Temperature analysis method

Abstract

To provide a method capable of avoiding a problem that a measured temperature of a device is separated from an actual reaction liquid temperature and a Tm value of a target nucleic acid is deviated from the actual temperature when a temperature change speed is increased in performing nucleic acid analysis such as melting curve analysis.SOLUTION: Melting curve data on a data plane are collected based on a high temperature change speed, and X coordinates of peak points of a plurality of kinds of markers are obtained. The plurality of types of markers include a target gene detection marker having a calculated target Tm0 value, a first standard marker having a calculated first Tm1 value that is different from the calculated target Tm0 value, and a second standard marker having a calculated second Tm2 value that is different from the calculated target Tm0 value and the calculated first Tm1 value, and the method includes using an X1 coordinate of a first peak point related to the first standard marker and an X2 coordinate of a second peak point related to the second standard marker on the data plane to calculate the following formula: An X coordinate obtained by normalizing the X0 coordinate of the target peak point is obtained.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to a method for identifying a target nucleic acid, and in particular to a technology for speeding up genetic testing carried out in clinical laboratories using small analyzers or rapid diagnostic kits. [Background technology]

[0002] In the diagnosis of infectious diseases, testing for the causative pathogenic microorganisms is extremely important, and pathogens are generally identified using methods such as microscopic examination, culture examination, immunological examination, and genetic testing.

[0003] In particular, genetic testing, which is a later technology among the testing methods mentioned above, became more important in 2020 due to the global spread of the new coronavirus (SARS-CoV-2), and testing was carried out in various places, resulting in widespread public recognition.

[0004] In Japan, various genetic testing reagents for detecting the novel coronavirus have been approved and are now on sale, including reverse transcription (RT)-PCR, which uses the polymerase chain reaction (PCR) method as the nucleic acid amplification principle, and Loop-mediated Isothermal Amplification (LAMP), Nicking Endonuclease Amplification Reaction (NEAR), SmartAmp, Transcription Mediated Amplification (TMA), and Transcription Reverse-transcription Concerted reaction (TRC), which use nucleic acid amplification principles other than PCR.

[0005] However, since most of the above nucleic acid amplification methods require 30 minutes to 5 hours for measurement, there is a need to significantly reduce the measurement time in the development of test kits using genetic testing methods.

[0006] Furthermore, genetic testing can test for multiple target genes at once, making it possible to simultaneously test for the presence of multiple pathogenic microorganisms or virus candidates that exhibit similar symptoms in a sample. In order to simultaneously test for the presence or absence of multiple target genes, it is necessary to simultaneously amplify the nucleic acids of the multiple target genes and identify each target gene from the nucleic acid amplification products.

[0007] One such method of discrimination is melting curve (dissociation curve) analysis, which is performed after nucleic acid amplification.

[0008] Melting curve analysis is a nucleic acid analysis method that utilizes the phenomenon that when a reaction solution containing double-stranded DNA is heated and the temperature is changed, it dissociates into single-stranded DNA at a temperature (Tm value) characteristic of the length and sequence of the base sequence. In melting curve analysis, the temperature of a reaction solution to which a fluorescent dye or a nucleic acid probe labeled with a fluorescent dye has been added is changed, and the presence or absence of a target gene is determined by whether or not there is a change in fluorescence intensity at a temperature characteristic of the length and sequence of the target sequence.

[0009] As described in Non-Patent Document 1 (Mei et al., Clinical Chemistry 60:6 864-872 (2014)), a temperature change rate of 0.005°C / sec to 0.100°C / sec is recommended for melting curve analysis using a general-purpose measuring device. Using such a temperature change rate prevents a temperature discrepancy between the measured temperature of the device and the actual reaction solution temperature (hereinafter, the term "temperature discrepancy" will be used in this sense). However, when measuring in a temperature range of 40°C to 95°C, the measurement time becomes long, ranging from approximately 3 hours to approximately 9 minutes.

[0010] Even a measurement time of 9 minutes would be considered too long, but as will be discussed later, simply increasing the rate of temperature change to shorten the measurement time would inevitably result in temperature discrepancies.

[0011] Currently, there are many measurement devices used for nucleic acid amplification reactions and melting curve analysis, including general-purpose PCR devices that can perform PCR and other nucleic acid amplification methods using standardized, general-purpose tubes and reaction vessels, as well as devices specialized for POCT. These measurement devices require strict temperature control of the reaction solution during nucleic acid amplification reactions and melting curve analysis. However, to prevent contamination and inhibition of nucleic acid amplification reactions, the reaction solution temperature is not measured directly, but is instead measured (estimated) indirectly via a temperature sensor installed near the reaction vessel. One example of a method for indirectly measuring the reaction solution temperature is to estimate it by measuring the temperature of an aluminum block located between the heat source and the reaction vessel.

[0012] Regarding the characteristics and issues related to melting curve analysis using a PCR device with such a device structure, Patent Document 1 (Patent Publication No. 5643196) discloses a technology for controlling the device temperature in real time by directly measuring the reaction solution temperature in order to avoid the problem of discrepancy between the actual temperature of the sample (reaction solution) and the temperature value of a physically separated temperature sensor in conventional PCR devices.

[0013] Specifically, Patent Document 1 discloses a method for accurately controlling the temperature during melting curve analysis by mixing a thermochromatic temperature reference substance into a solution expected to contain the target gene or into a nearby solution and measuring the change in its optical properties to obtain the reaction solution temperature without relying on a temperature sensor outside the sample container.

[0014] However, the technology disclosed in Patent Document 1 uses a special microfluidic device rather than a standardized general-purpose tube or reaction vessel, and furthermore, its implementation requires a dedicated device that controls the temperature based on changes in the fluorescence intensity value of a temperature reference substance.

[0015] In other words, the technology disclosed in Patent Document 1 cannot be said to be a highly versatile technology, and has the disadvantage that it is extremely difficult to reproduce using general-purpose PCR devices or other companies' specialized POCT devices.

[0016] Patent Document 2 (JP Patent Publication No. 2023-508843) discloses technology relating to a pouch-shaped dedicated device containing a microarray, which makes it possible to control the variation within and between measurements regarding the Tm values ​​of target genes and to correctly determine the results of melting curve analysis.

[0017] Specifically, Patent Document 2 discloses a technology for correcting the Tm value of a target gene by analyzing data obtained after melting curve analysis. The technology normalizes variations in Tm values ​​caused by subtle differences in reagent concentration, the amount of amplified product present in the reaction solution, characteristics of the measuring device, etc., using the Tm value of the target gene and the number of PCR cycles that resulted in a positive result, and corrects the Tm value from a relational equation, thereby disclosing a method for correctly determining the presence or absence of a target gene.

[0018] However, the technology described in Patent Document 2 is a technology related to microarrays or DNA arrays rather than standardized general-purpose tubes or reaction vessels, and has the disadvantage that it is very difficult to reproduce using general-purpose PCR devices and reaction vessels. [Patent Document 1] Patent No. 5643196 [Patent Document 2] Special Publication No. 2023-508843 [Non-Patent Document 1] Mei et al., Clinical Chemistry 60:6 864-872(2014) DISCLOSURE OF THE INVENTION [Problem to be solved by the invention]

[0019] Therefore, an object of the present invention is to provide a melting curve analysis method that can avoid the problem of the temperature measured by the device and the actual reaction solution temperature deviating when the temperature change rate is increased in nucleic acid analysis such as melting curve analysis, resulting in the displayed Tm value of the target nucleic acid differing from the actual temperature, and that can obtain a Tm value equivalent to that obtained when analysis is performed at a normal temperature change rate, and that is so versatile that it can be performed on general-purpose PCR devices and devices specialized for POCT. [Means for solving the problem]

[0020] A temperature analysis method according to a first aspect of the present invention is a temperature analysis method in which multiple types of markers are added to a reaction solution contained in a reaction vessel, information indicating data points is plotted on the horizontal axis X, and time derivatives of fluorescence intensity values ​​are plotted on the vertical axis Y. Melting curve data on a data plane is collected based on a temperature change rate that exceeds a normal temperature change rate (0.100°C / sec), and the X coordinates of the peak points of the multiple types of markers are determined, wherein the multiple types of markers include a target gene detection marker having a calculated target Tm0 value, a first standard marker having a calculated first Tm1 value that is different from the calculated target Tm0 value, and a second standard marker having a calculated second Tm2 value that is different from the calculated target Tm0 value and the calculated first Tm1 value. The X coordinate of the target peak point is normalized using the X1 coordinate of the first peak point for the first standard marker and the X2 coordinate of the second peak point for the second standard marker on the data plane.

[0021] The first and second standard markers may have the same or different wavelengths as long as they can be distinguished at different temperatures.

[0022] Furthermore, it is sufficient that the peak points of the first standard marker, second standard marker, and target gene detection marker are distinguishable by temperature or wavelength.

[0023] Preferably, the information indicative of a data point is any one of the measured temperature, the measured time, or the measured duration.

[0024] The temperature change rate, which is so high that the temperature measured by the device and the actual reaction liquid temperature diverge, is preferably 1.00 [°C / sec] or more and 2.00 [°C / sec] or less.

[0025] Preferably, the target Tm0 value is greater than the first Tm1 value and less than the second Tm2 value.

[0026] Such a magnitude relationship allows normalization to be performed with higher accuracy.

[0027] Furthermore, it is preferable to correct the X0 coordinate of the target peak point based on the normalized X coordinate of the X0 coordinate of the target peak point. [Effects of the Invention]

[0028] As described above, according to the present invention, by using multiple standard markers in high-speed melting curve analysis, it is possible to identify target genes while avoiding the problem of temperature deviation in a simple manner that can be performed even with a general-purpose PCR device, which has high practical effects such as being able to shorten measurement time without incurring large costs. DETAILED DESCRIPTION OF THE INVENTION

[0029] (Embodiment) Before describing specific embodiments of the present invention, an outline of the present invention will be described below. The present invention relates to a method for analyzing the presence and amplification of a target nucleic acid.

[0030] Specifically, in a system that changes the temperature of a reaction solution inside a device, such as a real-time PCR device, if a temperature difference is observed between the temperature sensor and the reaction solution inside the reaction vessel, the reaction solution temperature is estimated and used to analyze the target nucleic acid based on the estimated reaction solution temperature.

[0031] In a measuring device that performs nucleic acid amplification reactions and melting curve analysis, fluorescent markers (first and second standard markers) that serve as indicators for specific temperatures during temperature changes are added, in addition to the fluorescent signal related to the detection of the target nucleic acid, and a fluorescent signal for estimating the reaction solution temperature is detected.

[0032] The data obtained by detecting the fluorescent signal of the standard marker is used to determine the time it takes to reach at least two index temperatures, where the index temperatures are temperatures at which the temperature measured by the device does not deviate from the actual temperature of the reaction solution.

[0033] For the two standard markers used, the rate of temperature change is calculated from the relational expression between the index temperature and the time it takes for the reaction liquid temperature to reach the index temperature of the standard marker, and the reaction liquid temperature at the time of temperature change is calculated.

[0034] FIG. 1 is a schematic diagram showing a part of the heat source and the periphery of the reaction vessel of an aluminum block type thermal cycler in a small and simple real-time PCR device used in the present invention.

[0035] As shown in Figure 1, this device comprises a heat source 1 such as a thermal cycler, an aluminum block 2 that holds a reaction vessel 4 and transfers heat from the heat source 1, a temperature sensor 3 that measures the temperature of the aluminum block 2, and the reaction vessel 4 that contains a reaction solution 5 inside. The installation position of the temperature sensor 3 is typical for an aluminum block-type thermal cycler, and the temperature of the reaction solution 5 is measured indirectly via the aluminum block 2.

[0036] Furthermore, the method of directly measuring the temperature of the reaction solution with the temperature sensor 3 is avoided due to the risk of inhibiting the nucleic acid amplification reaction or of contamination of the amplified nucleic acid via the temperature sensor. Therefore, the usual method is to indirectly predict the temperature of the reaction solution from the temperature of the aluminum block 2 rather than directly measuring it. However, if possible, the temperature of the reaction solution 5 may also be measured directly.

[0037] In the present invention, the use of a standard marker allows the reaction solution temperature to be confirmed from changes in fluorescence intensity after melting curve analysis. Therefore, even in real-time PCR devices, dedicated POCT devices, and other similar devices with such a structure, the reaction solution temperature can be easily confirmed in the data after melting curve analysis by utilizing the data related to the fluorescence intensity used to confirm the presence of the target gene.

[0038] The high-speed melting curve analysis method according to the present invention is a highly versatile method that has three major features.

[0039] The first feature of the accelerated melting curve analysis method of the present invention is that, during accelerated melting curve analysis, two types of standard markers (a first standard marker and a second standard marker) are present in the reaction solution for estimating the reaction solution temperature.

[0040] The term "speeded-up" in a high-speed melting curve analysis method refers to, for example, a melting curve analysis method that acquires a temperature change in the range of 40°C to 95°C within 60 seconds, and specifically refers to a temperature change rate in the range of 1.00 [°C / sec] to 2.00 [°C / sec].

[0041] However, the upper limit of the temperature change rate is 2.00 [°C / sec], and this is merely an example, since a rate higher than this would result in a problem unrelated to the present invention, such as the inability to obtain the minimum number of data points required for melting curve analysis. Therefore, if the number of data points required for analysis can be secured in a melting curve analysis faster than 2.00 [°C / sec], the rate may be set higher.

[0042] The first and second standard markers are fluorescent markers having an index temperature in a temperature range that can be used for melting curve analysis (e.g., 40°C to 95°C), and are substances that are thermally responsive to the reaction solution temperature in the reaction solution, and whose fluorescence intensity changes significantly when the reaction solution temperature reaches a specific temperature.

[0043] The first and second standard markers are preferably nucleic acid probes modified with fluorescent dyes. The index temperature of the nucleic acid probes can be easily obtained as a Tm value by melting curve analysis.

[0044] An example of a nucleic acid probe modified with a fluorescent dye is QProbe (a trademark of Nippon Steel Environment Co., Ltd.), but there is no limitation as long as it is readily available to those skilled in the art and can be used for melting curve analysis. Preferably, the nucleic acid probe is a nucleic acid sequence that does not affect the detection of the target gene and exhibits a Tm value at any temperature.

[0045] Furthermore, the structure of the nucleic acid probe is not limited to ordinary nucleic acid bases, but may include artificial nucleic acids such as cross-linked artificial nucleic acids, structures that increase the Tm value, or structures that strengthen the binding affinity with the target nucleic acid sequence.

[0046] Furthermore, when the standard marker is a nucleic acid probe, a complementary strand having a complementary nucleic acid sequence to which the standard marker binds is required in order to cause a large change in fluorescence intensity in the reaction solution depending on the length of the sequence and the temperature characteristic of that sequence. The complementary strand may be either a non-amplified product or an amplified product, or an appropriate combination of these may be used.

[0047] When the complementary strand is a non-amplified product, it may be an artificially synthesized nucleic acid having a linear or cyclic structure, and its form may be completely separated from the nucleic acid probe modified with a fluorescent dye, or may be connected in a continuous manner via a linker or nucleic acid base.

[0048] When the complementary strand is a nucleic acid amplification product, the complementary strand may be any nucleic acid amplification product having a sequence that can be clearly distinguished from the target nucleic acid sequence of the target gene. For example, a primer set dedicated to a standard marker, or an amplification product derived from an artificially synthesized nucleic acid or specimen sample amplified with a primer set common to the target gene, or a nucleic acid amplification product used as an internal standard during a nucleic acid amplification reaction, can be used.

[0049] Furthermore, the primer set for the standard marker may be the same as the primer set for the target gene or internal standard, or both or either of the primers may be the same.

[0050] The at least two types of standard markers used to calculate the reaction solution temperature should be detectable and distinguishable from each other, and can be distinguished by the indicator temperature, the type of fluorescent dye (fluorescence wavelength), etc. Furthermore, it would be even better if they could be distinguished from each other by separating the indicator temperatures to an extent that does not affect fluorescence detection.

[0051] The same type of fluorescent dye (fluorescence wavelength) may be used in one reaction vessel.

[0052] The first and second standard markers only need to be distinguishable from the Tm value of the target gene detection marker, and are preferably used at temperatures higher and lower than the Tm value of the target gene detection marker. In this way, the Tm value of the target gene detection marker is positioned between the Tm values ​​of the first and second standard markers, allowing for accurate normalization. When multiple target genes are detected in a single measurement, a set of dedicated standard markers may be prepared for each target gene, and preferably one set for multiple target genes.

[0053] A second feature of the accelerated melting curve analysis method of the present invention is that the fluorescence of the standard marker and the target gene detection marker are detected simultaneously during the accelerated melting curve analysis. However, strict simultaneous detection is not required; if the fluorescence wavelengths are different, it is acceptable to sequentially detect the fluorescence of each wavelength while switching between multiple optical modules depending on the detection device. Furthermore, it is not a problem if the standard marker and the target gene detection marker are measured in different reaction vessels, but the fluorescence intensities must be obtained simultaneously using the same reagent composition and measurement device.

[0054] A third feature of the high-speed melting curve analysis method of the present invention is that the reaction solution temperature (correction temperature) during melting curve analysis is calculated using the measurement value of the standard marker and the index temperature, and the corrected temperature is used to analyze the target gene.

[0055] Each step of the temperature analysis method of the present invention will be described with reference to Figure 2. Step 1 is a step of extracting measurement data from the device after measurement of the melting curve analysis.

[0056] Many general-purpose PCR instruments display the results of melting curve analysis in a graph with temperature on the X axis and fluorescence intensity on the Y axis, but measurement data refers to the data on the X and Y axes in Figure 2. The X-axis data can be any different value that clearly distinguishes the acquired data points, and can include not only the temperature of the measurement instrument, but also the measurement time and duration. The Y-axis data is the fluorescence intensity value differentiated with respect to time.

[0057] Step 2 is a process in which the X-axis data obtained by the accelerated melting curve analysis is normalized, and an estimated value of the reaction solution temperature is calculated from the relationship between the value of this normalized X-axis data and the index temperature of the standard marker.

[0058] First, in order to normalize the X-axis data, the measured values ​​of the X-axis data at the inflection points of the first and second standard markers are extracted from the fluorescence intensity data.

[0059] The inflection point may be either the data point where the change in fluorescence intensity of the Y-axis data in the melting curve is greatest, or the data point that is the peak of the differentiated melting curve.

[0060] Next, the entire X-axis data is normalized using the measurements of the two extracted X-axis data. Here, the two standard markers used in the analysis are the one with the lower index temperature as the low-temperature standard marker (first standard marker) and the one with the higher index temperature as the high-temperature standard marker (second standard marker), and these measurements are used.

[0061] Next, the normalized X-axis data value is calculated by applying the measurement value of the X-axis data and the measurement value of the X-axis data corresponding to the inflection point of the standard marker to the following (Equation 1). The calculation using (Equation 1) is performed on the measurement values ​​of all X-axis data.

[0062]

number

[0063] Finally, the normalized X-axis data value calculated by (Equation 1) and the index temperature of the standard marker are applied to (Equation 2) to calculate the reaction solution temperature (corrected temperature). Moreover, the calculation using (Equation 2) is performed on all normalized X-axis data values.

[0064]

number

[0065] Step 3 is a process of calculating the Tm value (peak detection) of the target gene detection marker used to identify the target gene.

[0066] Using the reaction solution temperature (corrected temperature) calculated by (Equation 2) and the Y-axis data of the target gene marker, the Tm value of the target gene detection marker is calculated by a standard method.

[0067] In this case, a melting curve analysis diagram can be created with the reaction solution temperature (corrected temperature) on the X axis and the fluorescence intensity (change in fluorescence per unit time) on the Y axis, but this is optional as it is not necessarily required for identifying the target gene.

[0068] Step 4 is a step of identifying the target gene using the Tm value of the target gene detection marker calculated in step 3 and the Y-axis data of the target gene detection marker in the data after melting curve analysis.

[0069] The calculated Tm value of the target gene detection marker is compared with the previously confirmed criteria for identifying the target gene (Tm value range and Y-axis range) to determine whether or not the target gene is present, and the target gene is identified.

[0070] Alternatively, the target gene can be identified by visually determining whether a peak of the target gene detection marker is clearly present when creating a graph of the melting curve analysis, and whether the value at the apex of the peak (Tm value) is within the criteria for determining the target gene.

[0071] Step 5 is to display the desired results.

[0072] By performing the analysis using the melting curve analysis method of the present invention, it is possible to present whether or not a target gene is detected, and if the target gene is detected, the Tm value of the target gene, as well as the melting curve analysis diagram created in step 8. At the same time, the criteria used to determine the presence or absence of the target gene may be displayed numerically or diagrammatically, thereby providing the required results.

[0073] With these displayed, the data analysis by the high-speed melting curve analysis method according to the present invention is completed.

[0074] (Experiment 1) In melting curve analysis in which the temperature change rate during melting curve analysis is increased, it will be verified whether the increased melting curve analysis method of the present invention produces results equivalent to those of melting curve analysis performed by conventional methods.

[0075] <Conditions> As a fluorescent marker, QProbe (trademark), which is a nucleic acid probe modified with a fluorescent dye, is selected, and BODIPY FL is used as the fluorescent dye.

[0076] The target gene is the luciferase gene, the target nucleic acid sequence is selected from a part of the Lux A or B gene, and QProbe (trademark) with a calculated Tm value of 57°C is used as the target gene detection marker.

[0077] For the first standard marker, a low temperature standard marker, a QProbe™ made from a nucleic acid sequence having a calculated Tm value of 48°C, which is lower than the Tm value of 57°C of the target gene detection marker, is selected.

[0078] The second standard marker, the high temperature standard marker, is selected from QProbe™, which is made from a nucleic acid sequence with a calculated Tm value of 74°C, which is higher than the Tm value of 57°C of the target gene detection marker. Table 1 shows the information on the fluorescent markers used in Experiment 1.

[0079] [Table 1]

[0080] In this experiment, reaction solutions of the same composition were added to three different reaction vessels, and fluorescent markers with different uses and synthetic single-stranded DNA with complementary base sequences were added to each vessel, and melting curve analysis was simultaneously performed using one measurement device. (Table 2) shows the reaction solution composition used in Experiment 1.

[0081] [Table 2]

[0082] The measurement device used is the LightCycler Nano (Roche Diagnostics), a real-time PCR device that can set the temperature change rate in melting curve analysis from slow to fast (0.001°C / sec to 5.00°C / sec) and can perform multiple measurements simultaneously.

[0083] As mentioned above, when speeding up melting curve analysis, there is a concern that the temperature change of the reaction solution may not be able to keep up with the temperature change of the heat source shown in Figure 1, resulting in a discrepancy between the temperature at the temperature sensor attachment point (temperature of the measurement device) and the actual temperature of the reaction solution.

[0084] Therefore, in order to confirm the temperature change rate at which the temperature of the measurement device and the actual reaction solution temperature do not diverge in the LightCycler Nano, the temperature sensor of a data logger (midi LOGGER GL240 (Graphtec Corporation)) was immersed in 50 μl of reaction solution added to a reaction vessel, and melting curve analysis was performed while changing the set value of the temperature change rate to measure the reaction solution temperature.

[0085] As a result of a series of tests, no discrepancy was observed between the temperature of the measuring device and the actual reaction liquid temperature when the temperature change rate was set to 0.05°C / sec or less, so 0.05°C / sec was defined as the rate at which there was no temperature discrepancy.

[0086] (Melt curve analysis based on rate with no temperature deviation) A melting curve analysis was performed at a temperature change rate of 0.05°C / sec, which is a rate with no temperature deviation, and the measurement time required for the temperature change from 40°C to 95°C was 18 minutes and 20 seconds. Table 3 shows the reaction conditions for the melting curve analysis at a rate with no temperature deviation.

[0087] [Table 3]

[0088] For data analysis of the rate without temperature deviation, the LightCycler Nano analysis program is used.

[0089] The same measurement is repeated multiple times, and the index temperature of the standard marker and the Tm value of the target gene are determined from the Tm values ​​automatically calculated by the analysis program.

[0090] Figure 3 shows representative results from melting curve analysis data obtained from multiple measurements. Figure 3 summarizes the differentiated melting curves of each fluorescent marker in a single figure. The X axis represents temperature (temperature of the measurement device) and the Y axis represents fluorescence intensity (amount of fluorescence change per unit time). T (solid line) represents the curve for the target gene detection marker, M circled 1 (long-dashed line) represents the curve for the low-temperature standard marker, and M circled 2 (long-dashed line) represents the curve for the high-temperature standard marker.

[0091] The average Tm value for each marker was calculated from the data measured multiple times in the melting curve analysis, and the index temperatures for the low- and high-temperature standard markers used in subsequent verification, as well as the Tm value range for the target gene detection marker (Tm value and temperature range of allowable variation), were set. Table 4 shows the set values. The Tm value range for the target gene detection marker, 63.07°C ± 1.00°C, is also shown in strips in Figures 3 to 5.

[0092] [Table 4]

[0093] (Fast melting curve analysis) The accelerated melting curve analysis is performed using the same reaction solution with the same reagent composition (Table 2) and the same measuring equipment as the melting curve analysis without temperature deviation, with a temperature change rate of 1.00°C / sec. The time required for the accelerated melting curve analysis to change temperature from 40°C to 95°C is 55 seconds. Table 5 shows the reaction conditions.

[0094] [Table 5]

[0095] (Melting curve analysis according to the present invention)

[0096] Calculating the compensation temperature In order to perform data analysis according to the present invention, the reaction solution temperature (correction temperature) during melting curve analysis, which is necessary to calculate the Tm value of the target gene detection marker, is estimated from the data after melting curve analysis by calculating it using the measured value of the standard marker and the index temperature.

[0097] First, to normalize the temperature data of the measuring device as X-axis data, the inflection point of the fluorescence intensity is identified from the differentiated fluorescence intensity data of the standard marker, and the measured value of the X-axis data at the data point where the inflection point is found is calculated. The calculated measured values ​​are shown in Table 6.

[0098] [Table 6]

[0099] Next, each value (Xn) extracted from the X-axis data (temperature data of the measuring device) from the accelerated melting curve analysis and the measurement values ​​of the standard markers shown in (Table 6) are substituted into (Equation 1), and the temperature data is normalized to obtain the following results.

[0100]

number

[0101] Finally, the index temperature of the standard marker is substituted into (Equation 2), and all normalized values ​​(X.normalized.n) obtained by (Equation 1) are substituted into (Equation 2) in order, and the normalized values ​​are converted into reaction solution temperatures (corrected temperature values ​​(X.CalibrationTemp.n)).

[0102] By substituting the index temperatures of the standard markers shown in Table 4 into Equation 2, the following results are obtained.

[0103]

number

[0104] Using the measurement value of the standard marker and the index temperature, the X-axis data is analyzed in this manner to estimate the reaction solution temperature.

[0105] Calculation of Tm value of target gene The Tm value of the target gene is calculated using the reaction temperature data obtained from the standard marker and the data on the differentiated melting curve of the target gene detection marker, as per the standard method. At this time, a melting curve analysis diagram is also created (Figure 4).

[0106] Figure 4 is a melting curve analysis diagram that combines three different differentiated melting curves for each fluorescent marker into one. The X-axis represents temperature (temperature of the measuring device) and the Y-axis represents fluorescence intensity (amount of change in fluorescence per unit time).

[0107] In the figure, M circled 1 indicates the peak of the low-temperature standard marker, M circled 2 indicates the peak of the high-temperature standard marker, and T indicates the peak of the target gene detection marker. The range of Tm values ​​of the target gene detection marker (63.07°C ± 1.00°C), which was set at a rate with no temperature deviation, is also shown as a strip on the figure.

[0108] Table 7 shows the average Tm value of each marker after correction. The corrected Tm value of the target gene marker was 63.46°C (Figure 4-T).

[0109] [Table 7]

[0110] Identification of target genes The Tm value of the target gene detection marker, 63.46°C, calculated from the reaction solution temperature (corrected temperature), falls within the range of Tm values ​​(63.07°C ± 1.00°C) of the target gene detection marker set by melting curve analysis at a rate with no temperature deviation, making it possible to identify the target gene.

[0111] Furthermore, even on the melting curve analysis diagram that was drawn, the peak position of the target gene marker can be confirmed within the range of the strips indicating the Tm value range (63.07°C ± 1.00°C) of the target gene detection marker set in the melting curve analysis at a rate with no temperature deviation. Therefore, even with data from a faster melting curve, it is possible to display Tm values ​​and diagrams equivalent to those for data at a rate with no temperature deviation.

[0112] As described above, the high-speed melting curve analysis method using the standard markers according to the present invention can obtain results equivalent to those obtained when melting curve analysis is performed at a speed without temperature deviation.

[0113] (Melting curve analysis before data analysis according to the present invention) After the accelerated melting curve analysis is performed, a melting curve analysis diagram (FIG. 5) is prepared in a standard manner before the data analysis according to the present invention is performed.

[0114] Figure 5, like Figure 4, shows the differentiated melting curves of each fluorescent marker in a single diagram, with the X axis representing temperature (temperature of the measurement device) and the Y axis representing fluorescence intensity (amount of change in fluorescence per unit time). T (solid line) represents the curve for the target gene detection marker, M circled 1 (long-dashed line) represents the curve for the low-temperature standard marker, and M circled 2 (long-dashed line) represents the curve for the high-temperature standard marker. The range of Tm values ​​for the target gene detection marker, 63.07°C ± 1.00°C, which was set at a rate with no temperature deviation, is also shown as a strip.

[0115] The Tm value (71.75°C) of the target gene marker obtained from the melting curve analysis diagram shown in Figure 5 is a value that deviates from the range of Tm values ​​(63.07°C ± 1.00°C) of the target gene detection marker set at a rate with no temperature deviation.

[0116] 3 and 5, it can be seen that the peaks (T) of the target genes are not at the same position on the X-axis, and naturally, the obtainable Tm values ​​are also different. Without the data analysis according to the present invention, results equivalent to those obtained when melting curve analysis is performed at a speed without temperature deviation cannot be obtained. This indicates that even if the target gene is present in the reaction solution, the presence or absence of the target gene may be misjudged due to the influence of the high speed, making it impossible to identify the target gene.

[0117] (Experiment 2) In Experiment 2, we verified whether the accelerated melting curve analysis method of the present invention can obtain results equivalent to those of a melting curve analysis performed at a speed without temperature deviation, even if we perform an accelerated melting curve analysis by changing the temperature change in Experiment 1 from an increase in temperature (low temperature → high temperature) to a decrease in temperature (high temperature → low temperature).

[0118] <Conditions> The three fluorescent markers and complementary single-stranded DNA used in the measurement were the same as those in Experiment 1, and the reaction solution composition was also the same. Furthermore, the measurement device used was the same LightCycler Nano as in Experiment 1. Table 8 shows reaction conditions that differ from those in Experiment 1.

[0119] Data analysis is performed by extracting from the device temperature data indicating the temperature of the measuring device as X-axis data and fluorescence intensity data relating to the differentiated melting curve as Y-axis data.

[0120] [Table 8]

[0121] (Melt curve analysis based on rate with no temperature deviation) As in Experiment 1, melting curve analysis (18 minutes 20 seconds) is performed multiple times at a rate without temperature deviation using the three reaction vessels shown in the implementation conditions, and data is collected.

[0122] Figure 6 shows representative results from melting curve analysis data obtained from multiple measurements. Figure 6 summarizes the differentiated melting curves of each fluorescent marker in a single figure, with the X axis representing temperature and the Y axis representing fluorescence intensity. M circled 1 represents the peak of the low-temperature standard marker, M circled 2 represents the peak of the high-temperature standard marker, and T represents the peak of the target gene detection marker.

[0123] The average Tm value of each marker is calculated from the data of multiple melting curve analyses, and the index temperatures of the low-side standard marker and the high-side standard marker to be used in subsequent verification, as well as the range of Tm values ​​(acceptable temperature range of variation) of the target gene detection marker are set.

[0124] The set values ​​are shown in Table 9. The range of Tm values ​​of the target gene detection markers, 62.46°C ± 1.00°C, is shown in strips in Figures 6 to 8.

[0125] [Table 9]

[0126] (Fast melting curve analysis) The accelerated melting curve analysis was performed using the same reaction solution (Table 2) with the same reagent composition as in Experiment 1 and the same measurement equipment, with a temperature change rate of 1.00°C / sec. The measurement time required for the temperature change from 95°C to 40°C was 55 seconds. Table 10 shows the reaction conditions.

[0127] [Table 10]

[0128] (Melting curve analysis according to the present invention) Calculating the compensation temperature

[0129] Using the same procedure as in Experiment 1, the reaction solution temperature (corrected temperature) is estimated by calculating it from the X-axis data using the measurement value of the standard marker and the index temperature.

[0130] First, to normalize the temperature data of the measuring device as X-axis data, the inflection point of the fluorescence intensity is identified from the differentiated fluorescence intensity data of the standard marker, and the measured value of the X-axis data at the data point where the inflection point is found is calculated. Table 11 shows the calculated measured values.

[0131] [Table 11]

[0132] Next, each value (Xn) extracted from the X-axis data (temperature data of the measuring device) from the accelerated melting curve analysis and the measurement values ​​of the standard markers shown in (Table 11) are substituted into (Equation 1), the temperature data is normalized, and the following results are obtained.

[0133]

number

[0134] Finally, the index temperature of the standard marker is substituted into (Equation 2), and all normalized values ​​(X.normalized.n) obtained by (Equation 1) are substituted into (Equation 2) in order, and the normalized values ​​are converted into reaction solution temperatures (corrected temperature values ​​(X.CalibrationTemp.n)). The index temperatures of the standard markers shown in (Table 9) are substituted into (Equation 2) to obtain the following results.

[0135]

number

[0136] Using the standard marker measurement value and the index temperature, the reaction solution temperature is estimated by analyzing the X-axis data using the same procedure as in Experiment 1.

[0137] Calculation of Tm value of target gene As in Experiment 1, the Tm value of the target gene is calculated according to standard methods using the reaction solution temperature obtained from the standard marker and the data on the differentiated melting curve of the target gene detection marker. A melting curve analysis diagram is also created in the same way (Figure 7). Figure 7 shows the differentiated melting curves of each fluorescent marker in a single diagram.

[0138] M circled 1 indicates the low-temperature standard marker, M circled 2 indicates the high-temperature standard marker, and T indicates the peak of the target gene detection marker. The range of Tm values ​​of the target gene detection marker (62.46°C ± 1.00°C), which was set at a rate with no temperature deviation, is also shown as a strip on the diagram.

[0139] Table 12 shows the average Tm value of each marker after correction. The Tm value of the target gene marker after correction is 63.15°C (Figure 7-T).

[0140] [Table 12]

[0141] Identification of target genes The Tm value of the target gene detection marker, 63.15°C, calculated from the reaction solution temperature (corrected temperature), falls within the range of Tm values ​​(62.46°C ± 1.00°C) of the target gene detection marker set in the melting curve analysis at a rate without temperature deviation. Therefore, the target gene can be identified regardless of whether the temperature change during the melting curve analysis is an increase or decrease in temperature.

[0142] Furthermore, even on the melting curve analysis diagram that was drawn, the peak position of the target gene marker can be confirmed within the range of the strips indicating the Tm value range (62.46°C ± 1.00°C) of the target gene detection marker set in the melting curve analysis at a rate with no temperature deviation. Therefore, even with data from a faster melting curve, it is possible to display Tm values ​​and diagrams equivalent to those for data at a rate with no temperature deviation.

[0143] As described above, it was confirmed that the high-speed melting curve analysis method using the standard marker of the present invention can obtain results equivalent to those obtained when melting curve analysis is performed at a speed without temperature deviation, regardless of whether the temperature is rising or falling.

[0144] (Melting curve analysis before data analysis according to the present invention) After the accelerated melting curve analysis is performed, a melting curve analysis diagram (FIG. 8) is prepared in a standard manner before the data analysis according to the present invention is performed.

[0145] Similar to Figure 7, Figure 8 shows the differentiated melting curves of each fluorescent marker in a single graph. The X axis represents temperature (temperature of the measuring device) and the Y axis represents fluorescence intensity (amount of change in fluorescence per unit time). T (solid line) represents the curve of the target gene detection marker, M circled 1 (long-dashed line) represents the curve of the low-temperature standard marker, and M circled 2 (long-dashed line) represents the curve of the high-temperature standard marker.

[0146] In addition, the range of Tm values ​​of the target gene detection markers set at a rate with no temperature deviation, 62.46°C ± 1.00°C, is shown by a strip.

[0147] The Tm value (57.54°C) of the target gene marker obtained from the melting curve analysis diagram shown in Figure 8 is a value that deviates from the range of Tm values ​​(62.46°C ± 1.00°C) of the target gene detection marker set at a rate with no temperature deviation.

[0148] 6 and 8, it can be seen that the peaks (T) of the target genes are not at the same position on the X-axis, and naturally, the obtainable Tm values ​​are also different. Without the data analysis according to the present invention, results equivalent to those obtained when melting curve analysis is performed at a speed without temperature deviation cannot be obtained. Therefore, even if the target gene is present in the reaction solution, the influence of the increased speed leads to an erroneous determination of the presence or absence of the target gene, and the target gene cannot be identified, as in Experiment 1.

[0149] (Experiment 3) In Experiment 3, we verified whether the accelerated melting curve analysis method of the present invention can obtain results equivalent to those of melting curve analysis at a speed without temperature deviation, even when all three fluorescent markers are added to one reaction vessel and an accelerated melting curve analysis is performed.

[0150] <Conditions> The three fluorescent markers and complementary single-stranded DNA used in the measurement were the same as those in Experiment 1, and the reaction conditions were also the same. Table 13 also shows the reaction solution composition that differs from that in Experiment 1.

[0151] [Table 13]

[0152] The measuring device is Mizuho Medicine's Smart Gene (registered trademark). Data analysis is performed by extracting data on the temperature of the measuring device as X-axis data and data on the fluorescence intensity related to the melting curve as Y-axis data from the device.

[0153] As with the measurement devices used in Experiments 1 and 2, it has been confirmed in advance using a data logger that with Smart Gene (registered trademark), there is no discrepancy between the temperature of the measurement device and the actual reaction liquid temperature if the temperature change rate setting is 0.05 [°C / Sec] or less, and that the temperature range from 40°C to 95°C can be measured at a temperature change rate of 1.00 [°C / Sec] (measurement time 55 seconds).

[0154] (Melt curve analysis based on rate with no temperature deviation) As in Experiment 1, melting curve analysis (18 minutes 20 seconds) was performed multiple times at a rate of 0.05°C / sec without temperature deviation, and data was collected.

[0155] Figure 9 shows representative results from melting curve analysis data obtained from multiple measurements. Figure 9 shows the differentiated melting curves of each fluorescent marker in a single graph, with the X axis representing temperature and the Y axis representing fluorescence intensity (change in fluorescence per unit time).

[0156] The average Tm value of each marker was calculated from the peaks of multiple melting curve analyses, and the index temperatures of the low-side and high-side standard markers used in subsequent verification, as well as the Tm value range (acceptable temperature range of variation) of the target gene detection marker, were set. Table 14 shows the set values. The Tm value range of the target gene detection marker, 63.06°C ± 1.00°C, is also shown in Figures 9 to 11 as a strip.

[0157] [Table 14]

[0158] (Melting curve analysis according to the present invention) Calculating the compensation temperature

[0159] Using the same procedure as in Experiment 1, the reaction solution temperature (corrected temperature) is estimated by calculating it from the X-axis data using the measurement value of the standard marker and the index temperature.

[0160] First, to normalize the temperature data of the measuring device as X-axis data, the inflection point of the fluorescence intensity is identified from the differentiated fluorescence intensity data of the standard marker, and the measured value of the X-axis data at the data point where the inflection point is found is calculated. The calculated measured values ​​are shown in Table 15.

[0161] [Table 15] Next, each value (Xn) extracted from the X-axis data (temperature data of the measuring device) from the accelerated melting curve analysis and the measurement values ​​of the standard markers shown in (Table 15) are substituted into (Equation 1), the temperature data is normalized, and the following results are obtained.

[0162]

number

[0163] Finally, the index temperature of the standard marker is substituted into (Equation 2), and all normalized values ​​(X.normalized.n) obtained by (Equation 1) are substituted into (Equation 2) in order, and the normalized values ​​are converted into reaction solution temperatures (corrected temperature values ​​(X.CalibrationTemp.n)).

[0164] By substituting the index temperatures of the standard markers shown in Table 14 into Equation 2, the following results are obtained.

[0165]

number

[0166] Using the standard marker measurement value and the index temperature, the reaction solution temperature is estimated by analyzing the X-axis data using the same procedure as in Experiment 1.

[0167] Calculation of Tm value of target gene As in Experiment 1, the Tm value of the target gene was calculated in a standard manner using the reaction solution temperature obtained from the standard marker and the data on the differentiated melting curve of the target gene detection marker. A melting curve analysis diagram was also created in the same way (Figure 10). Figure 10 shows the differentiated melting curves of each fluorescent marker in a single diagram.

[0168] M circled 1 indicates the low-temperature standard marker, M circled 2 indicates the high-temperature standard marker, and T indicates the peak of the target gene detection marker. The range of Tm values ​​of the target gene detection marker (63.06°C ± 1.00°C), which was set at a rate with no temperature deviation, is also shown as a strip on the diagram.

[0169] Table 16 shows the average Tm value of each marker after correction. The Tm value of the target gene marker after correction is 63.48°C (Figure 10-T).

[0170] [Table 16]

[0171] Identification of target genes The Tm value of the target gene detection marker, 63.48°C, calculated from the reaction solution temperature (corrected temperature), falls within the range of Tm values ​​(63.06°C ± 1.00°C) of the target gene detection marker set in the melting curve analysis with no temperature deviation rate, so the presence of the target gene can be confirmed and identified.

[0172] Furthermore, even on the melting curve analysis diagram that was drawn, the peak position of the target gene marker can be confirmed within the range of the strips indicating the Tm value range (63.06°C ± 1.00°C) of the target gene detection marker set in the melting curve analysis at a speed with no temperature deviation. Therefore, even with data from the accelerated melting curve, it is possible to display Tm values ​​and diagrams equivalent to those for data at a speed with no temperature deviation.

[0173] As described above, it was confirmed that the high-speed melting curve analysis method using the standard marker of the present invention can obtain results equivalent to those obtained when melting curve analysis is performed at a speed without temperature deviation, even when multiple fluorescent markers are present in one container.

[0174] (Melting curve analysis before data analysis according to the present invention) After the accelerated melting curve analysis is performed, a melting curve analysis diagram (FIG. 11) is prepared in a standard manner before the data analysis according to the present invention is performed.

[0175] Like Figure 10, Figure 11 shows the differentiated melting curves of each fluorescent marker in a single graph, with the X axis representing temperature (temperature of the measuring device) and the Y axis representing fluorescence intensity (amount of change in fluorescence per unit time). T (solid line) represents the curve for the target gene detection marker, M circled 1 (long-dashed line) represents the curve for the low-temperature standard marker, and M circled 2 (long-dashed line) represents the curve for the high-temperature standard marker.

[0176] In addition, the range of Tm values ​​of the target gene detection markers set at a rate with no temperature deviation, 63.06°C ± 1.00°C, is shown by a strip.

[0177] The Tm value (71.75°C) of the target gene marker obtained from the melting curve analysis diagram shown in Figure 11 is a value that deviates from the range of Tm values ​​(63.07°C ± 1.00°C) of the target gene detection marker set at a rate without temperature deviation. Comparing Figures 9 and 11, it can be seen that the peaks (T) of the target genes are not at the same position on the X-axis, and naturally, the obtainable Tm values ​​are also different. Without the data analysis according to the present invention, the influence of high speed can lead to an erroneous determination of the presence or absence of the target gene even if it is present in the reaction solution, and results equivalent to those obtained when the melting curve analysis is performed at a rate without temperature deviation are not obtained, which indicates that the target gene cannot be identified, as in Experiments 1 and 2. [Brief explanation of the drawings]

[0178] [Figure 1] Schematic diagram showing the heat source and reaction vessel of the real-time PCR device according to the present invention. [Figure 2] 1 is a flowchart showing each step of a temperature analysis method according to the present invention. [Figure 3] Graph showing the melting curve (derivative value) of the rate without temperature deviation in Experiment 1 [Figure 4] Graph showing melting curves (derivative values) in Experiment 1 of the present invention [Figure 5] Graph showing melting curve (derivative value) before carrying out the present invention in Experiment 1 [Figure 6] Graph showing the melting curve (derivative value) of the rate without temperature deviation in Experiment 2 [Figure 7] Graph showing melting curves (derivative values) in Experiment 2 of the present invention [Figure 8] Graph showing melting curve (derivative value) before carrying out the present invention in Experiment 2 [Figure 9] Graph showing the melting curve (derivative value) of the rate without temperature deviation in Experiment 3 [Figure 10] Graph showing melting curves (derivative values) in Experiment 3 of the present invention [Figure 11] Graph showing melting curve (derivative value) before carrying out the present invention in Experiment 3 [Explanation of symbols]

[0179] 1 heat source 2 aluminum blocks 3 Temperature Sensor 4 Reaction vessel 5. Reaction solution

Claims

1. Adding multiple types of markers to a reaction solution contained in a reaction vessel, The melting curve data on a data plane, with the horizontal axis X representing the data points and the vertical axis Y representing the time derivative of the fluorescence intensity, is collected based on a temperature change rate exceeding the normal temperature change rate (0.100°C / sec), A temperature analysis method for determining the X coordinate of a peak point of the plurality of types of markers, The plurality of types of markers are a target gene detection marker having a calculated target Tm0 value; a first standard marker having a calculated first Tm1 value that is different from the calculated target Tm0 value; a second standard marker having a calculated second Tm2 value that is different from the calculated target Tm0 value and the calculated first Tm1 value; A temperature analysis method characterized by using the X1 coordinate of a first peak point related to the first standard marker and the X2 coordinate of a second peak point related to the second standard marker on the data plane to determine the X coordinate obtained by normalizing the X0 coordinate of the target peak point.

2. 2. The temperature analysis method according to claim 1, wherein the information indicating the data point is any one of a measured temperature, a measured time, and a measured duration.

3. 2. The temperature analysis method according to claim 1, wherein the exceeding temperature change rate is 1.00[°C / sec] or more and 2.00[°C / sec] or less.

4. 2. The temperature analysis method according to claim 1, wherein the target Tm0 value is greater than the first Tm1 value and less than the second Tm2 value.

5. 2. The temperature analysis method according to claim 1, further comprising correcting the X0 coordinate of the target peak point based on an X coordinate obtained by normalizing the X0 coordinate of the target peak point.

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