, and Tm3) are calculated from the fluorescence intensity graph (S104).

  Finally, the genotypes of the target genes (SNP1, SNP2, and SNP3) are determined (S105).

  Details of a series of steps from step S101 to S105 will be described with reference to a timing chart of a specific example of the first mode shown in FIG. 1B.

  Step S101 corresponds to the section indicated by the label of PCR1 in FIG. 1B. In step S101, the specimen is heated by repeating the periodic change a first number of times within a predetermined temperature range in the first cycle. More specifically, the sample is heated by repeating a 30-second periodic change in the temperature range from T1 to T3 10 times. That is, the temperature range from T1 to T3 is an example of the predetermined temperature range in the first mode, the 30-second cycle is an example of the first cycle in the first mode, and 10 times in the first mode. It is an example of the first number of times.

  Step S102 corresponds to the section indicated by the label PCR2 in FIG. 1B. In step S102, the specimen is heated by repeating a periodic change a second number of times in a predetermined temperature range and in a second cycle. More specifically, the specimen is heated by repeating a periodic change with a 20 second period in the temperature range from T1 to T3 40 times. That is, the temperature range from T1 to T3 is an example of the predetermined temperature range in the first aspect, 20 seconds is an example of the second period in the first aspect, and 40 times is the first period in the first aspect. It is an example of 2 times. Here, the second cycle is shorter than the first cycle. That is, unlike the first cycle and the second cycle, in this example, the second cycle applied in PCR2, which is a heating step performed later, is shorter than the first cycle.

  Step S103 corresponds to the section indicated by the labels SNP-1, SNP-2, and SNP-3 in FIG. 1B. In step S103, the specimen is heated at a constant rate at a constant temperature increase rate. Heating for 200 seconds from a temperature lower than T1 to a temperature close to T3 in this section of FIG. 1B is an example of this constant speed heating. In this section, the fluorescent dye (probe labeled with the fluorescent dye) bound to the PCR amplification products of a plurality of types of target genes contained in the specimen dissociates as the temperature rises and emits fluorescence.

  When this change in fluorescence is measured continuously, the probe labeled with a fluorescent dye is a completely complementary sequence in the genotype (1) having a sequence that is not completely complementary (for example, a sequence that differs by only one base). Dissociates from the PCR amplification product and emits light at a lower temperature than in the case of genotypes having On the other hand, in the genotype (2) having a completely complementary sequence, the probe labeled with a fluorescent dye dissociates at a higher temperature than in the case of the genotype (1) having a sequence that is not completely complementary, and emits light. To do. In addition, in the genotype (heterotype) having both the genotype (2) having a completely complementary sequence and the genotype (1) having a sequence not completely complementary, the low temperature and the high Dissociates and emits light at both temperatures. The dissociation temperature Tm value of the probe with such a genotype is calculated (S104), and the genotype is determined from the difference in the dissociation temperature (S105).

  As described above, in the genetic testing method according to the first aspect of the present embodiment, the heating step includes a first heating step of heating the specimen by repeating the periodic change a first number of times in the first cycle, And a second heating step of heating the specimen by repeating a periodic change a second number of times in a second period, which is different from the period of.

  Thus, by heating the specimen with periodic changes at different periods, each target gene of a plurality of types of target genes contained in the specimen can be amplified under more appropriate conditions. Thereby, the accuracy can be ensured while shortening the time required for the type (genotype) determination of a plurality of target genes.

  In the genetic testing method according to the first aspect of the present embodiment, the second period is shorter than the first period.

  Thereby, the target gene is amplified under appropriate conditions for each type, and the accuracy can be ensured while shortening the time required for the type (genotype) determination.

[A-1-2. Second aspect]
The genetic testing method according to the second embodiment will be described with reference to FIGS. 2A and 2B. FIG. 2A is a flowchart showing an example of the genetic testing method according to the second aspect, and FIG. 2B is a timing chart of an example of the genetic testing method according to the second aspect. In FIG. 2A, steps common to the flowchart of the first aspect are denoted by reference numerals common to FIG. 1A.

  The second aspect includes a first heating step and a second heating step having different heating cycles, and the heating cycle in the second heating step performed after the first heating step is the same as that in the first heating step. The point shorter than the period of heating is common to the first aspect. Hereinafter, a series of procedures of the second mode will be described while showing points different from the first mode.

  As shown in FIG. 3A, in the second embodiment, first, the genetic test chip into which the specimen has been injected is set in the genetic test system (S100).

  Next, in order to amplify the target gene, the first heating step (PCR1) that is the first heating step is performed (S111), and then the second heating step (PCR2) that is the second heating step is performed. (S112). In the second aspect, a third heating step is further performed following the second heating step (S113).

  Next, the fluorescence intensity of the target genes (SNP1, SNP2, and SNP3) is measured in the fluorescence detection step (S114). In the fluorescence detection step S114, the fluorescence detection circuit provided in the genetic test system is used. Each fluorescence detection circuit will be described later.

  Next, Tm values (Tm1, Tm2, and Tm3) are calculated from the fluorescence intensity graph (S115).

  Finally, the genotypes of the target genes (SNP1, SNP2, and SNP3) are determined (S116).

  Details of the flow from steps S111 to S116 will be described with reference to a timing chart of a specific example of the second mode shown in FIG. 2B.

  Step S111 corresponds to the section indicated by the label of PCR1 in FIG. 2B. In step S111, the sample is heated by repeating the periodic change a first number of times within a predetermined temperature range in the first cycle. More specifically, the specimen is heated by repeating a periodic change in a period of 35 seconds in the temperature range from T1 to T3 10 times. That is, the temperature range from T1 to T3 is an example of the predetermined temperature range in the second aspect, 35 seconds is an example of the first period in the second aspect, and 10 times is the second period in the second aspect. It is an example of 1 times.

  Step S112 corresponds to the section indicated by the label of PCR2 in FIG. 2B. In step S112, the sample is heated by repeating the periodic change a second number of times within a predetermined temperature range in the second cycle. More specifically, the sample is heated by repeating a periodic change with a 25-second period in the temperature range from T1 to T3 20 times. That is, the temperature range from T1 to T3 is an example of the predetermined temperature range in the second aspect, 25 seconds is an example of the second period in the second aspect, and 20 times is the second period in the second aspect. It is an example of 2 times.

  Step S113 corresponds to the section indicated by the label of PCR3 in FIG. 2B. In step S113, the sample is heated by repeating the periodic change a third number of times within a predetermined temperature range in the third cycle. More specifically, the specimen is heated by repeating a periodic change with a 15-second period in the temperature range from T1 to T3 20 times. That is, the temperature range from T1 to T3 is an example of the predetermined temperature range in the second aspect, 15 seconds is an example of the third period in the second aspect, and 20 times is the second period in the second aspect. It is an example of the number of times of 3.

  Step S114 corresponds to the section indicated by the labels SNP-1 and SNP-2 in FIG. 2B. In step S114, the specimen is heated at a constant rate at a constant temperature increase rate. Heating for 200 seconds from a temperature lower than T1 to a temperature close to T3 in this section of FIG. 2B is an example of this constant speed heating. In this section, among the multiple types of target genes contained in the specimen, the fluorescent dye (probe labeled with the fluorescent dye) bound to the PCR amplification product of the first target gene dissociates as the temperature increases, To emit. The determination of the genotype based on the change in the intensity of the fluorescence is as described for the first aspect, so the description is omitted here.

  Here, also in the second mode, as in the first mode, the second period is shorter than the first period. The heating step further includes a third heating step in which the specimen is heated by repeating the periodic change a third number of times in the third cycle, which is shorter than the second cycle.

  As a result, the number of stages of PCR is increased, and the time required for type (genotype) determination can be shortened as compared with the conventional method, and the accuracy can be ensured more reliably.

  Thus, unlike the first embodiment, the heating step is performed three times in the second embodiment. Even if the number of times is repeated, the target gene type can be accurately determined in a shorter time than the conventional method.

[A-1-3. Example]
Here, an example of genotype determination based on a fluorescence waveform by the genetic testing method according to the above embodiment will be shown. This determination was performed on three genes related to obesity (ADBR2 gene, ADBR3 gene, and UCP1 gene). In this method, the ADBR2 gene was treated as SNP1, the ADBR3 gene as SNP2, and the UCP1 gene as SNP3.

  First, as a genetic test method according to the first aspect, PCR1 is repeated 10 times in a predetermined temperature range (T3 = 95 ° C., T1 = 62 ° C.) with a 30-second periodical change (heating cycle). Table 1 shows the quality of the determination when a heating cycle with a period of 20 seconds in a predetermined temperature range (the same temperature range as PCR1) is repeated 30, 40, and 50 times.

  PCR2 in the table is the number of repetitions of periodic heating executed in PCR2. In Table 1, “x” indicates an erroneous determination, “△” indicates a low frequency but erroneous determination, “◯” indicates that the determination was possible, and “◎” indicates that the determination was accurate. .

  As can be seen from Table 1, in the genetic testing method according to the first aspect, it was possible to accurately determine any target gene when the heating cycle in PCR 2 was 40 times and 50 times. In this example, there was no (×) that had a high frequency of misjudgment.

  Next, as a genetic testing method of the second embodiment, PCR1 is subjected to a cyclic change (heating cycle) of 35 seconds in a predetermined temperature range (T3 = 95 ° C., T1 = 62 ° C.) 10 times, and then PCR2 Table 2 shows the quality of the determination when the heating cycle of 25 seconds is repeated 30 times, 40 times, and 50 times in a predetermined temperature range (the same temperature range as PCR1).

  As can be seen from Table 2, in the genetic testing method according to the second embodiment, when the heating cycle in PCR2 was set to 40 times and 50 times, it was possible to accurately determine any target gene.

  Moreover, even when the heating cycle of PCR2 was set to 30 times, all of the target genes could be determined. However, when the detection time is calculated, it is 1100 seconds (calculated only for the heating cycle time) in 40 times in the genetic testing method according to the first aspect and 30 times in the genetic testing method according to the second aspect. Therefore, the target gene can be more accurately determined by increasing the number of times in a short cycle.

  In the second embodiment, a genetic test method including a procedure for determining the number of such heating cycles will be described.

[A-2. Second Embodiment]
As described above, the genetic testing method according to the present embodiment measures the analysis conditions (specifically, the temperature range, period, and periodic change of heating in PCR) while measuring the fluorescence intensity of multiple types of target genes. Cycle number), PCR (heating step) number, etc.).

  In addition, the gene testing method according to the present embodiment can perform the following steps, and is configured to include first to third fluorescence detection circuits for detecting SNP1, SNP2, and SNP3, respectively. Implemented using inspection system. The configuration of this genetic test system is basically the same as the configuration of the genetic test system used in the first embodiment, and details will be described later.

  Hereinafter, the genetic testing method according to the second embodiment will be described with reference to FIGS. FIG. 3 is a flowchart showing a genetic testing method according to the second embodiment, and FIGS. 4A to 4C show the fluorescence intensities of a plurality of types of target genes (SNP1, SNP2, and SNP3) in the second embodiment, respectively. It is an example of the graph used for a change point detection.

  4A, FIG. 4B, and FIG. 4C show the fluorescence changes measured in the preliminary PCR described later for the ADBR2 gene as SNP1, the ADBR3 gene as SNP2, and the UCP1 gene as SNP3, respectively. For example, referring to FIG. 4A, it is shown that the tendency of fluorescence change has changed greatly in the vicinity of the 25th heating cycle. The present embodiment is based on the confirmation that there is a certain relationship between the number of heating cycles and the optimum number of PCR amplifications.

  As shown in FIG. 3, first, the genetic test chip into which the specimen has been injected is set in the genetic test system (S100).

  Next, the first, second, and third fluorescence detection circuits are activated (S130).

  Next, while performing PCR (hereinafter referred to as “preliminary PCR”) set to a predetermined temperature range and a predetermined number of heating cycles Pn-0 (for example, 50 times), a plurality of types of target genes ( Changes in the fluorescence intensity of SNP1, SNP2, and SNP3) are measured (S141).

  Next, change points (also referred to as inflection points) Pth-1, Pth-2, and Pth-3 of the fluorescence intensity of each target gene are detected (S142).

  Next, the number of heating cycles in the preliminary PCR at the change point of the fluorescence intensity of each target gene detected in step S142 is multiplied by a predetermined coefficient, and each of the PCR1, PCR2, and PCR3 is performed until completion. Pn-1, Pn-2, and Pn-3, which are cumulative numbers of heating cycles, are calculated (S143).

  Next, the number of remaining heating cycles to be performed before the completion of PCR1 is calculated by subtracting Pn-0 which is the number of performed heating cycles from the calculated number of Pn-1 cycles ( S144).

  The flow from step S141 to S144 will be described in more detail using a specific example.

  As shown in FIGS. 4A to 4C, during the preliminary PCR (Pn-0 = 50) (S141), the change in the fluorescence intensity of multiple types of target genes (SNP1, SNP2, and SNP3) is continuously measured. (S142). In this example, among multiple types of target genes, SNP1 is in the 25th cycle (Pth-1 = 25), SNP2 is in the 25th cycle (Pth-2 = 25), and SNP3 is in the 35th cycle (Pth-3 = 35). A change point is seen in (S142).

  Next, these change points are multiplied by a predetermined coefficient for each target gene, and the cumulative number of heating cycles Pn-1, Pn-2, and Pn- that are performed until each of PCR1, PCR2, and PCR3 is completed. 3 is calculated (S143). This coefficient is determined based on, for example, a certain relationship between the number of heating cycles described above and the optimum number of PCR amplifications confirmed by the inventors.

  Thereafter, the number Pn-0 of heating cycles of the preliminary PCR is subtracted from Pn-1 calculated in step S143 to calculate the number of remaining cycles to be performed in PCR1 (S144). When the number of remaining cycles is 0, it is assumed that PCR1 (S151) is completed by performing preliminary PCR (S141). If the remaining cycle number is greater than 0, PCR1 is completed by repeating the heating cycle for the remaining cycle number (S151).

  Moreover, in PCR2, the heating cycle of the cycle number of Pn-2 calculated by step S143 is implemented (S251).

  Moreover, in PCR3, the heating cycle of the cycle number of Pn-3 calculated by step S143 is implemented (S351). Thereafter, Tm values (Tm1, Tm2, and Tm3) are calculated from the fluorescence change of each fluorescence detector (S352), and the genotype of each target gene (SNP1, SNP2, and SNP3) is determined (S353).

  After the flow from step S131 to S353 is completed (genotype determination for all types of target genes is completed), the first to third fluorescence detection circuits 27 to 29 are stopped (S190), and the second The genetic testing method according to the embodiment is completed.

  In the above description, in step S144, when the number of remaining cycles calculated for PCR1 is 0, PCR1 has been completed, but the same applies to PCR2 or PCR3. In other words, PCR1 to PCR3 are not performed when the calculated remaining cycle number is zero. As described above, the number of PCR implementations (one of PCR1 to PCR3) may be determined by the genetic testing method according to the second embodiment.

  When the cycle of PCR1 is short, some of PCR1 and PCR2 may be included in the preliminary PCR. FIG. 5A shows a change in fluorescence in SNP3 when preheating PCR1 is performed 10 times. In this range, there is no change point in the fluorescence change. Subsequently, FIG. 5B shows the fluorescence change when PCR2 is performed 30 times, and the change point of the fluorescence change can be seen at the 26th cycle. By multiplying this change point by a predetermined coefficient, the cumulative number Pn-2 or Pn-3 of heating cycles to be performed until the completion of PCR2 or PCR3 is calculated (S143). The coefficient in this case is determined in advance based on the period of the heating cycle described above, the optimum number of PCR amplifications, and the results of other genes (SNP1, SNP2).

  As described above, the genetic testing method according to the present embodiment measures changes in the fluorescence intensity of a plurality of types of target genes while heating a specimen by repeating a periodic change a first number of times in a first cycle, During the heating of the specimen by determining at least one of the first number of times and the number of times after the second number based on the change in the fluorescence intensity, or repeating the periodic change for the second number of times in the second period. There may be included a heating cycle determination step of measuring a change in the fluorescence intensity of each type of target gene and determining at least one of the second number and the second and subsequent times based on the change in the fluorescence intensity.

  This makes it possible to set heating conditions for realizing genotype determination in a shorter time even for new types of target genes that do not have existing analysis data (analysis conditions and the like). Moreover, since the number of heating cycles can be changed according to individual differences between samples, the accuracy of genotype determination can be improved.

  In the genetic testing method according to the present embodiment, changes in the fluorescence intensity of a plurality of types of target genes are measured while the specimen is heated in the heating cycle determination step, and subsequent heating is performed based on the changes in fluorescence intensity. A temperature range different from the temperature range in the heating cycle may be set as the temperature range in the cycle. Thereby, the target gene is amplified under appropriate conditions for each type, and the accuracy can be ensured while shortening the time required for the type (genotype) determination.

[B. Genetic testing chip and genetic testing method using genetic testing chip]
Hereinafter, a genetic testing chip and a genetic testing method using this genetic testing chip in the present disclosure will be described as a third embodiment with reference to FIGS. 6 and 7. FIG. 6 is a perspective view showing an example of a genetic test chip according to the present embodiment. FIG. 7 is an example of a genetic testing method using the genetic testing chip according to this embodiment.

  As shown in FIG. 6, the genetic test chip 10 according to the present embodiment includes a chip unit 1 and a chip folder 2. The chip unit 1 repeats a periodic change in a predetermined temperature range, heats the sample, amplifies a plurality of types of target genes contained in the sample, a sample holding unit 3 that holds the sample, and a chip folder 2 Comprises a medium 4 for holding an identifier associated with the number of times of heating of the specimen for amplifying a plurality of types of target genes.

  Since the genetic test chip 10 according to the present embodiment has the above-described identifier, an operation such as setting a genetic test condition for each specimen becomes unnecessary, and the test can be automated. Therefore, determination of genotypes (types) of a plurality of types of target genes can be realized in a short time. In addition, other information associated with the identifier (for example, sample ID, coefficients necessary for determining the number of PCR cycles, etc.) eliminates the need for complicated operations such as setting conditions suitable for multiple types of target genes. Can be automated. Furthermore, the accuracy of determination of the genotypes of a plurality of types of target genes can be improved.

  Examples of the medium 4 that holds the identifier include a two-dimensional barcode and an IC chip.

  As an example of an association between the identifier by the medium 4 and the number of times of heating of the specimen, for example, the medium 4 may directly indicate the number of times of heating of each specimen that can be read by the genetic test system. Alternatively, the medium 4 indicates a sample ID, and the genetic testing system holds data such as a table in which the sample ID of each specimen is associated with the number of times of heating in a storage device, and is based on the sample ID read from the medium 4 You may acquire the frequency | count of a heating of the applicable specimen. Details of this genetic test system will be described later as a fourth embodiment.

  The genetic test chip 10 may include a sample injection port 5 and a flow path (not shown) that connects the sample injection port 5 and the sample holding unit 3.

  As shown in FIG. 7, an example of genetic testing using the genetic testing chip 10 is as follows. First, a sample is injected into the genetic testing chip 10 and the chip is set in the apparatus (S100), and then the first associated with the identifier. Information such as the number of times 1 (the number of heating cycles of PCR1) and the second number of times (the number of heating cycles of PCR2) is read (S170). Next, target genes (SNP1, SNP2, SNP3) are amplified in the first heating step (PCR1) (S171). Subsequently, in order to further amplify the target gene, a second heating step (PCR2) is performed (S172). Next, the fluorescence intensity of the target genes (SNP1, SNP2, SNP3) is measured in the fluorescence detection step (S173). Next, Tm values (Tm1, Tm2, Tm3) are calculated from the fluorescence intensity graph (S174). Next, the genotype of the target gene (SNP1, SNP2, SNP3) is determined (S175).

  That is, in the genetic testing method according to the present embodiment, the specimen is held on the chip 10 including the medium 4 that holds the first period and the first number of times, and the identifier indicating the second period and the second number of times. In the first heating step, the sample is heated by repeating the periodic change a first number of times in the first period corresponding to the identifier, and in the second heating step, the periodic change is performed in the second period corresponding to the identifier. May be repeated a second number of times to heat the specimen. In this way, determination of genotypes of multiple types of target genes can be realized in a short time by reading identifiers such as analysis conditions suitable for multiple types of target genes contained in the specimen and performing genetic testing. it can.

  The identifier may be further associated with a temperature range for heating the specimen for amplifying a plurality of target genes. Thereby, the target gene is amplified under appropriate conditions for each type, and the accuracy can be ensured while shortening the time required for the type (genotype) determination.

[C. Genetic testing system]
Hereinafter, the genetic testing system according to the present disclosure will be described with reference to FIGS. 7 and 8 as a fourth embodiment. FIG. 8 is a block diagram showing a genetic test system according to this embodiment.

  As shown in FIG. 8, the genetic test system according to the present embodiment mixes a detection reagent containing a plurality of types of fluorescent dyes and a biological sample, and each of a plurality of types of target genes includes a plurality of types of fluorescent dyes. A sample obtained by modifying different fluorescent dyes is heated in a predetermined temperature range by repeating periodic changes (Peltier element 25), and the sample is heated at a constant rate of temperature while the wavelength of the sample is adjusted. In the fluorescence detection unit 30 (the first fluorescence detection unit 31, the second fluorescence detection unit 32, and the third fluorescence detection unit 33) that irradiates a plurality of different excitation lights and detects the fluorescence change of the specimen, A determination unit that performs a type determination of a plurality of types of target genes based on a change in the obtained fluorescence intensity, and the heating unit 25 heats the specimen by repeating a periodic change a first number of times in a first period. , The first period and Becomes, the periodic variation in the second period repeated the second number of heating the specimen.

  As shown in FIG. 7, the genetic test system according to the present embodiment reads an identifier with the identifier reader 21 from the genetic test chip 10 including the medium 4 that holds the identifier described above (S170), and the controller inside the personal computer 22a. The information of the identifier is transmitted to 22. By the transmission from the controller 22, the Peltier drive circuit 24 is activated through the temperature setting circuit 23, and the Peltier element 25 heats or cools the specimen holding unit 3. Thereby, PCR1 (S171), PCR2 (S172), and constant-speed heating (S173) shown in FIG. 7 are performed.

  Fluorescence detection is performed at once for all of the multiple types of target genes. The fluorescence detection unit 30 differs depending on the type of target gene to be determined. That is, as described above, each of the plurality of types of target genes is modified with a different one of the plurality of types of fluorescent dyes, so that the fluorescence detection unit 30 detects the wavelength range of detection according to the plurality of types of fluorescent dyes. Are provided with a plurality of fluorescence detection units. For example, a case where the genotype of a target gene labeled with a predetermined fluorescent dye is determined will be described.

  For example, the first fluorescence detection unit 31 activates the first fluorescence detection circuit 27 through the fluorescence detector switching circuit 26 in accordance with an instruction from the controller 22. Then, the light having a predetermined wavelength emitted from the LED of the first fluorescence detection unit 31 passes through the filter, so that the wavelength band of the light is narrowed, reflected by the dichroic mirror, and irradiated onto the specimen holding unit 3 ( Excitation light). Fluorescence from the sample holder 3 is received by a photodiode (PD) through a dichroic mirror. When the fluorescence is received by the PD, a light reception signal is transmitted from the PD to the controller 22 through the PCR fluorescence detection circuit 34. After the first fluorescence detection step is completed, a Tm value is calculated by the Tm calculation circuit 35 according to an instruction from the controller 22, and the Tm value is transmitted to the controller 22. Based on this signal, the determination unit of the controller 22 determines the genotype of the target gene labeled with a predetermined fluorescent dye that can be detected by the first fluorescence detection unit 31.

  Since the second fluorescence detection unit 32 and the third fluorescence detection unit 33 are the same as described above, detailed description thereof is omitted.

  Thereby, the type (genotype) determination of multiple types of target genes can be realized in a short time. Furthermore, genetic testing can be automated, and genotypes of multiple types of target genes can be determined quickly and accurately.

[D. Others]
In the embodiment described above, in each heating step (PCR1, PCR2, and PCR3) that repeats a periodic change in a predetermined temperature range, an example of changing the length of the period and the number of repetitions of the periodic change (the number of heating cycles) Although described, for example, as shown in FIGS. 9A and 9B, the temperature range may be further changed.

  FIG. 9A is an example of a timing chart of a genetic testing method according to another embodiment, and FIG. 9B is another example of a timing chart of the genetic testing method according to another embodiment.

  As shown in FIG. 9A, in PCR2, the specimen may be heated by repeating a periodic change in the second temperature range, which is different from the first temperature range in PCR1, a second number of times. In addition, as shown in FIG. 9B, in PCR3, the sample is heated by repeating a periodic change in the third temperature range, which is different from the first temperature range in PCR1 and the second temperature range in PCR2, by the third number of times. May be.

  In this way, by heating the specimen with periodic changes in different temperature ranges, each target gene of a plurality of types of target genes contained in the specimen can be amplified under more appropriate conditions. Thereby, the accuracy can be ensured while shortening the time required for the type (genotype) determination of a plurality of target genes.

  For example, the upper limit temperature T3b of the second temperature range is higher than the upper limit temperature T3a of the first temperature range, and the upper limit temperature T3c of the third temperature range is higher than the upper limit temperature T3b of the second temperature range. Also good. Thereby, the target gene is amplified under appropriate conditions for each type, and the accuracy can be ensured while shortening the time required for the type (genotype) determination.

(Summary)
As described above, the genetic testing method according to the above embodiment mixes a detection reagent containing a plurality of types of fluorescent dyes and a biological sample containing a plurality of types of target genes, and a plurality of types of each of the plurality of types of target genes. A heating step for heating samples obtained by modifying different fluorescent dyes among the above-mentioned fluorescent dyes by repeating periodic changes within a predetermined temperature range, and the wavelength of the sample while heating the sample at a constant temperature increase rate. A fluorescence detection step of irradiating a plurality of different excitation lights and detecting a fluorescence change of the specimen, and a determination step of performing a type determination of a plurality of types of target genes based on a change in fluorescence intensity obtained in the fluorescence detection step. The heating step is different from the first heating step in which the specimen is heated by repeating the periodic change a first number of times in the first cycle, and the periodic change in the second cycle, which is different from the first cycle. Comprising a second heating step of heating the second repeat count sample, the.

  In this way, by heating the specimen with periodic changes in different temperature ranges, each target gene of a plurality of types of target genes contained in the specimen can be amplified under more appropriate conditions. Thereby, the accuracy can be ensured while shortening the time required for the type (genotype) determination of a plurality of target genes.

  For example, in the genetic testing method according to the above embodiment, the second period may be shorter than the first period.

  Thereby, the target gene is amplified under appropriate conditions for each type, and the accuracy can be ensured while shortening the time required for the type (genotype) determination.

  In the genetic testing method according to the above-described embodiment, the heating step further includes a third heating step of heating the specimen by repeating the periodic change a third number of times in the third cycle, which is shorter than the second cycle. May be included.

  As a result, the number of stages of PCR is increased, and the time required for type (genotype) determination can be shortened as compared with the conventional method, and the accuracy can be ensured more reliably.

  In the genetic testing method according to the above embodiment, the first heating step further has a first temperature range, and the second heating step further has a second temperature different from the first temperature range. You may have a range.

  Thereby, the target gene is amplified under appropriate conditions for each type, and the accuracy can be ensured while shortening the time required for the type (genotype) determination.

  In addition, the genetic testing method according to the above embodiment further measures changes in the fluorescence intensity of a plurality of types of target genes while heating a specimen by repeating a periodic change a first number of times in a first cycle. Determining at least one of the first number and the second and subsequent number based on the change in fluorescence intensity, or repeating the periodic change a second number in the second period and heating the specimen, There may be included a heating cycle determination step of measuring a change in the fluorescence intensity of a plurality of types of target genes and determining at least one of the second number and the second and subsequent times based on the change in the fluorescence intensity.

  This makes it possible to determine the genotype of a new type of target gene that does not have existing analysis data (analysis conditions or the like). Moreover, since the number of heating cycles can be changed according to individual differences between samples, the accuracy of genotype determination can be improved.

  In the genetic testing method according to the above-described embodiment, the specimen is held on the chip 10 including the medium 4 that holds the first period and the first number of times, and the identifier indicating the second period and the second number of times. In the first heating step, the sample is heated by repeating the periodic change a first number of times in the first period corresponding to the identifier, and in the second heating step, the periodic change is performed in the second period corresponding to the identifier. The specimen may be heated by repeating the second number of times.

  As a result, even when analysis conditions suitable for each type of target gene contained in a specimen are used, genotypes of multiple types of target genes can be obtained by performing a genetic test using an identifier indicating the analysis conditions of each specimen. It is possible to reduce the time required for the determination or to shorten the time.

  The genetic test chip 10 according to the above embodiment heats a sample by repeating periodic changes in a predetermined temperature range, and a sample holding unit for holding a sample for amplifying a plurality of types of target genes contained in the sample 3 and a medium 4 that holds an identifier associated with the heating cycle and the number of times of heating for amplifying a plurality of types of target genes.

  Since the genetic test chip according to the present disclosure has the identifier described above, operations such as setting conditions for genetic test are not required, and the test can be automated. Therefore, determination of genotypes (types) of a plurality of types of target genes can be realized in a short time. In addition, other information associated with the identifier (for example, sample ID, coefficients necessary for determining the number of PCR cycles, etc.) eliminates the need for complicated operations such as setting conditions suitable for multiple types of target genes. Can be automated. Furthermore, the accuracy of determination of the genotypes of a plurality of types of target genes can be improved.

  The genetic test system according to the above embodiment mixes a detection reagent containing a plurality of types of fluorescent dyes and a biological sample, and modifies different types of fluorescent dyes among the plurality of types of fluorescent dyes to each of the plurality of types of target genes. The specimen obtained by heating the specimen 25 by repeating periodic changes in a predetermined temperature range, and irradiating the specimen with a plurality of excitation lights having different wavelengths while heating the specimen at a constant rate of temperature rise. A fluorescence detection unit 30 that detects a change in fluorescence of the fluorescent light, and a determination unit that performs type determination of a plurality of types of target genes based on a change in fluorescence intensity obtained in the fluorescence detection unit 30, and the heating unit 25 includes a first detector After the sample is heated by repeating the periodic change for the first number of times in the cycle, the sample is heated by repeating the periodic change for the second number of times in a second cycle different from the first cycle.

  Thereby, the type (genotype) determination of multiple types of target genes can be realized in a short time. Furthermore, genetic testing can be automated, and genotypes of a plurality of types of target genes can be determined quickly and accurately.

  As described above, the genetic testing method, the genetic testing chip, and the genetic testing system according to the present disclosure have been described based on the embodiments. However, the present disclosure is not limited to these embodiments. Unless it deviates from the gist of the present disclosure, various modifications conceived by those skilled in the art and other forms constructed by combining some components in the embodiments are also within the scope of the present disclosure. include.

  The genetic testing method, the genetic testing chip, and the genetic testing system according to the present disclosure are, for example, foods, cosmetics, supplements, training proposals suitable for an individual's constitution based on the obtained genetic information, or diagnosis of disease prevalence It can be applied to the types and effects of administered drugs and prediction of side effects.

4 Medium 10 Genetic testing chip (chip)
25 Peltier element (heating unit)
30 Fluorescence detector

Claims (8)

Obtained by mixing a detection reagent containing multiple types of fluorescent dyes and a biological sample containing multiple types of target genes, and modifying each of the multiple types of target genes with a different fluorescent dye among the multiple types of fluorescent dyes A heating step of repeatedly heating the specimen in a predetermined temperature range with periodic changes;
A fluorescence detection step of irradiating the specimen with a plurality of excitation lights having different wavelengths while heating the specimen at a constant heating rate, and detecting a fluorescence change of the specimen;
A determination step of performing a type determination of the plurality of types of target genes based on a change in fluorescence intensity obtained in the fluorescence detection step,
The heating step includes
A first heating step of heating the specimen by repeating the periodic change a first number of times in a first period;
A second heating step that heats the specimen by repeating the periodic change a second number of times in a second period different from the first period,
Genetic testing method. The second period is shorter than the first period.
The genetic test method according to claim 1. The heating step further includes a third heating step of heating the specimen by repeating the periodic change a third number of times in a third cycle that is shorter than the second cycle.
The genetic test method according to claim 1 or 2. The first heating step further has a first temperature range;
The second heating step further has a second temperature range different from the first temperature range.
The genetic test method according to any one of claims 1 to 3. Further, while the sample is heated by repeating the periodic change in the first period for the first number of times, a change in fluorescence intensity of the plurality of types of target genes is measured, and based on the change in the fluorescence intensity Determining at least one of the first number of times and the second and subsequent number of times, or repeating the periodic change for the second number of times in the second period and heating the specimen, Measuring a change in the fluorescence intensity of a plurality of types of target genes, and determining at least one of the second number and the second and subsequent number based on the change in the fluorescence intensity,
The genetic test method according to any one of claims 1 to 4. The specimen is held in a chip including a medium that holds an identifier indicating the first period and the first number of times, and the second period and the second number of times,
In the first heating step, the periodic change is repeated the first number of times in the first period corresponding to the identifier to heat the specimen,
In the second heating step, the periodic change is repeated the second number of times in the second period corresponding to the identifier to heat the specimen.
The genetic test method according to any one of claims 1 to 5. A sample holder for holding the sample for heating the sample by repeating periodic changes in a predetermined temperature range, and amplifying a plurality of types of target genes contained in the sample;
A medium for holding an identifier associated with the heating cycle and the heating frequency of the specimen for amplifying the plurality of types of target genes,
Genetic testing chip. A specimen obtained by mixing a detection reagent containing a plurality of types of fluorescent dyes and a biological sample, and modifying each of the plurality of types of target genes with different fluorescent dyes among the plurality of types of fluorescent dyes, A heating unit that heats by repeating a periodic change in a temperature range;
Irradiating the specimen with a plurality of excitation lights having different wavelengths while heating the specimen at a constant heating rate, and detecting a fluorescence change of the specimen;
A determination unit that performs type determination of the plurality of types of target genes based on a change in fluorescence intensity obtained in the fluorescence detection unit,
The heating unit is
After heating the specimen by repeating the periodic change a first number of times in a first period,
Heating the specimen by repeating the periodic change a second number of times in a second period, different from the first period,
Genetic testing system.