JP2020103095A - Melt temperature analyzer - Google Patents

Abstract

To provide a fusion temperature analyzer which hardly receives an affection due to an uneven temperature of a temperature variable part for heating or cooling an observed sample and which is produced in consideration of above mentioned situations.SOLUTION: The invention relates to a fusion temperature analyzer comprising: a temperature variable part for heating or cooling an observed sample; a fluorescence measuring part for measuring a fluorescent brightness of the observed sample; a distribution measuring part for measuring by an infrared ray, temperature distribution of the temperature variable part; an actual measurement part for measuring an actual measurement temperature of the temperature variable part, and the fusion temperature analyzer acquires data of the fluorescent brightness, data of the temperature distribution, and data of the actual measurement temperature at a prescribed time.SELECTED DRAWING: Figure 1

Description

The present invention relates to a melting temperature analyzer for quantitatively analyzing a plurality of target nucleic acids.

Conventionally, a digital PCR (dPCR; digital Polymerase Chain Reaction) method has been proposed as a method for quantitatively analyzing the concentration of a nucleic acid (target nucleic acid) containing a specific base sequence from a sample in which various nucleic acids are mixed.

In digital PCR, a sample containing a target nucleic acid is mixed with an amplification reagent for amplifying the target nucleic acid, a fluorescent reagent for detecting the target nucleic acid, etc. and diluted to obtain a sample (FIG. 5(a)). Is divided into a plurality of physically independent minute sections (FIG. 5B). Then, PCR is independently generated in each of the plurality of micro-compartments to amplify the target nucleic acid so that it can be detected (FIG. 5(c)).

The number of microcompartments in which a fluorescent signal was detected after amplification (the number of positive compartments) and/or the number of microcompartments in which no fluorescence signal was detected after the amplification (the number of negative compartments) is counted. The concentration of the target nucleic acid in the sample can be quantified by statistically processing the counting result based on a predetermined probability distribution such as Poisson distribution (FIG. 5).

As a method of dividing a reaction solution into a plurality of minute sections in digital PCR, there is a method of forming a droplet of the reaction solution in oil. That is, a method of forming a water-in-oil emulsion (W/O emulsion) (Patent Document 1), a method of dividing a reaction liquid by using a chip provided with a plurality of minute compartments on a substrate (Patent Document 2), etc. There is.

The concentration of the target nucleic acid is quantified by treating the counted micro-compartments with a statistical Poisson distribution. Therefore, it is preferable that there is no variation in the probability that the target nucleic acid enters each of the micro-compartments. Therefore, it is preferable to use a dividing method that is closer to monodisperse as the minute partitions.

On the other hand, the high resolution melting curve (High Resolution Melting: HRM) analysis is a method of detecting melting mutations of a plurality of target nucleic acids after amplification of the target nucleic acid by PCR. HRM analysis is a more sensitive method compared to the conventional melting curve method and monitors the temperature at which a double-stranded nucleic acid dissociates into a single-stranded nucleic acid. This temperature is known as the melting temperature (Tm) of the amplification product.

The method for detecting Tm in HRM analysis is to amplify a gene fragment of about 80 to 250 base pairs by PCR in a reaction solution containing a highly functional double-stranded nucleic acid binding dye. The amplified product is annealed and placed in the standby state where the fluorescence intensity is the highest, and the temperature is slowly raised, while the fluorescence data from the amplicon is recorded. When the PCR product begins to denature (or melt), the fluorescence of the amplification product slowly decreases as it approaches the Tm as the fluorescent dye is released. Near the Tm, a sharp decrease in fluorescence is observed as the sample changes from double-stranded to single-stranded nucleic acid (FIG. 6).

As a method for measuring the temperature of the micro section, there is a method of using a standard fluorescent dye having known temperature information. Utilizing the fact that the fluorescence intensity decreases as the temperature rises due to the decrease in quantum yield, the standard fluorescent dye is dispensed into each microcompartment and the fluorescence intensity is measured, which is used as the data of the temperature distribution. Method (Patent Document 3).

Infrared thermography is a typical non-contact method for imaging temperature. Infrared thermography is a method of detecting infrared rays emitted by an object and displaying the temperature distribution on the surface of the object two-dimensionally for visualization. The advantage of using heat radiation is that it can be measured in a non-contact manner, does not affect the object to be measured, has excellent responsiveness, and can measure rapid temperature changes (Patent Document 4).

Special table 2012-503773 gazette Japanese Patent Publication No. 2015-516802 Japanese Patent No. 5703377 Japanese Patent Laid-Open No. 10-2803

In the Tm detection method using HRM analysis, extremely high temperature accuracy is required for the temperature variable unit in order to detect a slight difference in melting temperatures Tm of plural kinds of target nucleic acids. Therefore, a temperature varying unit using a Peltier element is used for accurate temperature control of heating/cooling. However, there was a measurement error of the temperature sensor. Further, in the Peltier device itself and in the mounting portion of the sample observing section (observation sample) provided with the minute sections and the temperature varying section, temperature nonuniformity due to contact resistance occurs.

Due to these fluctuation factors, an error in the observation temperature may occur between the hottest position and the coldest position in the micro-compartment. When identifying a slight difference in the melting temperature Tm as the observation temperature for the sample in the micro-compartment. There was a problem. Therefore, the observation temperature of the sample in each micro-compartment needs to be measured in all the compartments in time units.

However, the method of performing the temperature calibration of the temperature variable unit using the standard fluorescent dye, which is the conventional example described in Patent Document 3, is one of the methods used for the accuracy control of the real-time PCR device. This method has a problem in that it is not possible to deal with the nonuniform temperature due to the mounting portion between the observation sample provided with the minute sections and the temperature varying portion.

That is, since the mounting portion of the observation sample to the temperature varying portion has a temperature transfer structure by surface contact, the thermal resistance changes depending on the mounting state, resulting in non-uniformity in temperature transfer to the observation sample.

On the other hand, the conventional example described in Patent Document 4 is an optical device that can simultaneously obtain an image of a temperature distribution by an infrared camera and an optical image by a visible light camera for an observation sample, but continuously while heating and cooling the sample. It does not have a temperature variable function to measure by. Therefore, non-uniformity of temperature was not a problem.

However, the infrared camera is easily affected by the outside air temperature, which causes an error when measuring the temperature of the object. Normally, temperature correction is performed using a mechanical shutter, the shutter is closed and the influence of the outside air temperature at that time is read out, and the correction is performed by subtracting by image processing. The measured value immediately after the shutter correction is an accurate value, but when the temperature measurement is continuously repeated, an error occurs with the actual sample temperature.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a melting temperature analysis device that is less likely to be affected by temperature nonuniformity of a temperature varying unit that heats or cools an observation sample.

The present invention provides a temperature varying unit for heating or cooling an observation sample, a fluorescence measuring unit for measuring the fluorescence brightness of the observation sample, a distribution measuring unit for measuring the temperature distribution of the temperature varying unit with infrared rays, and the temperature varying unit. An actual measurement unit that measures an actual measurement temperature of the unit, and acquires the fluorescence brightness data, the temperature distribution data, and the actual measurement temperature data at a predetermined time.

It is possible to provide a melting temperature analyzer which is less likely to be affected by the temperature nonuniformity of the temperature varying unit that heats or cools the observation sample.

It is a figure explaining the structure of the apparatus (melting temperature analyzer) which concerns on the Example of this invention. It is a figure which shows the operation|movement flow of the apparatus (melting temperature analyzer) which concerns on the Example of this invention. It is a block diagram of the apparatus (melting temperature analysis apparatus) concerning the Example of this invention. It is a figure which shows the perspective view of a sample observation part (observation sample) and a temperature variable part. It is the figure which showed the quantification principle of a target nucleic acid. It is the figure which showed the characteristic of the melting curve of a target nucleic acid. It is the figure which showed the temperature correction method of a melting curve. It is a figure explaining each measurement data and measurement time. It is the figure which showed the temperature value of a reference point, and the relationship of measurement time.

[Example 1]
FIG. 1 is a drawing best showing the features of the apparatus (melting temperature analyzer) according to the present invention.

In the figure, 1 is a fluorescence camera (fluorescence measuring unit) 1 for measuring the fluorescence brightness of a sample. Reference numeral 2 is a fluorescence filter 2 which is an optical long-pass filter that attenuates the wavelength of the excitation light and passes only the fluorescence of the sample. Reference numeral 3 is an excitation light source 3 that generates excitation light for irradiating the sample. Reference numeral 4 is an excitation filter 4 of an optical short-pass filter that passes only the wavelength required for excitation.

Reference numeral 5 is an infrared thermography (distribution measuring unit) 5 that detects infrared rays and visualizes the temperature distribution on the sample surface. Reference numeral 6 is a dichroic mirror 6 that reflects excitation light and transmits fluorescence. Reference numeral 7 is a hot mirror 7 that reflects long-wavelength infrared light and transmits only visible light. Reference numeral 8 denotes a sample observation section (observation sample) 8 in which the sample is divided into a plurality of minute sections.

Reference numeral 9 denotes a plate 9 that transfers heat to the observation sample 8 to perform heating or cooling (at least one of heating and cooling). Reference numeral 10 denotes a contact-type temperature sensor (actual measurement unit) 10 that measures an actual measurement temperature with respect to a reference point of the plate 9. Reference numeral 11 denotes a Peltier element 11, which is a heat transfer element that heats or cools the plate 9. Reference numeral 12 is a heat sink 12 that radiates heat on the heat radiation side of the Peltier element 11. Reference numeral 13 denotes a fan 13 forcibly discharging the heat of the heat sink 12.

The unit in which the units related to temperature control 9 to 13 are put together is put together as a temperature varying unit 14.

FIG. 2 is a diagram showing an operation flow of the device (melting temperature analyzer) according to the present invention.

In step S<b>1, the output of the excitation light source 3 is turned on, and the excitation light having the wavelength that has passed through the excitation filter 4 is reflected by the dichroic mirror 6 and is applied to the observation sample 8. In step S2, the fluorescence emitted from the sample divided into minute sections by the excitation light passes through the hot mirror 7 and the dichroic mirror 6, and the fluorescence filter 2 removes the wavelength of the excitation light as a fluorescence image. Measured at 1. In step S3, the infrared rays radiated from the sample are reflected by the hot mirror 7 and measured by the distribution measuring unit 5 as an image of the temperature distribution.

In step S4, the actual measurement unit 10 mounted on the plate 9 measures the contact temperature at the reference point. In step S5, the temperature of the sample is increased by +n° C. by controlling the temperature varying unit 14. In step S6, the temperature varying unit 14 controls the heating in the temperature range of 65 to 95° C. required for the HRM analysis, and therefore steps S2 to S5 are repeated until the temperature reaches 95° C. In step S7, a waiting time of m seconds is set in order to adjust the ramp speed by the temperature varying unit 14. At the branch of step S6, when the temperature reaches 95° C., the excitation light source is turned off in step S8 to end the operation.

In the figure, in steps S2 to S4, time data at the time of measurement can be acquired at the same time so that they can be treated as time series data mutually.

When fading of the sample is a problem, the excitation light source may be turned on and off only during the time when the measurement by the fluorescence measurement unit is performed. By inserting the step of turning on/off the excitation light source before and after step S2, it is possible to limit the irradiation of the excitation light to the minimum necessary.

Further, the measurement timings of the fluorescence measuring unit and the distribution measuring unit may not be measured in the order of step S2 and step S3 as shown in FIG. If the time (predetermined time) of each measured timing is managed, there is no problem whether it is asynchronous control or synchronous control. The same applies to the temperature measurement in step S4 as long as the measurement timing time (predetermined time) is managed.

FIG. 3 is a block diagram of an apparatus (melting temperature analyzer) according to the present invention.

In the figure, the main controller 15 is responsible for main control of the entire apparatus, and manages measurement time by the fluorescence measurement unit 1 and the distribution measurement unit 5, acquires images, and controls on/off of the excitation light source 3. The temperature control controller 16 controls the temperature of the sample in the temperature varying unit 14, and switches the temperature detected by the actual measurement unit 10 and the current of the Peltier element 11 to perform PID control of heating and cooling, and a heat sink. The on/off control of the fan 13 is performed for the heat radiation of the fan 12.

From the main controller 15 of the main control, the temperature control of the sample is executed by instructing the set temperature to the temperature control controller 16 of the temperature varying unit 14. The temperature data of the reference point is measured in response to a request from the main controller 15 to the temperature controller 16, and the measured temperature and time data are provided together.

FIG. 4 is a perspective view of the temperature varying unit 14 and the observation sample 8.

In the figure, the observation sample 8 is a transparent member such as glass or resin, and is used to divide the sample into a plurality of minute compartments by using fine holes or droplets. Fixed.

An actual measurement unit 10 is embedded in the plate 9, and the temperature is measured using a contact platinum sensor, a thermocouple, or the like. A black area serving as a reference point 17 is provided in the vicinity of the actual measurement unit 10 of the plate 9.

The reference point 17 has a structure that can be directly observed from the distribution measuring unit 5 even when the observation sample 8 is fixed to the plate 9. Then, the difference between the actual temperature measured by the actual measurement unit 10 and the temperature measured by the distribution measurement unit 5 in the black region of the reference point 17 is stored as the actual temperature data whose time is managed.

The position of the reference point 17 only needs to be in the vicinity of the actual measurement unit 10, and it is also possible to provide it at the four corners of the plate 9 outside the range of the observation sample 8 or to provide a plurality of pairs of the reference point and the actual measurement unit. is there.

Below the plate 9, a Peltier element 11 and a heat sink 12 are stacked with a member having high thermal conductivity such as grease or a silicon sheet interposed therebetween. Two electrodes are connected to the Peltier element 11, and by switching the polarity of the direct current, heating and cooling can be switched to perform temperature control.

The temperature varying unit 14 controls heating within a temperature range of 65 to 95° C. required for HRM analysis. The explanation will be given along the operation flow shown in FIG. 2. When the sample is measured at a ramp rate (1° C./sec) of +1° C. (n=1) every second (m=1), the observation time is Can be completed in 30 seconds. However, at the ramp speed as described above, the nonuniformity of the temperature generated in the Peltier element remarkably occurs, and an error occurs in the sample temperature of the minute section.

In order to reduce the error of the sample temperature, a method of reducing the error factor by decreasing the ramp rate of temperature rise is also taken. However, when the temperature is increased at 0.1° C./sec, which is 1/10 times, the observation time is required to be 5 times, which is 10 times, and the throughput of HRM analysis is deteriorated.

Therefore, by recording the times measured by the fluorescence measurement unit that measures the fluorescence brightness and the distribution measurement unit that measures the temperature distribution, it is possible to measure the fluorescence brightness data and the temperature distribution data of the observation sample 8 in the same time series. Is possible. At the same time, the actual temperature data of the reference point 17 in which the actual measurement unit 10 is embedded in the plate 9 of the temperature variable unit 14 is also acquired in the same time series, so that the temperature of the reference point 17 measured by the distribution measurement unit at the same time is obtained. The difference is used as the temperature correction value.

FIG. 7 is a diagram showing a method for correcting the temperature of the melting curve. FIG. 7A is a melting curve in which the vertical axis represents the luminance of fluorescence and the horizontal axis represents the temperature. It can be seen that when the melting curves of the minute sections are overlapped, there are large variations in the luminance and the temperature. FIG. 7B is a melting curve obtained by normalizing the luminance on the vertical axis and converting it to 0 to 100%, but the variation in the temperature on the horizontal axis remains because the variation in the luminance is suppressed. FIG. 7C shows a melting curve in which the temperature on the horizontal axis is corrected by using the temperature correction value in which the measured temperature data is taken into consideration. As a result, it is possible to suppress the variation between the minute compartments on both the vertical axis and the horizontal axis, and to obtain the same melting curve regardless of the location of the minute compartments for the same sample. That is, it becomes possible to accurately read the melting temperatures Tm of a plurality of target nucleic acids.

8 and 9 are diagrams illustrating a method of calculating the temperature correction value.

FIG. 8 shows the following contents. The fluorescence luminance data Emit01 to Emit07 by the fluorescence measurement unit and the measured time time are shown. Further, the temperature distribution data Therm01 to Therm07 by the distribution measuring unit and the measured time time are also shown. Also, the measured temperatures Temp01 to Temp07 of the actual measurement unit are shown as the measured time. It is possible to confirm the timing (predetermined time) of the data measured by each in the same time axis.

Since the fluorescence brightness data and the temperature distribution data are two-dimensional image data (Raw data, Bitmap data, etc.), the fluorescence brightness value and the temperature of each area of the observation sample divided into minute sections from the image data. The values are extracted and a melting curve is generated. However, since it is necessary to extract the fluorescence brightness value and the temperature value at the same time, it is necessary to correct the time value for the temperature value acquired asynchronously, but it is not necessary to correct the time value in the system where the measurement is performed by the synchronous control. Lost.

In FIG. 9, the temperature value 18 at the reference point read from the distribution measuring unit 5 and the actually measured temperature value 19 at the reference point by the contact thermometer are plotted on the vertical axis, and the measurement time (time) at which each data is acquired is plotted on the horizontal axis. It is the graph that I made. Since the temperature difference becomes ΔTemp1 at time t1, the temperature measured by the infrared camera image at that time is corrected by −ΔTemp1 as a whole. Similarly, since the temperature difference becomes ΔTemp2 at time t2, the temperature measured by the infrared camera image at that time is corrected by −ΔTemp2 as a whole.

In this way, by calculating the temperature correction value for each measurement time, it is possible to know the actually measured temperature for each minute section in accordance with the measurement timing (predetermined time) of the fluorescence brightness data. Can be output.

Therefore, in the Tm detection method using HRM analysis, the temperature distribution data can be corrected by the temperature correction values measured in the same time series even when the heating lamp speed is increased and the temperature nonuniformity of the observation portion occurs. The melting temperature Tm suitable for the actually measured temperature can be calculated.

1 Fluorescence camera (fluorescence measurement unit)
5 Infrared thermography (distribution measurement section)
10 Temperature sensor (actual measurement unit)
14 Temperature variable part

Claims (6)

A temperature variable unit for heating or cooling the observation sample,
A fluorescence measurement unit that measures the fluorescence brightness of the observation sample,
A distribution measurement unit that measures the temperature distribution of the observation sample with infrared rays,
An actual measurement unit for measuring the actual temperature of the temperature variable unit,
A melting temperature analyzer, which acquires the data of the fluorescence brightness, the data of the temperature distribution, and the data of the actually measured temperature at a predetermined time. The melting temperature analyzer according to claim 1, wherein the fluorescence brightness data, the temperature distribution data, and the measured temperature data at a plurality of the predetermined times are acquired. Obtaining the data of the fluorescent brightness, the data of the temperature distribution, and the data of the measured temperature together with the time data,
Obtained, from the data of the fluorescent brightness and the data of the temperature distribution, the data of the measured temperature and the data of the time, at the predetermined time, the data of the fluorescent brightness and the data of the temperature distribution and the measured temperature The melting temperature analyzer according to claim 1 or 2, wherein data is acquired. By synchronously controlling the fluorescence measurement unit, the distribution measurement unit, and the actual measurement unit, at the predetermined time, the fluorescence brightness data, the temperature distribution data, and the actual measurement temperature data are acquired. The melting temperature analyzer according to claim 1 or 2. The melting temperature analyzer according to any one of claims 1 to 4, wherein the temperature of the melting curve is corrected based on the difference between the data of the temperature distribution at the predetermined time and the data of the actually measured temperature. .. The melting temperature analyzer according to any one of claims 1 to 5, wherein the fluorescence measuring unit measures the fluorescence brightness with visible light.

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