A method for calibrating the melting curve temperature of a scanning qPCR instrument and the scanning qPCR instrument.

Abstract

This invention, entitled "A Method for Molting Curve Temperature Calibration of a Scanning qPCR Instrument and a Scanning qPCR Instrument," belongs to the field of PCR medical technology. The technical problem to be solved is to reduce actual temperature measurement errors and improve the reliability of high-throughput detection. The key technical solution is: a method for calibrating the melting curve temperature of a scanning qPCR instrument, comprising the following steps: linear calibration of well site temperature to obtain the actual temperature T of N well sites within the target temperature range of the melting curve at at least three different TEC temperature control module feedback temperatures. i , denoted as T i A temperature estimation experiment of the TEC temperature control module was conducted during sampling to obtain the actual TEC temperature control module temperature at the nth sampling point; melting curve data processing was performed to obtain T. m .

Description

Technical Field

[0001] This invention provides a melting curve temperature calibration method for a scanning qPCR instrument and a scanning qPCR instrument, belonging to the field of PCR medical technology. Background Technology

[0002] Real-time quantitative PCR (qPCR) is a core technology in the field of molecular biology for the quantitative detection of nucleic acids. It achieves quantitative nucleic acid concentration by monitoring changes in fluorescence signals during the PCR reaction. Melting curve experiments are an important part of qPCR technology. By analyzing the curve characteristics of fluorescence signals changing with temperature, the melting temperature (Tm value) of nucleic acid fragments can be determined, which can be used in scenarios such as genotyping and mutation detection.

[0003] Scanning qPCR instruments are widely used in high-throughput detection scenarios (such as 96-well plate assays) due to their compact optical path structure and high detection efficiency. Their working principle involves sequentially scanning N wells with a fiber optic scanning mechanism to acquire fluorescence signals one well at a time. In melting curve experiments, the instrument controls the TEC (thermal condenser) temperature control module to raise the temperature at a set rate while simultaneously acquiring fluorescence signals from each well. Finally, a melting curve is plotted based on the temperature-fluorescence value correlation, and the Tf value is calculated. m value.

[0004] In existing technologies, temperature sampling of melting curves has two major drawbacks: TEC temperature control module and orifice temperature deviation: Existing instruments use the feedback temperature of the TEC temperature control module as the sampling temperature of all orifices. However, due to the difference in the uniformity of heat conduction of the N-well plate (the heat dissipation conditions of edge holes and central holes are different), there is a deviation between the actual orifice temperature and the feedback temperature of the TEC temperature control module. Moreover, the deviation pattern is different for different orifices, resulting in inaccurate temperature measurement. Sampling timing temperature difference: The melting curve stage is a continuous heating process (the heating rate is usually 0.01-0.08℃ / second), while scanning instruments need to collect fluorescence signals well by well (it takes 3-5 seconds to complete the N-well scan). The sampling time of each well position is different, which causes the actual temperature of the TEC temperature control module to change when sampling at different well positions. However, the existing technology uniformly calculates the sampling temperature of all well positions according to the temperature at the time of feedback of the TEC temperature control module, ignoring the timing temperature difference.

[0005] The aforementioned defects cause a mismatch between the recorded temperature and the actual temperature at each well site, resulting in a drift in the melting curve and ultimately causing T m Value calculation deviation (usually ≥ ±0.5℃) affects the reliability of the test results, especially in scenarios with extremely high temperature accuracy requirements such as high-resolution melting (HRM), where this problem is more prominent.

[0006] Relevant patent documents retrieved: This document, published in China (CN203855583U) on October 1, 2014, discloses a PCR instrument temperature calibration system. The system includes a PCR instrument and sequentially connected temperature-sensing elements, a data acquisition card circuit, and a PC. This design utilizes high-precision sensors and acquisition modules to build a calibration hardware platform, establishing an online automatic temperature calibration system for PCR instruments based on multi-sensor fusion technology. This improves the system's testing accuracy, and the temperature calibration uncertainty is evaluated to be better than 0.03℃. Experimental results show that the system has the characteristics of high accuracy, simple algorithm, and high efficiency, and can be applied to the online calibration of reaction tube temperatures in most currently used PCR instruments.

[0007] Relevant non-patent literature retrieved: The journal or book title is *China Testing*, the document title is "A Calibration Method for Real-Time Quantitative PCR Instruments Based on Melting Curves", Volume 44, Supplement 1, Publication Date: December 2018. This document discloses: Addressing the imperfections in optical calibration methods for real-time quantitative PCR instruments, this method analyzes the necessity, basic structure, and mathematical principles of real-time quantitative PCR instrument calibration, and proposes a calibration method based on melting curves from C... t Uniformity of values, C t The calibration method of real-time quantitative PCR instrument was analyzed in terms of precision, channel peak height consistency, linear sensitivity coefficient, melting temperature drift, and melting temperature ratio.

[0008] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: Some patents attempt to improve temperature accuracy through a single compensation method (such as linear correction only for orifice temperature), but fail to address the time-series temperature discrepancies inherent in scanning instruments. Other technologies improve accuracy through hardware modifications (such as adding orifice temperature sensors), but this significantly increases instrument costs and is unsuitable for the compact structure of fiber optic scanning. Therefore, there is an urgent need for a calibration method that balances accuracy and cost, specifically addressing the dual temperature deviations in scanning instruments. Summary of the Invention

[0009] The purpose of this invention is to provide: A melting curve temperature calibration method for a scanning qPCR instrument and a scanning qPCR instrument, and related technologies, to solve technical problems such as reducing actual temperature measurement errors and improving the reliability of throughput detection, or a combination thereof.

[0010] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0011] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0012] The definitions of standard terms can be found in the reference "National Metrological Technical Specifications of the People's Republic of China".

[0013] Unless specifically defined herein, the use of various commercially available products herein employs standard techniques. For example, they may be implemented in a manner known in the art or as described in this invention. The techniques and methods described herein are generally implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0014] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0015] In a first aspect, the present invention provides: a method for calibrating the melting curve temperature of a scanning qPCR instrument, comprising the following steps: (1) Linear calibration of borehole temperature was performed to obtain the actual temperature of N boreholes, denoted as T. i ; (2) Conduct a sampling timing TEC temperature control module temperature estimation experiment to obtain the actual TEC temperature control module temperature when sampling at the nth orifice; (3) Processing the melting curve data to obtain the melting temperature T m .

[0016] Where n represents the hole number of the nth hole, which is an integer greater than or equal to 1; N is the total number of holes, which is an integer greater than or equal to 1.

[0017] The method for linear calibration of the orifice temperature is as follows: using a high-precision temperature acquisition device, before the instrument leaves the factory or during periodic calibration, the actual temperature of N orifices is measured at at least 3 different TEC temperature control module feedback temperatures within the target temperature range of the melting curve.

[0018] The accuracy of the high-precision temperature acquisition device is 0.01℃.

[0019] The target temperature range for the melting curve is 55-95℃.

[0020] Wherein, the actual temperature T i The measurement method is as follows: Select the target temperature within the target temperature range of the melting curve, and after the temperature of the TEC temperature control module of the scanning qPCR instrument stabilizes, record the T value of each well. i The temperature fed back by the corresponding TEC temperature control module is denoted as T. _TEC T for each hole position _TEC Linear fitting was performed to obtain the linear temperature relationship for each pore location: T i =K i ×T _TEC +B i , where K i Let B be the slope. i The intercept is given, and K is used for each hole position. i B i The data is stored in the storage unit of the scanning qPCR instrument's control system for subsequent actual temperature calculations.

[0021] The linear fitting method is the least squares method.

[0022] The method for temperature estimation experiment of the sampling timing TEC temperature control module is as follows: The heating rate of the melting curve stage is preset, denoted as v; the time to complete the scanning sampling of N well positions is measured by the instrument timing module, denoted as t; and T is calibrated. _TEC The sampling hole position corresponding to the nth sampling point is designated as hole position 1. The remaining holes are sequentially marked as 2-n according to a fixed scanning order. The actual TEC temperature control module temperature at the nth sampling point is obtained as: T n_TEC =T _TEC +(n-1)×v×t / N, where N is the total number of holes.

[0023] Wherein, the range of v is: 0.01-0.08℃ / second.

[0024] The method for measuring t is as follows: record the time for one sampling cycle with an accuracy of ≤ ±0.001 seconds.

[0025] The fixed scanning sequence is: a pre-set scanning path for the hole positions that cannot be changed arbitrarily.

[0026] The method for processing the melting curve data is as follows: the fluorescence module of the scanning qPCR instrument acquires the fluorescence signal of each well in a fixed scanning order, and the T... n_TEC Substituting T into the linear temperature relationship for each orifice location_TEC , get T i , with T i Plot the melting curve for each well site with the x-axis representing the fluorescence signal value and the y-axis representing the melting curve. Calculate T using the first derivative method. m value.

[0027] The first derivative method involves using the first derivative to determine the temperature corresponding to the maximum rate of change of the fluorescence signal.

[0028] In the n-sorting, when the actual TEC temperature control module temperature is obtained, the corresponding sampling hole position is hole position 1, and the remaining holes are marked as 2-n in a fixed scanning order; while in the i-sorting, the top-left hole position is hole position 1; therefore, when the actual TEC temperature control module temperature is obtained and the corresponding sampling hole position is not the top-left hole position, the hole position sorting of n and i is different; therefore, when T n_TEC Substituting T into the linear temperature relationship for each orifice location _TEC , get T i At that time, it was based on the physical location of the hole, which was substituted one-to-one.

[0029] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes: The first preferred option: a method for linear calibration of orifice temperature.

[0030] Second preferred option: Actual temperature T i The measurement method.

[0031] The third preferred option: a method for calculating the temperature of the TEC temperature control module based on sampling timing.

[0032] The above technical solutions not only solved the technical problem of "reducing the actual temperature measurement error", but also further solved the technical problem of "improving the reliability of throughput detection".

[0033] Secondly, the present invention provides a scanning qPCR instrument, comprising: a reaction module, a temperature detection module, a fluorescence module, a control system, and a human-computer interaction module; the reaction module is connected to the temperature detection module and the fluorescence module, the temperature detection module is connected to the control system and the human-computer interaction module, the fluorescence module is connected to the control system and the human-computer interaction module, and the control system is connected to the human-computer interaction module, for implementing the melting curve temperature calibration method.

[0034] The reaction module consists of an N-hole heating element and a TEC temperature control module connected to each other, supporting precise temperature rise of 0.01-0.08℃ / second.

[0035] The temperature detection module consists of a TEC temperature sensor and a high-precision calibration probe to collect T data in real time. _TEC .

[0036] The fluorescence module comprises: an optical fiber scanning mechanism, an excitation module, and a fluorescence detection module.

[0037] The control system consists of interconnected storage units, computing units, and timing modules.

[0038] The human-computer interaction module consists of a parameter setting module and a result display module.

[0039] The N-hole heating element is a metal body used to heat N holes.

[0040] The TEC temperature control module is used to regulate the temperature of the N-hole heating element.

[0041] The TEC temperature sensor outputs T _TEC The signal is sent to the control system.

[0042] The high-precision calibration probe has a pluggable structure. During calibration, it is inserted into the hole of the N-hole heating element to measure T. i And transmit it to the control system.

[0043] The excitation module is connected to the fiber optic scanning mechanism, and the fluorescence detection module is connected to the fiber optic scanning mechanism.

[0044] The fiber optic scanning mechanism is used for scanning operation, thereby scanning each aperture in a fixed scanning sequence.

[0045] The excitation module is used to excite the sample at each well site using fluorescence.

[0046] The fluorescence detection module is used to receive fluorescence from the samples at each well.

[0047] The excitation light emitted by the excitation module is transmitted to the target aperture via the fiber optic scanning mechanism. The fluorescence signal generated by the excitation of the fluorescent material in the aperture is returned to the fluorescence detection module via the fiber optic cable, converted into an electrical signal, and then transmitted to the control system. The fiber optic scanning mechanism is driven by a motor and switches to different apertures in a preset sequence.

[0048] The storage unit stores K i coefficient, B i Coefficients, v, t, and scanning order.

[0049] The timing module records the time t it takes for the fiber optic scanning mechanism to complete the scanning and sampling of each aperture.

[0050] The calculation unit performs temperature calculation, curve plotting, and T... m Value calculation.

[0051] After setting the corresponding parameters in the parameter setting module, the corresponding results can be obtained in the result display module.

[0052] The present invention has at least the following beneficial effects: Compared with existing technologies, the present invention has better technical effects in reducing actual temperature measurement errors and improving the reliability of throughput detection.

[0053] According to experimental tests, the present invention reduces the CV value from 0.95% in the prior art to below 0.12%. Attached Figure Description

[0054] Figure 1 This is a diagram of the reaction module of the scanning qPCR instrument described in this invention.

[0055] like Figure 1 As shown, the module includes an N-hole heating element (101) and a TEC temperature control module (102). The TEC temperature control module (102) is attached to the bottom of the N-hole heating element (101) and is used to regulate the temperature of the N-hole heating element.

[0056] Figure 2 This is a diagram of the temperature detection module of the scanning qPCR instrument described in this invention.

[0057] like Figure 2 As shown, the module includes: a TEC temperature sensor (201) and a high-precision calibration probe (202); the TEC temperature sensor (201) is attached to the surface of the TEC temperature control module (102) and outputs T... _TEC The signal is transmitted to the control system; the high-precision calibration probe (202) is a pluggable structure, inserted into the hole of the N-hole heater (101) during calibration, and T is measured. i And transmit it to the control system.

[0058] Figure 3 This is a diagram of the fluorescence module of the scanning qPCR instrument described in this invention.

[0059] like Figure 3 As shown, the module includes: an optical fiber scanning mechanism (301), an excitation module (302), and a fluorescence detection module (303); the excitation light emitted by the excitation module (302) is transmitted to the target aperture through the optical fiber scanning mechanism (301), and the fluorescence signal generated by the fluorescent material in the aperture is returned to the fluorescence detection module (303) through the optical fiber, converted into an electrical signal, and transmitted to the control system; the optical fiber scanning mechanism (301) is driven by a stepper motor and switches to different apertures in a preset sequence.

[0060] Figure 4 This is a diagram of the human-computer interaction module of the scanning qPCR instrument described in this invention.

[0061] like Figure 4 As shown, this module is the TK-7000 medical PCR analysis software, used to set parameters such as v and temperature range, and to display melting curves and T. m Value result. Detailed Implementation

[0062] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0063] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0064] Example 1 Step 1: Linear calibration of orifice temperature.

[0065] Experimental preparation: A temperature acquisition device with an accuracy of 0.01℃ was selected as a high-precision temperature acquisition device (202). The temperature acquisition device was inserted into each hole of the 96-hole heating element to ensure that the probe was in full contact with the heating element. Temperature point setting: The target temperature of the TEC temperature control module (102) can be set to 60℃, 75℃ and 90℃ through the human-machine interaction module. After each temperature point is reached, it is maintained to ensure stability. Data acquisition: The control system records the temperature T fed back by the TEC temperature control module at each temperature point. _TEC (Collected by TEC temperature sensor 201) and the actual temperature Ti of each hole (collected by high-precision temperature acquisition device 202); Linear fitting: Three sets (T) for each pore location _TEC The data for Ti were fitted using the least squares method to obtain K. i and B i .For example: Hole 1 (Central Hole): T i =0.996×T _TEC -0.34 Hole position 96 (edge ​​hole): T i =1.003×T _TEC -0.69 Store 96 sets of coefficients in the storage unit.

[0066] Step 2: Temperature calibration and testing of melting curve.

[0067] Experimental parameter settings: The melting curve parameters are set through the human-computer interaction module: heating rate v = 0.05℃ / second, temperature range 55-95℃, and scanning sequence is a pre-set, non-changeable well scanning path (in this embodiment, it is divided into 4 parts, each part has 24 wells (3 columns and 8 rows). The scanning sequence is: first the 24 wells of part 1, then parts 2, 3, and 4. In the first part, the first row has wells 1-3 → the second row has wells 4-6 → ... → the eighth row has wells 22-24, and so on for the remaining parts). Sampling timing parameter measurement: Start the pre-scan, the timing module measures the time t=3.840 seconds for the fiber optic scanning mechanism (301) to complete the scanning of 96 holes, calculate the time difference between adjacent holes Δt=3.840 / 96=0.04 seconds, and the temperature difference ΔT=0.05×0.04=0.002℃; Formal experiment: The TEC temperature control module (102) heats up at a rate of 0.05℃ / second. When the TEC temperature sensor (201) reports T... _TEC At 60℃, the control system triggers the fluorescence module to start scanning and calibrates the sampling well position at this time as well position 1 (such as the first well in the first row). TEC temperature control module temperature estimation: The control system records the sampling times of orifice positions 2-96 sequentially to estimate the actual TEC temperature control module temperature. Hole position 2: T n_TEC =60+(2-1)×0.002=60.002℃; Hole position 3: T n_TEC =60+(3-1)×0.002=60.004℃; ... Hole position 96: T n_TEC =60+(96-1)×0.002=60.19℃; Actual temperature calculation at the borehole: The calculation unit (402) calls the stored K... i and B i Substitute the above T n_TEC Calculate T i : Hole position 2 (assuming its K) i =1.008, B i =-0.45):T i =1.008×60.002-0.45≈60.032℃ ... Hole position 96: T i =1.003×60.19-0.69≈59.681℃ Melting curve plotting and T m Value calculation: The fluorescence detection module (303) collects fluorescence signals (such as FAM dye fluorescence) at each well site, and the calculation unit calculates the value in T. iPlot a curve with the x-axis and fluorescence value as the y-axis, and calculate T using the first derivative method. m Value. The results show that the T value for each hole m The CV value is ≤0.12%, which is significantly better than the traditional method (0.8-1.5%).

[0068] The traditional method involves directly using test values ​​for calculation without any temperature compensation.

[0069] The above embodiments demonstrate that the present invention can effectively improve the accuracy of melting curves and well uniformity through dual temperature compensation, making it suitable for high-precision detection scenarios of scanning qPCR instruments.

[0070] T for each hole m The CV values ​​are shown in Table 1.

[0071] Table 1

[0072] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for calibrating the melting curve temperature of a scanning qPCR instrument, characterized in that, Includes the following steps: (1) Linear calibration of borehole temperature was performed to obtain the actual temperature of N boreholes, denoted as T. i ; (2) Conduct a sampling timing TEC temperature control module temperature estimation experiment to obtain the actual TEC temperature control module temperature when sampling at the nth orifice; (3) Processing the melting curve data to obtain the melting temperature T m . Where n represents the hole number of the nth hole, which is an integer greater than or equal to 1; N is the total number of holes, which is an integer greater than or equal to 1.

2. The melting curve temperature calibration method according to claim 1, characterized in that, The method for linear calibration of the orifice temperature is as follows: using a high-precision temperature acquisition device, before the instrument leaves the factory or during periodic calibration, the actual temperature of N orifices is measured at at least 3 different TEC temperature control module feedback temperatures within the target temperature range of the melting curve.

3. The melting curve temperature calibration method according to claim 2, characterized in that, The accuracy of the high-precision temperature acquisition device is 0.01℃.

4. The melting curve temperature calibration method according to claim 2, characterized in that, The target temperature range for the melting curve is 55-95℃.

5. The melting curve temperature calibration method according to claim 2, characterized in that, The method for measuring the actual temperature is as follows: select a target temperature within the target temperature range of the melting curve, and after the temperature of the TEC temperature control module of the scanning qPCR instrument stabilizes, record the T value for each well. i The temperature fed back by the corresponding TEC temperature control module is denoted as T. _TEC T for each hole position _TEC Linear fitting was performed to obtain the linear temperature relationship for each pore location: T i =K i ×T _TEC +B i , where K i Let B be the slope. i The intercept is K, and the K values ​​for each hole position are... i B i The data is stored in the storage unit of the control system of the scanning qPCR instrument.

6. The melting curve temperature calibration method according to claim 5, characterized in that, The method for calculating the temperature of the sampling timing TEC temperature control module is as follows: The heating rate of the preset melting curve is denoted as v. The time to complete the scanning sampling of N wells is measured using the timing module of the scanning qPCR instrument and denoted as t. T is then calibrated. _TEC The sampling hole position corresponding to the sampling time is hole position 1. The remaining holes are marked as 2-n in a fixed scanning order. The actual TEC temperature control module temperature at the sampling time of the nth hole position is obtained and denoted as T. n_TEC T n_TEC =T _TEC +(n-1)×v×t / N.

7. The melting curve temperature calibration method according to claim 6, characterized in that, The range of v is: 0.01-0.08℃ / second; The method for measuring t is as follows: record the time for one sampling cycle, with an accuracy of ≤ ±0.001 seconds; The fixed scanning sequence is: a pre-set scanning path for the hole positions that cannot be changed arbitrarily.

8. The melting curve temperature calibration method according to claim 6, characterized in that, The method for processing the melting curve data is as follows: the fluorescence module of the scanning qPCR instrument collects the fluorescence signal values ​​of each well in a fixed scanning order, and then processes the T... n_TEC Substituting T into the linear temperature relationship for each orifice location _TEC , get T i , with T i Plot the melting curve for each well site with the x-axis representing the fluorescence signal value and the y-axis representing the melting curve. Calculate T using the first derivative method. m .

9. A scanning qPCR instrument, characterized in that, include: The reaction module, temperature detection module, fluorescence module, control system, and human-computer interaction module are used to implement the melting curve temperature calibration method according to any one of claims 1-8; the reaction module is connected to the temperature detection module and the fluorescence module, the temperature detection module is connected to the control system and the human-computer interaction module, the fluorescence module is connected to the control system and the human-computer interaction module, and the control system is connected to the human-computer interaction module.

10. The scanning qPCR instrument according to claim 9, characterized in that, The reaction module includes an N-hole heating element and a TEC temperature control module connected to each other, supporting precise temperature rise of 0.01-0.08℃ / second; The temperature detection module includes a TEC temperature sensor and a high-precision calibration probe to collect the temperature feedback from the TEC temperature control module in real time. The fluorescence module includes: a fiber optic scanning mechanism, an excitation module, and a fluorescence detection module; The control system includes: an interconnected storage unit, a computing unit, and a timing module; The human-computer interaction module includes a parameter setting module and a result display module.

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