WO2009081966A1 - 核酸増幅判定方法および核酸増幅判定装置 - Google Patents
核酸増幅判定方法および核酸増幅判定装置 Download PDFInfo
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- WO2009081966A1 WO2009081966A1 PCT/JP2008/073536 JP2008073536W WO2009081966A1 WO 2009081966 A1 WO2009081966 A1 WO 2009081966A1 JP 2008073536 W JP2008073536 W JP 2008073536W WO 2009081966 A1 WO2009081966 A1 WO 2009081966A1
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
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- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
- G16B40/10—Signal processing, e.g. from mass spectrometry [MS] or from PCR
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- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
- G16B40/20—Supervised data analysis
Definitions
- the present invention relates to a nucleic acid amplification determination method, a nucleic acid amplification determination system, a nucleic acid amplification determination device, a computer program capable of executing the determination method on a computer, and an electronic medium storing the computer program.
- melting curve analysis method a method for analyzing a melting curve of a double-stranded nucleic acid formed from a target nucleic acid and a probe. According to the melting curve analysis method, by analyzing the melting curve for the presence or absence of a peak at the melting temperature (Tm: melting temperature) of the double strand, determination of gene polymorphism (genotype) and mutation The presence or absence can be detected.
- Tm melting temperature
- Tm is generally defined as follows. When the solution containing the double-stranded nucleic acid is heated, the absorbance at 260 nm increases. This is because hydrogen bonds between both strands in a double-stranded nucleic acid are unwound by heating and dissociated into single-stranded nucleic acids (melting of double-stranded nucleic acids). When all the double-stranded nucleic acids are dissociated into single-stranded nucleic acids, the absorbance is about 1.5 times the absorbance at the start of heating (absorbance of only double-stranded nucleic acids), thereby melting. It can be judged that it has been completed. Based on this phenomenon, the melting temperature Tm (° C.) is generally defined as the temperature at which the absorbance reaches 50% of the total increase in absorbance.
- a double-stranded nucleic acid is formed by forming a double-stranded nucleic acid between a single-stranded nucleic acid to be analyzed and the probe using a mutant-type detection probe complementary to a target nucleic acid sequence including a mutant-type target site.
- Heat treatment is performed on the strand nucleic acid, and the dissociation of the double strand accompanying temperature rise is detected by measuring the signal such as absorbance, and the presence or absence of mutation at the target site is determined based on the behavior of the signal at the Tm value based on this detection result (See Non-Patent Document 1 and Patent Document 1).
- the Tm value is higher as the homology of the double-stranded nucleic acid is higher, and lower as the homology is lower.
- Tm values serving as evaluation criteria are obtained in advance.
- the higher the homology the higher the Tm value. Therefore, the former, that is, the Tm value when the target site is a mutant type (hereinafter also referred to as “Tm m value”) is relatively high, and the latter That is, the Tm value (hereinafter also referred to as “Tm w value”) when the target site is wild type is relatively low.
- a melting curve of a double-stranded nucleic acid formed from the single-stranded nucleic acid to be analyzed and the mutant detection probe is prepared, and a signal peak exists in any of the Tm m value and Tm w value obtained in advance. Confirm whether to do.
- the single-stranded nucleic acid to be analyzed can be determined to be a mutant polymorphism because it is 100% matched with the mutant detection probe.
- the single-stranded nucleic acid to be analyzed can be determined to be a wild-type polymorphism because it has a one-base mismatch with the mutation-type mutation probe. It can also be determined whether the polymorphism is homozygous or heterozygous. That is, in the analysis of a pair of alleles, if there is a peak near both the Tm m value and the Tm w value, it can be judged as heterozygous, whereas if a peak exists only near the Tm m value, homozygous mutant, when a peak only in the vicinity of Tm w value is present, it can be determined that the homozygous wild-type.
- the sample containing RNA or DNA is subjected to amplification treatment of the target nucleic acid sequence, and the obtained amplification product is subjected to double strand formation with the probe as described above. Dissociation by heating is performed.
- the target nucleic acid sequence cannot be sufficiently amplified in the amplification process, there is a problem that a polymorphism is erroneously determined. Therefore, in the conventional method, with respect to the sample after the amplification treatment, the relationship between each temperature and a signal value representing the melting state of the sample at each temperature or a differential value of the signal value (hereinafter referred to as “signal differential value”) is as follows.
- an object of the present invention is to provide an amplification determination method capable of determining whether or not amplification of a target nucleic acid has been performed on a sample that has been subjected to nucleic acid amplification processing. Furthermore, an object of the present invention is to provide an amplification determination system, an amplification determination device, a program, and an electronic medium for executing such an amplification determination method.
- the amplification determination method of the present invention is an amplification determination method for determining whether or not amplification of a target nucleic acid has been performed on a sample that has been subjected to nucleic acid amplification processing,
- a signal value preparation step of preparing a signal value indicating a melting state of the post-treatment sample at each temperature;
- a maximum value (A) search step of searching for a maximum value (A) from the signal value at each temperature;
- a differential operation step of obtaining a differential value by performing differentiation between successive signal values,
- a differential operation step for obtaining a differential value by performing differentiation between successive differential values For the differential value obtained in the step, a differential operation step for obtaining a differential value by performing differentiation between successive differential values, Among the double differential values obtained in the step, the maximum double differential value (D max ′) and the minimum double differential value from the double differential value in a predetermined temperature range including the Tm value of the target nucleic acid.
- the present invention it is possible to easily determine whether or not the target nucleic acid has been amplified by using the above-described operation such as double differentiation. For this reason, for example, it is possible to avoid problems such as the difference in the determination criteria of individuals performing the determination and the need for specialized knowledge as in the prior art.
- the presence or absence of amplification of the target nucleic acid can be easily determined, for example, for a sample showing an amplification failure that causes a genotype misjudgment, the subsequent melting curve analysis can be stopped. Thereby, finally, it becomes possible to perform melting curve analysis more easily and with high reliability.
- the system of the present invention into a conventional gene analyzer or the like, for example, it is possible not only to confirm the presence or absence of amplification, but also to fully automate from nucleic acid amplification to genotype determination. Become. Therefore, the present invention is extremely useful particularly in the field of gene analysis, for example, because it can be used in general analysis and diagnosis sites and can easily analyze a large amount of samples. It can be said that technology.
- FIG. 1 is an overall configuration diagram of an example of a stand-alone type apparatus using the system of the present invention.
- FIG. 2 is an overall configuration diagram of an example of a network utilization type apparatus using the system of the present invention.
- FIG. 3 is a block diagram illustrating an example of a device configuration of the stand-alone type apparatus.
- FIG. 4 is a block diagram illustrating an example of a device configuration of the network type device.
- FIG. 5 is an example of a flowchart for implementing the system of the present invention.
- FIG. 6 is a graph showing a melting curve in the embodiment of the present invention.
- FIG. 7 is a graph showing a differential curve in the embodiment of the present invention.
- FIG. 8 is a graph showing a second derivative curve in the embodiment of the present invention.
- the signal indicating the melting state of the sample may be generated by, for example, non-melting of the sample and suppressed by the melting of the sample. Conversely, the signal may be generated by non-melting of the sample. It may be suppressed and generated by melting of the sample.
- the signal differential value may be represented by “dF / dT” or “ ⁇ dF / dT”, for example. dF represents the amount of change in signal value, and dT represents the amount of change in temperature.
- the melting curve in which the signal differential value is expressed by “dF / dT” has a valley shape, and the melting curve in which the signal differential value is expressed by “ ⁇ dF / dT”.
- the peak has a mountain shape.
- the peak has a mountain shape, and the melting curve in which the signal differential value is represented by “ ⁇ dF / dT”.
- the peak is valley-shaped.
- the type of signal value is not particularly limited, and examples thereof include absorbance (absorption intensity) and fluorescence intensity.
- Specific examples of the signal include absorbance at 260 nm, which increases as a result of double-strand melting as described above.
- the signal value may be the intensity of the fluorescence emitted by the excitation light corresponding to the fluorescent material when the fluorescent material is used.
- the fluorescent substance may emit fluorescence by forming a double strand (non-melted) or may emit fluorescence by melting a double strand.
- Specific examples of the fluorescent material include intercalators such as ethidium bromide and SYBR (registered trademark) Green.
- the fluorescent substance may be bound to, for example, at least one single-stranded nucleic acid constituting a double-stranded nucleic acid.
- the single-stranded nucleic acid to which the fluorescent substance is bound include a so-called fluorescence quenching probe such as QProbe (registered trademark) known as a guanine quenching probe.
- the fluorescence quenching probe generally quenches fluorescence by forming a double strand, and generates fluorescence by melting the double strand.
- the present invention is characterized by signal value processing, and is not limited to the type of signal.
- the target nucleic acid to be amplified (hereinafter referred to as “target nucleic acid”) is a nucleic acid having a polymorphism at the target site, and the Tm value of the target nucleic acid is polymorphic at the target site.
- Tm value of a double-stranded nucleic acid composed of a nucleic acid having a nucleic acid and a nucleic acid capable of hybridizing to the target site hereinafter referred to as “detection nucleic acid”.
- detection nucleic acid An example of a Tm value of a double-stranded nucleic acid composed of a nucleic acid having a nucleic acid and a nucleic acid capable of hybridizing to the target site
- the temperature interval of the signal value is not particularly limited, but is, for example, an interval of 0.1 to 5 ° C., preferably an interval of 0.2 to 3 ° C., more preferably an interval of 0.8 to 1.2 ° C. It is.
- the temperature intervals may be different, for example, but are preferably equal intervals.
- the signal value is preferably prepared for a temperature range including, for example, a Tm value.
- a temperature range including, for example, a Tm value.
- the Tm value of the double-stranded nucleic acid composed of the target nucleic acid and the detection nucleic acid is relative to the Tm H value at a relatively high temperature.
- a low Tm L value is assumed. Therefore, it is preferable to prepare signal values for a wide temperature range including, for example, both Tm H and Tm L values.
- the temperature range for example, lower limit is preferably 1 ⁇ 20 ° C. lower temperature than Tm L value, more preferably, a lower temperature 1 ⁇ 10 ° C.
- Tm L value the upper limit is, Tm
- the temperature is preferably 1 to 20 ° C. higher than the H value, and more preferably 1 to 10 ° C. higher than the Tm H value.
- the temperature range is preferably [Tm L value ⁇ 5] ° C. to [Tm H value + 5] ° C.
- the target nucleic acid includes a target nucleic acid in which the target site is a wild type (hereinafter referred to as “wild type target nucleic acid”), and a target nucleic acid in which the target site is a mutant type ( Hereinafter, it is considered as “mutant target nucleic acid”). Since the wild-type target nucleic acid and the mutant-type target nucleic acid usually differ by only one base at the target site, the Tm value of the double-stranded nucleic acid is, for example, 2% that matches 100% with the nucleic acid for detection.
- Examples thereof include a Tm H value of a single strand and a Tm L value of a double strand that mismatches with the detection nucleic acid by one base.
- the Tm value becomes higher as the homology of the double strand is relatively higher, and becomes lower as it is lower. Therefore, the former Tm H value is higher than the latter Tm L value.
- mutant detection nucleic acid a detection nucleic acid capable of hybridizing to a mutant target site
- the mutant target nucleic acid and the mutant detection nucleic acid And the Tm value of the double-stranded nucleic acid of the wild-type target nucleic acid and the mutant-type detection nucleic acid.
- the former Tm value becomes the Tm H value
- the latter Tm value becomes the Tm L value
- a detection nucleic acid capable of hybridizing to a wild-type target site hereinafter referred to as “wild-type detection nucleic acid”
- wild-type detection nucleic acid a detection nucleic acid capable of hybridizing to a wild-type target site
- a wild-type target nucleic acid and the wild-type detection nucleic acid examples thereof include the Tm value of a double-stranded nucleic acid and the Tm value of a double-stranded nucleic acid of a mutant target nucleic acid and the wild-type detection nucleic acid.
- the former Tm value becomes the Tm H value
- the latter Tm value becomes the Tm L value.
- the Tm value can be appropriately determined according to the sequence of the target nucleic acid, the sequence of the nucleic acid for detection, and the like. Specifically, for example, conventionally known MELTCALC software (http://www.meltcalc.com) /) Etc., and can be determined by the nearest neighbor method (hereinafter the same).
- the signal value indicating the melting state of the treated sample is, for example, a double strand comprising an amplification product of the target nucleic acid in the treated sample and a detection nucleic acid that can hybridize to the target nucleic acid. It can also be called a signal value indicating the melting state of the nucleic acid.
- Maximum value (A) search step Next, the maximum value (A) is searched from the signal value at each temperature. At this time, if the maximum value (A) cannot be searched, it can be determined that the amplification is defective.
- the differentiation is not particularly limited, but is preferably, for example, a differentiation between 2 consecutive points to 10 points, more preferably between 2 consecutive points to 5 points, and particularly preferably 2 continuous points. Differentiation between points.
- the differential value (D p ) at an arbitrary point (p) is the signal value (P + 1) of a point (p + 1) adjacent to the signal value (S p ) of the arbitrary point (p).
- p is a positive integer
- p is a positive integer of 2 or more.
- Second derivative operation step Further, the derivative value obtained in the above step is differentiated between a plurality of consecutive points to obtain a twice differentiated value.
- the differentiation of the differential value is preferably performed, for example, between 2 to 10 consecutive points, more preferably between 3 to 7 points, still more preferably between 3 to 5 points, and particularly preferably 4 points. It is between points.
- the double differential value (D p ′) at an arbitrary point (p) may be, for example, any one of Examples 1 to 3 below. In the same manner, it is preferable to obtain a differential value twice.
- the double differential value (D p ′) at an arbitrary point (p) is calculated by, for example, differentiation between a total of four points (p), (p + 1), (p + 2), and (p + 3) as Example 1.
- the point (p-1), (p), (p + 1), (p + 2) may be calculated by differentiation between a total of four points.
- the point (p-2) , (P-2), (p), (p + 1) may be calculated by differentiation between a total of four points.
- p is a positive integer.
- p is a positive integer of 2 or more.
- p is a positive integer of 3 or more.
- the predetermined temperature range only needs to include the Tm value of the nucleic acid to be amplified.
- the temperature range includes both the Tm H value and the Tm L value. It is.
- the lower limit of the temperature range is preferably than Tm L value is 1 ⁇ 20 ° C. lower temperature, more preferably from 1 ⁇ 10 ° C. lower temperature than Tm L value, more preferably, [Tm L value -5] ° C.
- the upper limit of the temperature range is preferably than Tm H value is 1 ⁇ 20 ° C. higher temperatures, more preferably a temperature higher 1 ⁇ 10 ° C. than Tm H value, more preferably, [Tm H value +5] ° C.
- the maximum value and the minimum value may be selected from the twice differential values in the predetermined temperature range. For this reason, for example, in the above-mentioned double differential calculation process, it is sufficient to obtain a double differential value in the predetermined temperature range. In the differential calculation step described above, it is sufficient to obtain a differential value within a range where a double differential value within the predetermined temperature range can be obtained.
- amplification is normal, and if X satisfies [X ⁇ predetermined threshold], it is determined that the amplification is defective.
- normal amplification means, for example, a state in which the target nucleic acid is amplified and it is considered possible to perform reliable gene analysis
- poor amplification means for example, amplified. It means that the gene is not amplified, hardly amplified, or a different nucleic acid is amplified, and a reliable genotype analysis cannot be performed or there is a possibility of such a situation.
- the threshold includes, for example, the type of signal, the detection wavelength of the signal, the type of fluorescent substance that emits a signal (fluorescence), the type of nucleic acid to be amplified and the polymorphism in the target site of the nucleic acid, the sequence of the nucleic acid for detection, It can be set as appropriate according to the composition of the reaction solution when forming the strand nucleic acid.
- the present invention is not characterized by a specific value of the threshold and a setting method thereof, and is not limited by them. An example of the threshold setting method will be described later.
- the amplification determination method of the present invention preferably further includes, for example, an output step of outputting information on the obtained determination result.
- Examples of the determination result include an item of whether amplification is normal.
- the determination result may be output, the value of X, the graph of the melting curve in which the temperature and the differential value are plotted, the temperature and the double differential value are plotted.
- a graph or the like may be output together with the determination result.
- the signal value in the signal value preparation step may be, for example, data obtained by detection in advance.
- the signal value is prepared by detecting the signal value prior to the signal value preparation step.
- a temperature change step for changing the temperature of the sample after amplification (for example, a double-stranded nucleic acid), and a melting state of the sample at the time of the temperature change You may have the detection process which detects the signal value to show continuously or intermittently.
- the temperature changing step may be, for example, a heating step for heating the sample or a cooling step for cooling the heated sample, but the heating step is preferable.
- a nucleic acid amplification step for performing nucleic acid amplification treatment of the sample may be included prior to the temperature changing step.
- the nucleic acid for detection described above may be added to the sample after the nucleic acid amplification treatment, for example, but since the treatment can be performed continuously, prior to the nucleic acid amplification treatment, The detection nucleic acid is preferably added to the sample.
- FIGS. 6 to 8 are graphs showing the relationship between temperature and signal value
- FIG. 7 is a graph (differential curve) showing the relationship between temperature and signal differential value
- FIG. 8 is a graph showing the relationship between temperature and double differential value. It is a graph (double differential curve) which shows the relationship of these.
- the Y axis is a signal value
- the Y axis is a signal differential value
- the Y axis is a double differential value
- the X axis is temperature.
- the relatively low Tm L value is 50 ° C.
- the relatively high Tm H value is 56 ° C.
- the temperature range in the maximum difference (B) calculation step is 45 ° C. (Tm L value ⁇ 5 ° C.) to 61 ° C. (Tm H value + 5 ° C.).
- a graph showing the relationship between the temperature and the signal value at each temperature is prepared, and the maximum value (A) is searched.
- the signal value is differentiated to create a graph showing the relationship between the temperature and the differential value at each temperature.
- the differential value is differentiated, and a graph showing the relationship between the temperature range of 45 to 61 ° C. and the double differential value at each temperature is created.
- the amplification is determined to be normal, and when [X ⁇ predetermined threshold] is satisfied, the amplification is determined to be defective. Only for the sample after the nucleic acid amplification process, in which amplification is determined to be normal, for example, a melting curve analysis as described later may be performed to determine the polymorphism.
- FIGS. 6 to 8 are examples in which the signal value increases with the dissociation of double-stranded nucleic acid due to a temperature increase, but in the case where the signal value decreases with the dissociation of double-stranded nucleic acid due to a temperature increase.
- A maximum value
- B maximum difference
- the threshold value of X is not limited at all, and can be appropriately determined according to the type of target nucleic acid (gene), the type of polymorphism, and the like.
- the threshold value setting method will be exemplified below, but the present invention is not limited to this.
- the value of X is clearly different between the nucleic acid sample ( ⁇ ) with normal amplification and the nucleic acid sample ( ⁇ ) with poor amplification. Therefore, the critical value of X between normal amplification and poor amplification may be obtained from this graph and determined as the threshold value.
- the target nucleic acid is not limited to the target nucleic acid having a polymorphism at the target site where two Tm values are assumed as described above.
- the temperature range including the Tm value in the maximum difference (B) calculation step is preferably a temperature whose lower limit is 1 to 20 ° C. lower than the Tm value, and more preferably, The temperature is 1 to 10 ° C. lower than the Tm value, and more preferably [Tm value ⁇ 5] ° C.
- the upper limit of the temperature range is preferably a temperature that is 1 to 20 ° C. higher than the Tm value, more preferably a temperature that is 1 to 10 ° C. higher than the Tm value, and even more preferably [Tm value + 5] ° C. It is.
- the amplification determination method of the present invention may further include a melting curve analysis step of analyzing the melting curve of the sample after the nucleic acid amplification process to determine whether or not a peak exists in a predetermined temperature range.
- melting curves can be analyzed only for samples determined to have normal amplification, for example, polymorphism analysis (genotype analysis) can be performed.
- polymorphism analysis gene analysis
- the present invention includes a melting curve analysis step, for example, from a determination of nucleic acid amplification to a melting curve analysis and a polymorphism (genotype) determination of a sample determined to be normal in amplification are continuously performed. be able to. Therefore, the amplification determination method of the present invention can also be referred to as, for example, a melting curve analysis method or a polymorphism analysis method (or genotype analysis method).
- the amplification determination method of the present invention can be realized, for example, by executing the amplification determination system of the present invention described later, the amplification determination apparatus of the present invention, the execution of the computer program of the present invention, and the like.
- the amplification determination system of the present invention is an amplification determination system that determines whether or not amplification of a target nucleic acid has been performed on a sample that has been subjected to nucleic acid amplification processing.
- a signal value input unit for inputting a signal value indicating a melting state of the sample after treatment at each temperature;
- a maximum value (A) search unit for searching for a maximum value (A) from the signal value at each temperature input in the signal value input step;
- a differential operation unit that obtains a differential value by performing differentiation between successive signal values,
- a differential operation unit that obtains a differential value twice by performing differentiation between successive differential values,
- the largest double differential value (D max ′) from the double differential value in a predetermined temperature range including the Tm value of the nucleic acid to be amplified is
- a maximum difference (B) calculation unit that selects the smallest double differential value (D min ′) and obtains the largest difference (B) of the double differential values from the following equation:
- B (D max ')-(D min ')
- a calculation unit that performs the following calculation using the maximum value (A) and the maximum difference (B):
- the amplification determination system of the present invention it is preferable that, for example, differentiation between two consecutive points is performed in the differential calculation unit to obtain a differential value, and in the double differential calculation unit, for example, four consecutive points It is preferable to obtain a differential value twice by differentiating between them.
- the target nucleic acid is preferably a nucleic acid having a polymorphism at a target site.
- the Tm value of the target nucleic acid is preferably the Tm value of a double-stranded nucleic acid composed of a nucleic acid having a polymorphism at the target site (target nucleic acid) and the detection nucleic acid.
- the detection nucleic acid is a wild-type detection nucleic acid
- the Tm value of the nucleic acid to be amplified is, for example, the Tm of a double-stranded nucleic acid composed of the wild-type target nucleic acid and the wild-type detection nucleic acid.
- the H value and the Tm L value of a double-stranded nucleic acid composed of the mutant target nucleic acid and the wild-type detection nucleic acid are, for example, a double-stranded nucleic acid composed of the mutant target nucleic acid and the mutation detection nucleic acid.
- the predetermined temperature range including the Tm value of the nucleic acid to be amplified is, for example, a temperature whose lower limit is 1 to 20 ° C. lower than the Tm L value, and whose upper limit is lower than the Tm H value. Also, the temperature is higher by 1 to 20 ° C., preferably in the temperature range of [Tm L value ⁇ 5] ° C. to [Tm H value + 5] ° C.
- the amplification determination system of the present invention further includes a temperature change part that changes the temperature of the sample after the nucleic acid amplification process, and a detection that continuously or intermittently detects a signal value indicating a melting state of the sample at the time of the temperature change.
- the temperature changing unit may be, for example, a heating unit that heats the sample or a cooling unit that cools the heated sample.
- Examples of the temperature changing unit include a temperature controller capable of adjusting temperature, a heater, a thermal cycler, and the like.
- the detecting unit include a spectrophotometer and a fluorometer.
- the signal is, for example, fluorescence, and the detection unit preferably detects fluorescence.
- the amplification determination system of the present invention may further include a nucleic acid amplification unit that performs nucleic acid amplification processing of the sample.
- the amplification determination system of the present invention may have an addition unit for adding a nucleic acid capable of hybridizing to the target site to the sample.
- the amplification determination system of the present invention may further include a melting curve analysis unit that analyzes the melting curve of the sample after the nucleic acid amplification process and analyzes whether or not a peak exists in a predetermined temperature range.
- the amplification determination system of the present invention preferably further includes, for example, an output unit that outputs information on the obtained determination result.
- Examples of the determination result include an item of whether amplification is normal.
- the value of X the value of X
- the graph of the melting curve in which the temperature and the differential value are plotted the temperature and the double differential value are plotted.
- a graph or the like may be output together with the determination result.
- the amplification determination system of the present invention may be a network system having the following terminals and servers. Unless otherwise indicated, the system is the same as the amplification determination system described above. That is, the amplification determination system of the present invention is a network amplification determination system that analyzes whether or not amplification of a target nucleic acid has been performed on a sample that has been subjected to nucleic acid amplification processing, A terminal and a server, The terminal and the server can be connected via a communication network outside the system, The terminal A signal value input unit for inputting a signal value indicating a melting state of the sample after treatment at each temperature; A terminal-side transmitter that transmits information in the terminal to the server via the communication network; and A terminal-side receiving unit that receives information transmitted from the server via the communication network; The server A server-side transmitter that transmits information in the server to the terminal via the communication network; A server-side receiving unit that receives information transmitted from the terminal via the communication network; A maximum value
- the terminal of the present invention is a terminal used in the network amplification determination system of the present invention,
- the terminal A differential value input unit for inputting a signal value indicating the melting state of the sample at each temperature, A terminal-side transmitter that transmits information in the terminal to the server via the communication network; and A terminal-side receiving unit that receives information transmitted from the server via the communication network; At least the signal value at each temperature is transmitted from the terminal-side transmitting unit to the server-side receiving unit, and information on a determination result of whether the amplification is normal or defective is the server-side transmitting unit. To the terminal side receiving unit.
- the amplification determination apparatus of the present invention is an amplification determination apparatus that determines whether or not amplification of a target nucleic acid has been performed on a sample that has been subjected to nucleic acid amplification processing, and includes the amplification determination system of the present invention. It is characterized by that.
- the program of the present invention is a computer program capable of executing the amplification determination method of the present invention on a computer.
- the electronic medium of the present invention is an electronic medium storing the computer program of the present invention.
- FIG. 1 shows an overall configuration diagram of a stand-alone type which is an example of a system configuration of the present invention.
- the system shown in FIG. 1 includes an amplification determination system 11 according to the present invention, and the amplification determination system 11 includes a data input / output unit 12 and an amplification determination calculation unit 13.
- FIG. 3 shows an example of the hardware configuration of a stand-alone type amplification determination apparatus.
- the amplification determination system 11 includes a data input / output unit 12, an amplification determination calculation unit 13, and a storage device 37.
- the data input / output unit 12 is configured by computer equipment having a CPU 31 for executing a program, an input / output I / F (interface) 32, an input device 33 for inputting data, and an output device 34 for outputting data.
- Examples of the input device 33 include a keyboard and a mouse.
- Examples of the output device 34 include a printer, an LED, a liquid crystal display, and the like.
- the amplification determination calculation unit includes a computer device having a program storage unit 36 storing a program and a CPU 35 executing the program.
- the storage device 37 includes, for example, a signal value at each temperature, a signal differential value, a double differential value, a Tm value (Tm H value, Tm L value), a predetermined temperature range including the Tm value, an array of detection probes, Data such as the type (whether wild type detection or mutation type detection) is stored in a callable state.
- Examples of the storage device 37 include a ROM, an HDD, and an HD.
- the storage device 37 controls reading / writing and stores data under the control of the CPU. Note that the data input / output unit 12, the amplification determination calculation unit 13, and the storage device 37 are merely functional, and may be configured integrally with, for example, one computer device or a plurality of computer devices. You may comprise separately.
- the system of the present invention further detects a temperature change portion (for example, a heat treatment portion that performs heat treatment) that changes the temperature of the sample, and a signal value that indicates the melting state of the sample at the time of temperature change continuously or intermittently.
- a temperature change portion for example, a heat treatment portion that performs heat treatment
- You may have a detection part. And the signal value detected in the said detection part may be input by the said data input / output part.
- An example of the temperature changing portion is a heating device.
- the detection unit include an optical photometer and a fluorometer.
- each of the heat treatment unit and the detection unit may be configured integrally with one computer device, or may be individually configured with a plurality of computer devices.
- the system of the present invention further includes a melting curve analysis unit for analyzing the melting curve of the sample after the nucleic acid amplification treatment and analyzing whether or not a peak exists in a predetermined temperature range. Also good.
- the system of the present invention not only determines whether the amplification is normal or defective, but also analyzes the melting curve for the sample determined to be normal, for example, the presence or absence of a peak.
- the polymorphism (genotype) of the target nucleic acid can be determined.
- a nucleic acid extraction unit for extracting nucleic acid from a biological sample, an amplification processing unit for performing a nucleic acid amplification reaction, and the like may be provided.
- nucleic acid amplification system that can automatically perform from nucleic acid amplification to amplification determination or nucleic acid amplification to polymorphism (genotype) determination in one system. Can provide.
- FIG. 2 shows an overall configuration diagram of a network type system processed by a server.
- the system according to the present embodiment includes an amplification determination system 21 according to the present invention and a server system 24 including an amplification determination calculation unit 23.
- the amplification determination system 21 includes a data input / output unit 22.
- the amplification determination system 21 and the server system 24 are connected via a communication line 100 such as a public network functioning as the Internet based on TCP (Transmission Control Protocol) / IP (Internet Protocol) or a dedicated line.
- FIG. 4 shows an example of the configuration of the apparatus of the network type system.
- the amplification determination system 21 includes a data input / output unit 22 and a communication I / F (interface) 47 and is connected to the communication line 100 via the communication I / F 47.
- the server system 24 includes an amplification determination calculation unit 23 and a communication I / F 48, and is connected to the communication line 100 via the communication I / F 48.
- the data input / output unit 22 includes a CPU 41 that executes a program, an input / output I / F 42, an input device 43 that inputs data, and an output device 44 that outputs data.
- the data input / output unit 22 and the communication I / F 47 are merely functional.
- the data input / output unit 22 and the communication I / F 47 may be configured integrally with one computer device or may be configured individually with a plurality of computer devices.
- the amplification determination calculation unit 23 includes a CPU 45 that executes a program and a program storage unit 46 that stores the program.
- the amplification determination calculation unit 23 and the communication I / F 48 are merely functional, and may be configured integrally with, for example, one computer device, or may be individually configured with a plurality of computer devices. .
- each processing step in the system of the present invention includes, for example, a CPU, a main memory, a bus, a secondary storage device, a hardware device such as a printing device or a display, other external peripheral devices, or an external peripheral device thereof.
- This can be executed by appropriately using input / output (I / O) ports, driver programs for controlling these hardware, and other application programs.
- [1] Enter the signal value at each temperature. [2] The largest value (A) is searched. [3] Differentiate the signal value at each temperature. [4] Differentiate the signal derivative further. [5] From the double differential value, a maximum double differential value and a minimum double differential value are searched. [6] The difference between the maximum double differential value and the minimum double differential value, that is, the maximum difference (B) is obtained. [7] It is determined whether X calculated from the maximum value (A) and the maximum difference (B) satisfies [X> threshold]. [8: Yes] When [7] is Yes, it is determined that the amplification is normal. [9: No] When [7] is No, it is determined that the amplification is defective.
- the present invention it is possible to easily determine whether or not amplification of a target nucleic acid has been performed by utilizing the above-described operation such as double differentiation. For this reason, for example, it has become possible to avoid problems such as the difference in the determination criteria of individuals performing the determination and the need for specialized knowledge, as in the prior art. In this way, since the presence or absence of amplification of the target nucleic acid can be easily determined, for example, for a sample showing an amplification failure that causes a genotype misjudgment, further melting curve analysis can be stopped. For this reason, it was finally possible to perform melting curve analysis more easily and with high reliability.
- the present invention can be used, for example, in general analysis and diagnosis, and enables easy analysis of a large amount of specimens. This is a useful technique.
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Abstract
Description
クリニカル・ケミストリー、2000年、第46号、第5号、p631‐635、
各温度における前記処理後サンプルの融解状態を示すシグナル値を準備するシグナル値準備工程、
前記各温度における前記シグナル値から、最大値(A)を検索する最大値(A)検索工程、
前記各温度における前記シグナル値について、連続するシグナル値間の微分を行って微分値を得る微分演算工程、
前記工程において得られた微分値について、連続する微分値間の微分を行って二回微分値を得る二回微分演算工程、
前記工程において得られた二回微分値のうち、前記目的核酸のTm値を含む所定の温度範囲における二回微分値から、最大の二回微分値(Dmax’)と最小の二回微分値(Dmin’)とを選択し、下記式から二回微分値の最も大きい差(B)を得る最大差(B)演算工程、
B=(Dmax’)-(Dmin’)
前記最大値(A)および前記最大差(B)を用いて下記式の演算を行う演算工程
X=(B)/(A)
および
前記Xが[X>所定閾値]を満たす場合、増幅が正常であると判断し、前記Xが[X≦所定閾値]を満たす場合、増幅が不良であると判断する判定工程
を含むことを特徴とする。
本発明の増幅判定方法について、一例として、増幅させる目的核酸(以下、「標的核酸」という)が標的部位に多型を有する核酸であり、前記目的核酸のTm値が、前記標的部位に多型を有する核酸と前記標的部位にハイブリダイズ可能な核酸(以下、「検出用核酸」という)とから構成される二本鎖核酸のTm値である例をあげて説明する。なお、本発明は、これには制限されない。
まず、各温度における前記処理後サンプルの融解状態を示すシグナル値を準備する。
つぎに、前記各温度における前記シグナル値から、最大値(A)を検索する。この際、最大値(A)が検索できない場合は、増幅不良と判断できる。
つぎに、前記各温度における前記シグナル値について、連続するシグナル値間の微分を行って微分値を得る。前記微分は、特に制限されないが、例えば、連続する2点間~10点間の微分であることが好ましく、より好ましくは、連続する2点間~5点間であり、特に好ましくは連続する2点間の微分である。例えば、2点間の微分を行う場合、任意の点(p)における微分値(Dp)は、任意の点(p)のシグナル値(Sp)と近接する点(p+1)のシグナル値(Sp+1)との間における微分値でもよいし、任意の点(p)のシグナル値(Sp)と近接する点(p-1)のシグナル値(Sp-1)との間における微分値でもよいが、各点において、同様にして微分値を求めることが好ましい。前者の場合、pは、正整数であり、後者の場合、pは、2以上の正整数である。
さらに、前記工程において得られた微分値について、連続する複数点間の微分を行って二回微分値を得る。前記微分値の微分は、例えば、連続する2~10点間で行うことが好ましく、より好ましくは、3~7点間であり、さらに好ましくは、3~5点間であり、特に好ましくは4点間である。具体例として、4点間の微分を行う場合、任意の点(p)における二回微分値(Dp’)は、例えば、下記例1~例3のいずれであってもよいが、各点について、同様にして二回微分値を求めることが好ましい。任意の点(p)の二回微分値(Dp’)は、例1として、例えば、点(p)、(p+1)、(p+2)、(p+3)の計4点間の微分により算出してもよく、例2として、点(p-1)、(p)、(p+1)、(p+2)の計4点間の微分により算出してもよく、例3として、点(p-2)、(p-2)、(p)、(p+1)の計4点間の微分により算出してもよい。前記pは、前記例1の場合、正整数であり、前記例2の場合、pは、2以上の正整数であり、前記例3の場合、pは、3以上の正整数である。
前記工程において得られた二回微分値のうち、前記増幅目的の核酸のTm値を含む所定の温度範囲における二回微分値から、最大の二回微分値(Dmax’)と最小の二回微分値(Dmin’)とを選択し、下記式から最大差(B)を得る。
(B)=(Dmax’)-(Dmin’)
前記最大値(A)および前記最大差(B)を用いて下記式の演算を行う。
X=(B)/(A)
前記Xが[X>所定閾値]を満たす場合、増幅が正常であると判断し、前記Xが[X≦所定閾値]を満たす場合、増幅が不良であると判断する。なお、本発明において、増幅が正常とは、例えば、標的核酸が増幅されており、信頼性ある遺伝子解析を行うことが可能と思われる状態を意味し、増幅が不良とは、例えば、増幅されていない、ほとんど増幅されていない、または、異なる核酸が増幅されている場合等であって、信頼性ある遺伝子型解析を行うことができない、もしくは、そのおそれがある状態を意味する。
本発明の増幅判定システムは、核酸増幅処理が行われた処理後のサンプルについて、目的の核酸の増幅が行われたか否かを判定する増幅判定システムであって、
各温度における前記処理後サンプルの融解状態を示すシグナル値を入力するシグナル値入力部、
前記シグナル値入力工程により入力された前記各温度における前記シグナル値から、最大値(A)を検索する最大値(A)検索部、
前記各温度における前記シグナル値について、連続するシグナル値間の微分を行って微分値を得る微分演算部、
前記微分演算部により得られた微分値について、連続する微分値間の微分を行って二回微分値を得る二回微分演算部、
前記二回微分演算部により得られた二回微分値のうち、前記増幅目的の核酸のTm値を含む所定の温度範囲における二回微分値から、最も大きい二回微分値(Dmax’)と最も小さい二回微分値(Dmin’)とを選択し、下記式から二回微分値の最も大きい差(B)を得る最大差(B)演算部、
B=(Dmax’)-(Dmin’)
前記最大値(A)および前記最大差(B)を用いて下記式の演算を行う演算部
X=(B)/(A)
および
前記Xが[X>所定閾値]を満たす場合、増幅が正常であると判断し、前記Xが[X≦所定閾値]を満たす場合、増幅が不良であると判断する判定部
を含むことを特徴とする。なお、特に示さない限り、前述の本発明の増幅判定方法と同様である。
本発明の増幅判定システムは、以下に示す端末とサーバーとを有するネットワークシステムであってもよい。なお、特に示さない限りは、前述の増幅判定システムと同様である。すなわち、本発明の増幅判定システムは、核酸増幅処理が行われた処理後のサンプルについて、目的の核酸の増幅が行われたか否かを解析するネットワーク増幅判定システムであって、
端末と、サーバーとを有し、
前記端末および前記サーバーは、システム外の通信網を介して接続可能であり、
前記端末は、
各温度における前記処理後サンプルの融解状態を示すシグナル値を入力するシグナル値入力部、
前記端末内の情報を前記通信網を介して前記サーバーに送信する端末側送信部、および、
前記サーバーから送信された情報を前記通信網を介して受信する端末側受信部を有し、
前記サーバーは、
前記サーバー内の情報を前記通信網を介して前記端末に送信するサーバー側送信部、
前記端末から送信された情報を前記通信網を介して受信するサーバー側受信部、
前記サーバー側受信部により受信した前記各温度における前記シグナル値から、最大値(A)を検索する最大値(A)検索部、
前記各温度における前記シグナル値について、連続するシグナル値間の微分を行って微分値を得る微分演算部、
前記微分演算部により得られた微分値について、連続する微分値間の微分を行って二回微分値を得る二回微分演算部、
前記二回微分演算部により得られた二回微分値のうち、前記増幅目的の核酸のTm値を含む所定の温度範囲における二回微分値から、最大の二回微分値(Dmax’)と最小の二回微分値(Dmin’)とを選択し、下記式から二回微分値の最も大きい差(B)を得る最大差(B)演算部、
(B)=(Dmax’)-(Dmin’)
前記最大値(A)および前記最大差(B)を用いて下記式の演算を行う演算部
X=(B)/(A)
および
前記Xが[X>所定閾値]を満たす場合、増幅が正常であると判断し、前記Xが[X≦所定閾値]を満たす場合、増幅が不良であると判断する判定部を含み、
少なくとも前記各温度における前記シグナル値が、前記端末側送信部から前記サーバー側受信部に送信され、且つ、前記増幅が正常であるか不良であるかの判定結果の情報が、前記サーバー側送信部から前記端末側受信部に送信されることを特徴とする。
前記端末は、
各温度におけるサンプルの融解状態を示すシグナル値を入力する微分値入力部、
前記端末内の情報を前記通信網を介して前記サーバーに送信する端末側送信部、および、
前記サーバーから送信された情報を前記通信網を介して受信する端末側受信部を有し、
少なくとも前記各温度における前記シグナル値が、前記端末側送信部から前記サーバー側受信部に送信され、且つ、前記増幅が正常であるか不良であるかの判定結果の情報が、前記サーバー側送信部から前記端末側受信部に送信されることを特徴とする。
本発明の増幅判定装置は、核酸増幅処理が行われた処理後のサンプルについて、目的の核酸の増幅が行われたか否かを判定する増幅判定装置であって、本発明の増幅判定システムを含むことを特徴とする。
本発明のプログラムは、本発明の増幅判定方法をコンピュータ上で実行可能なコンピュータプログラムである。
本発明の電子媒体は、本発明のコンピュータプログラムを格納した電子媒体である。
図1に、本発明のシステムの構成の一例であるスタンドアローン型の全体構成図を示す。図1に示すシステムは、本発明の増幅判定システム11から構成され、増幅判定システム11は、データ入出力部12と増幅判定計算部13から構成される。図3に、スタンドアローン型の増幅判定装置のハードウェア構成の一例を示す。図示のように、増幅判定システム11は、データ入出力部12、増幅判定計算部13および記憶装置37から構成されている。前記データ入出力部12は、プログラムを実行するCPU31、入出力I/F(インターフェース)32、データの入力を行う入力装置33、データの出力を行う出力装置34を有するコンピュータ機器で構成される。入力装置33としては、例えば、キーボードやマウス等があげられ、出力装置34としては、例えば、プリンターや、LEDまたは液晶ディスプレイ等があげられる。増幅判定計算部は、プログラムが格納されたプログラム格納部36およびプログラムを実行するCPU35を有するコンピュータ機器で構成される。記憶装置37には、例えば、各温度におけるシグナル値、シグナル微分値、二回微分値、Tm値(TmH値、TmL値)、Tm値を含む所定の温度範囲、検出用プローブの配列や種類(野生型検出用であるか変異型検出用であるか)等のデータが呼び出し可能な状態で記憶される。記憶装置37としては、例えば、ROM、HDD、HD等があげられ、CPUの制御下、読みこみ/書きこみを制御し、データを記憶する。なお、データ入出力部12、増幅判定計算部13、記憶装置37は、あくまでも機能上のものであり、例えば、1台のコンピュータ機器で一体に構成してもよいし、複数台のコンピュータ機器で個別に構成してもよい。
図2に、サーバーで処理するネットワーク型のシステムの全体構成図を示す。図2に示すように、本実施形態のシステムは、本発明の増幅判定システム21、および、増幅判定計算部23から構成されるサーバーシステム24で構成される。増幅判定システム21は、データ入出力部22から構成される。増幅判定システム21とサーバーシステム24は、例えば、TCP(Transmission Control Protocol)/IP(Internet Protocol)に基づくインターネットとして機能する公衆網や専用線等の通信回線100を介して接続されている。図4に、前記ネットワーク型システムの装置の構成の一例を示す。増幅判定システム21は、データ入出力部22および通信I/F(インターフェース)47から構成され、通信I/F47を介して通信回線100に接続されている。サーバーシステム24は、増幅判定計算部23および通信I/F48から構成され、通信I/F48を介して通信回線100に接続されている。データ入出力部22は、プログラムを実行するCPU41、入出力I/F42、データの入力を行う入力装置43およびデータの出力を行う出力装置44から構成される。前記データ入出力部22および通信I/F47は、あくまでも機能上のものであり、例えば、1台のコンピュータ機器で一体に構成してもよいし、複数台のコンピュータ機器で個別に構成してもよい。増幅判定計算部23は、プログラムを実行するCPU45およびプログラムが格納されたプログラム格納部46で構成される。増幅判定計算部23および通信I/F48は、あくまでも機能上のものであり、例えば、1台のコンピュータ機器で一体に構成してもよいし、複数台のコンピュータ機器で個別に構成してもよい。
本発明の増幅判定システムの基本的な処理の例を、図5のフローチャートに示す。以下、同図にしたがって、処理の流れを説明する。なお、本発明のシステムにおける各処理ステップは、例えば、CPU、主メモリ、バス、あるいは、二次記憶装置、印刷装置やディスプレイ、その他の外部周辺装置等のハードウェア構成部や、その外部周辺機器用の入出力(I/O)ポート、それらハードウェアを制御するためのドライバプログラムやその他のアプリケーションプログラムなどを適宜利用することで実行できる。
各温度におけるシグナル値を入力する。
[2]
最も大きい値(A)を検索する。
[3]
各温度におけるシグナル値を微分する。
[4]
シグナル微分値をさらに微分する。
[5]
前記二回微分値から、最大の二回微分値と最小の二回微分値を検索する。
[6]
前記最大二回微分値と最小二回微分値の差、すなわち最大差(B)を得る。
[7]
前記最大値(A)および前記最大差(B)から演算したXが、[X>閾値]を満たすか判断する。
[8:Yes]
前記[7]がYesの場合、増幅は正常と判断する。
[9:No]
前記[7]がNoの場合、増幅は不良と判断する。
Claims (19)
- 核酸増幅処理が行われた処理後のサンプルについて、目的核酸の増幅が行われたか否かを判定する増幅判定方法であって、
各温度における前記処理後サンプルの融解状態を示すシグナル値を準備するシグナル値準備工程、
前記各温度における前記シグナル値から、最大値(A)を検索する最大値(A)検索工程、
前記各温度における前記シグナル値について、連続するシグナル値間の微分を行って微分値を得る微分演算工程、
前記工程において得られた微分値について、連続する微分値間の微分を行って二回微分値を得る二回微分演算工程、
前記工程において得られた二回微分値のうち、前記目的核酸のTm値を含む所定の温度範囲における二回微分値から、最大の二回微分値(Dmax’)と最小の二回微分値(Dmin’)とを選択し、下記式から二回微分値の最も大きい差(B)を得る最大差(B)演算工程、
(B)=(Dmax’)-(Dmin’)
前記最大値(A)および前記最大差(B)を用いて下記式の演算を行う演算工程
X=(B)/(A)
および
前記Xが[X>所定閾値]を満たす場合、増幅が正常であると判断し、前記Xが[X≦所定閾値]を満たす場合、増幅が不良であると判断する判定工程
を含む増幅判定方法。 - 前記微分演算工程において、連続する2点間の微分を行って微分値を得る、請求の範囲1記載の増幅判定方法。
- 前記二回微分演算工程において、連続する4点間の微分を行って二回微分値を得る、請求の範囲1記載の増幅判定方法。
- 前記目的核酸が、標的部位に多型を有する核酸である、請求の範囲1記載の増幅判定方法。
- 前記目的核酸のTm値が、前記標的部位に多型を有する核酸と前記標的部位にハイブリダイズ可能な核酸とから構成される二本鎖核酸のTm値である、請求の範囲4記載の増幅判定方法。
- 前記標的部位にハイブリダイズ可能な核酸が、野生型の前記標的部位にハイブリダイズ可能な核酸である場合、
前記目的核酸のTm値は、前記標的部位が野生型の核酸と前記野生型の標的部位にハイブリダイズ可能な核酸とから構成される二本鎖核酸のTmH値、および、前記標的部位が変異型の核酸と前記野生型の標的部位にハイブリダイズ可能な核酸とから構成される二本鎖核酸のTmL値であり、
前記標的部位にハイブリダイズ可能な核酸が、変異型の前記標的部位にハイブリダイズ可能な核酸である場合、
前記目的核酸のTm値は、前記標的部位が変異型の核酸と前記変異型の標的部位にハイブリダイズ可能な核酸とから構成される二本鎖核酸のTmH値、および、前記標的部位が野生型の核酸と前記変異型の標的部位にハイブリダイズ可能な核酸とから構成される二本鎖核酸のTmL値である、請求の範囲5記載の増幅判定方法。 - 前記最大差(B)演算工程において、前記目的核酸のTm値を含む所定の温度範囲が、下限がTmL値よりも1~20℃低い温度であり、上限がTmH値よりも1~20℃高い温度である、請求の範囲6記載の増幅判定方法。
- 前記温度範囲が、[TmL値-5]℃~[TmH値+5]℃である、請求の範囲7記載の増幅判定方法。
- 前記シグナル値準備工程に先立って、さらに、
前記核酸増幅処理後のサンプルの温度を変化させる温度変化工程、
および、
温度変化時における前記処理後のサンプルの融解状態を示すシグナル値を連続的または断続的に検出する検出工程を有する、請求の範囲1記載の増幅判定方法。 - 前記目的核酸が、標的部位に多型を有する核酸であり、
前記温度変化工程に先立って、前記サンプルに、前記標的部位にハイブリダイズ可能な核酸を添加する添加工程を有する、請求の範囲9記載の増幅判定方法。
- 核酸増幅処理が行われた処理後のサンプルについて、目的核酸の増幅が行われたか否かを判定する増幅判定システムであって、
各温度における前記処理後サンプルの融解状態を示すシグナル値を入力するシグナル値入力部、
前記シグナル値入力工程により入力された前記各温度における前記シグナル値から、最大値(A)を検索する最大値(A)検索部、
前記各温度における前記シグナル値について、連続するシグナル値間の微分を行って微分値を得る微分演算部、
前記微分演算部により得られた微分値について、連続する微分値間の微分を行って二回微分値を得る二回微分演算部、
前記二回微分演算部により得られた二回微分値のうち、前記目的核酸のTm値を含む所定の温度範囲における二回微分値から、最大の二回微分値(Dmax’)と最小の二回微分値(Dmin’)とを選択し、下記式から二回微分値の最も大きい差(B)を得る最大差(B)演算部、
B=(Dmax’)-(Dmin’)
前記最大値(A)および前記最大差(B)を用いて下記式の演算を行う演算部
X=(B)/(A)
および
前記Xが[X>所定閾値]を満たす場合、増幅が正常であると判断し、前記Xが[X≦所定閾値]を満たす場合、増幅が不良であると判断する判定部
を含む増幅判定システム。 - 前記微分演算部において、連続する2点間の微分を行って二回微分値を得る、請求の範囲11記載の増幅判定システム。
- 前記二回微分演算部において、連続する4点間の微分を行って二回微分値を得る、請求の範囲11記載の増幅判定システム。
- 前記目的核酸が、標的部位に多型を有する核酸である、請求の範囲11記載の増幅判定システム。
- 前記目的核酸のTm値が、前記標的部位に多型を有する核酸と前記標的部位にハイブリダイズ可能な核酸とから構成される二本鎖核酸のTm値である、請求の範囲14記載の増幅判定システム。
- 前記標的部位にハイブリダイズ可能な核酸が、野生型の前記標的部位にハイブリダイズ可能な核酸である場合、
前記目的核酸のTm値は、前記標的部位が野生型の核酸と前記野生型の標的部位にハイブリダイズ可能な核酸とから構成される二本鎖核酸のTmH値、および、前記標的部位が変異型の核酸と前記野生型の標的部位にハイブリダイズ可能な核酸とから構成される二本鎖核酸のTmL値であり、
前記標的部位にハイブリダイズ可能な核酸が、変異型の前記標的部位にハイブリダイズ可能な核酸である場合、
前記目的核酸のTm値は、前記標的部位が変異型の核酸と前記変異型の標的部位にハイブリダイズ可能な核酸とから構成される二本鎖核酸のTmH値、および、前記標的部位が野生型の核酸と前記変異型の標的部位にハイブリダイズ可能な核酸とから構成される二本鎖核酸のTmL値である、請求の範囲15記載の増幅判定システム。 - 前記最大差(B)演算部において、前記目的核酸のTm値を含む所定の温度範囲が、下限がTmL値よりも1~20℃低い温度であり、上限がTmH値よりも1~20℃高い温度である、請求の範囲16記載の増幅判定システム。
- 前記温度範囲が、[TmL値-5]℃~[TmH値+5]℃である、請求の範囲17記載の増幅判定システム。
- 核酸増幅処理が行われた処理後のサンプルについて、目的の核酸の増幅が行われたか否かを判定する増幅判定装置であって、
請求の範囲11記載の増幅判定システムを含む増幅判定装置。
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JP2000333700A (ja) * | 1999-04-27 | 2000-12-05 | Univ Utah | リアルタイム核酸増幅の自動分析 |
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