CN113528315A

CN113528315A - Method for simulating gene amplification standard curve

Info

Publication number: CN113528315A
Application number: CN202110885962.XA
Authority: CN
Inventors: 祝天宇; 李征; 薛诚; 朱娟; 郑子伟; 原霖
Original assignee: Beijing Lindian Weiye Electronic Technology Co ltd
Current assignee: Beijing Lindian Weiye Electronic Technology Co ltd
Priority date: 2021-08-03
Filing date: 2021-08-03
Publication date: 2021-10-22

Abstract

The invention provides a method for simulating a gene amplification standard curve, which is used for detecting and calibrating a real-time fluorescence quantitative PCR instrument, wherein the standard device comprises a detection end and a circuit board, and the detection end comprises a standard light source; starting the real-time fluorescent quantitative PCR instrument and the standard device, setting a corresponding qPCR program and a calibration test program, and putting the standard device into the real-time fluorescent quantitative PCR instrument; operating the qPCR program, and acquiring temperature data of a heating module of the real-time fluorescent quantitative PCR instrument by a detection end; the standard device controls the luminance according to the temperature data acquired by the calibration test program and the preset luminance proportion, so that the luminance change of the fluorescent group amplification reaction is simulated; the brightness change of the standard device is detected by a real-time fluorescence quantitative PCR instrument to generate detection data simulating the fluorescent group amplification reaction, and finally, a standard curve is drawn according to the detection data.

Description

Method for simulating gene amplification standard curve

Technical Field

The invention relates to the technical field of real-time fluorescent quantitative PCR instrument detection, in particular to a method for simulating a gene amplification standard curve, and a matched standard device for molecular diagnosis and detection is adopted. The invention also relates to a molecular diagnosis and detection instrument, in particular to a matching calibration device of a real-time fluorescence quantitative PCR instrument, belongs to a molecular biological information analysis and processing system in the molecular diagnosis and detection instrument, and also relates to various reagents and test paper for in-vitro diagnosis in the in-vitro diagnosis and detection instrument, matching equipment and consumables thereof.

Background

The real-time fluorescent quantitative PCR instrument is used for monitoring the fluorescence of the cyclic process, a computer connected with real-time equipment collects fluorescence data, and the data is displayed in a standard curve form through developed real-time automatic analysis software. The real-time fluorescent quantitative PCR instrument mainly adopts an external standard curve method to carry out quantitative analysis on a specific DNA sequence in a sample to be detected. The current real-time fluorescence quantitative PCR instrument is widely applied to the fields of gene expression research, transgene research, gene polymorphism research, drug efficacy assessment, pathogen detection and the like.

The temperature field module and the optical system of the real-time fluorescence quantitative PCR instrument need to realize the calibration of the instrument parameters before the gene amplification reaction and the real-time monitoring in the reaction process through monitoring.

At present, physical monitoring of a temperature field part of a real-time fluorescence quantitative PCR instrument can be realized, but a light path system of the real-time fluorescence quantitative PCR instrument mainly adopts a biochemical method and lacks a perfect physical method. Common biochemical methods are: the fluorescence intensity precision, the sample precision, the fluorescence linear correlation and the sample linear correlation of the test plate are detected by adopting a biological reagent test plate or plasmid DNA standard substances, ribonucleic acid standard substances and the like provided by manufacturers. This biochemical approach has several common problems: (1) the detection in this way is essentially a comparison and judgment of the detection result of the instrument itself, and the process and related parameters cannot be traced back, and it cannot be judged whether the deviation of the result is caused by the temperature control system or the optical path system. The influence relationship between the temperature and the fluorescence of the real-time fluorescence quantitative PCR instrument on the final quantitative result, the error amount, the error source and the like cannot be explained. (2) The combined linearity between the wells can be measured only, and the obtained result only represents the average result between the wells of the fluorescence quantitative PCR instrument and cannot represent the linearity of a single well. (3) Has no direct parameter traceability. (4) The adopted reagent and standard substance belong to consumables, and can only be used once, so that the long-term use cost is high; meanwhile, the reagent and the standard substance generally need to be stored in an environment of-80 ℃ to keep the characteristics of the reagent and the standard substance unchanged, and once taken out, the reagent and the standard substance need to be used and cannot be repeatedly frozen and thawed.

For the calibration of the metering performance of a module-heated Polymerase Chain Reaction (PCR) analyzer, a clear specification is made in JJF1527-2015 polymerase chain reaction analyzer calibration specification and a pharmaceutical industry specification YY/T1173-. For the detection of the optical path system, the specification explicitly describes that the sample linearity and fluorescence linearity are detected by performing gradient dilution by using a plasmid DNA standard substance or a fluorescent dye standard substance. The specification requires that sample linearity is linear regression of the logarithm of the concentration and Ct value of amplified Ct value of standard substance (at least 5) in serial dilution, and the linear regression coefficient is calculated. The current standard generally dilutes the standard substance with a concentration of S1-S77 gradients, which may be 5 or 6, each gradient having multiple replicate wells (e.g., 6 wells), and then the average Ct value of the 6 replicate wells is used as the Ct value result of the concentration. NTC is a negative control, i.e., no initial DNA, the fluorescence signal of the whole process is 0 or very low in theoretical value, and the emphasis is that no change occurs. When the standard substance is used for concentration dilution, certain errors are certainly introduced by manual operation, and the error value is possibly larger, so that the final detection result is directly influenced.

Disclosure of Invention

The present disclosure is directed to overcoming, at least in part, the deficiencies of the prior art by providing a method of simulating a gene amplification standard curve for use in the detection and calibration of a real-time fluorescence quantitative PCR instrument.

The present disclosure is also directed to a method for simulating a gene amplification standard curve, which is used for simultaneously detecting and calibrating a light path system and a temperature field of a real-time fluorescence quantitative PCR instrument by a physical method.

The present disclosure is also directed to provide a method for simulating a gene amplification standard curve, which overcomes the problem that duplicate wells must be set during calibration of a standard sample, and the average value is calculated by using duplicate well detection.

The present disclosure is also directed to provide a method for simulating a gene amplification standard curve, which can effectively reduce the use cost of biochemical methods, and simultaneously overcome the problems that calibration materials cannot be reused and the requirement on storage conditions is high.

In order to achieve one of the above purposes, the present disclosure provides the following technical solutions:

a method of simulating a gene amplification standard curve for use in the detection and calibration of a real-time fluorescent quantitative PCR instrument, the method comprising: constructing a standard device which comprises a circuit board and a plurality of detection ends, wherein the detection ends are used for collecting temperature and simulating the luminous brightness of a fluorescent group, and each detection end comprises a standard light source, a temperature probe and a shell; starting the real-time fluorescent quantitative PCR instrument and the standard device, selecting the arrangement of the standard device, setting a qPCR program matched with the real-time fluorescent quantitative PCR instrument, setting a calibration test program matched with the standard device, and putting the standard device into the real-time fluorescent quantitative PCR instrument; operating the qPCR program, collecting the temperature data of a heating module of the real-time fluorescent quantitative PCR instrument by a detection end, and transmitting the data to standard device software; the standard device controls the brightness of the emitted light according to a preset light proportion of temperature data acquired by a calibration test program, so that the brightness change of the fluorescent group amplification reaction is simulated; a calibration test program for simulating the brightness change of the fluorescent group amplification reaction is operated until the brightness of the standard light source is 100%, and the simulation is closed; the luminous brightness of the standard light source is detected by a real-time fluorescence quantitative PCR instrument, and simulated fluorescence detection data is generated; and drawing a standard curve according to the detection data output by the real-time fluorescence quantitative PCR instrument.

According to a preferred embodiment of the present invention, the standard device is arranged in an arrangement in which the number of the plurality of detection ends is in a range of at least 5 and at most not more than the number of plate wells of the maximum configuration plate type of the real-time fluorescence quantitative PCR instrument.

According to a preferred embodiment of the present invention, the arrangement of the selection standard device is selected according to different concentrations of simulated target standard samples, and at least 5 concentration gradients are simulated, wherein each concentration gradient is simulated for 1 time or multiple times; the standard sample includes standard substances, quality control substances, sample-type standard samples, and the like.

According to a preferred embodiment of the present invention, when each concentration gradient of the target standard sample is simulated for 1 time, the arrangement of the standard device is provided in a plurality of types; each concentration gradient of the target standard sample is simulated for multiple times, and the arrangement of the standard device is also multiple.

According to a preferred embodiment of the present invention, when each concentration gradient of the target standard sample is simulated for a plurality of times, the deviation of the data detected for a plurality of times reflects the performance deviation of the hole in which the deviation data is located.

According to a preferred embodiment of the present invention, the qPCR program is set according to a sample type of the simulation standard sample, and the calibration test program is set according to the sample type of the simulation standard sample and the arrangement of the selection standard device.

According to a preferred embodiment of the present invention, the calibration test procedure is to realize the simulation of the gene amplification standard curve of the standard samples with different concentrations by controlling the variation of the light-emitting brightness of the standard light source according to the variation of the fluorescence signal intensity of the amplification reaction of the standard samples with different concentrations.

According to a preferred embodiment of the present invention, the arrangement of the standard device includes setting an unknown sample with a certain concentration or not setting an unknown sample with a certain concentration.

According to a preferred embodiment of the present invention, the standard device is arranged and configured to include a negative control, or not to include a negative control.

According to a preferred embodiment of the present invention, when the negative control is set, the calibration test procedure corresponding to the standard device of the well has no change in simulated brightness all the time, and is always 20%.

According to a preferred embodiment of the present invention, the sensing end comprises a standard device, a temperature probe and a housing; the shell is wrapped on the outer side of the standard light source and made of a light-transmitting material; the standard light source can be traced to national optical standards in China, and has traceability; the temperature probe consists of a thermistor element and a metal shell wrapped outside; the thermistor element and the metal shell are fixed through heat-conducting sealant; the shell made of the light-transmitting material is connected with the metal shell through threads; the detection end is fixedly connected with the circuit board through screws; a light-transmitting point with the diameter of 2-3mm is arranged at the joint of the detection end and the circuit board; the material of the light-transmitting points can be consistent with that of the shell wrapping the standard light source, and can be PE light-transmitting material, porous organic silicon material or acrylic material. According to the standard device used in the invention, the position settings of the standard light source and the light-transmitting point are not limited to the positions described above, and the adjustment can be made according to the actual situation so as to meet the purposes of accurately controlling the light-emitting brightness of the standard light source and detecting the light-emitting brightness.

According to a preferred embodiment of the present invention, the emission light of the standard light source is mixed light with a spectral range of 320-780 nm; the standard light source is matched with a filter with fixed wavelength, and can emit standard light with fixed wavelength; the detection range of the thermistor element of the temperature probe is 0-120 ℃, and the accuracy can be +/-0.05 ℃ by a five-point six-section correction method.

According to a preferred embodiment of the present invention, the detection end is attached to a PCR reaction well of a real-time fluorescence quantitative PCR instrument.

The invention has the beneficial effects that:

the invention provides a method for simulating a gene amplification standard curve, which adopts a standard light source to simulate a fluorescent group amplification reaction, and specifically comprises the steps of simulating the fluorescent group amplification reaction by carrying out program control on the brightness of the standard light source, generating a detection result after the change provided by a standard device is collected by a real-time fluorescent quantitative PCR instrument, analyzing the detection result and comparing the detection result with the result data of the standard device, and detecting whether a light path system of the real-time fluorescent quantitative PCR instrument is accurate and reliable.

According to the method, the brightness change of the standard light source is controlled according to the temperature change collected by the temperature field of the real-time fluorescence quantitative PCR instrument as a control signal, the detection result is analyzed, so that the result detection can be carried out on the temperature field and the light path system of the instrument, the deviation of the measurement result can be analyzed whether the deviation is caused by the temperature field or the light path system of the real-time fluorescence quantitative PCR instrument, the influence relationship of the temperature and the fluorescence of the real-time fluorescence quantitative PCR instrument on the final quantitative result, the error quantity, the error source and the like are explained, and the correlation influence relationship on the final quantitative result is given.

The constructed standard device belongs to a small instrument, does not belong to consumables relative to consumables such as reagents, standard substances and the like, can be used for a long time after being purchased once, is convenient to use, and has low long-term use cost; meanwhile, the instrument equipment has low requirement on storage environment, does not need to be placed in low-temperature storage instrument equipment such as a refrigerator and the like, does not have shorter effective period and stricter storage conditions similar to standard substances, can be repeatedly used, and can reduce the influence of human errors besides avoiding the inherent defects existing in the biochemical detection.

According to the method, the standard light source has international standards and national standards, the specific problem that direct parameters of the standard light source used for simulating the brightness change of the gene amplification standard curve can be traced is solved, and the method has tracing performance.

According to the method, the standard device can only comprise one detection end, so that the problem of linearity of a single hole of a fluorescence quantitative PCR instrument can be solved, and repeated hole detection is not needed to calculate an average value.

The invention provides a novel method for simulating a gene amplification standard curve, which can realize correlation simulation of fluorescent signal changes of four periods in a standard PCR amplification curve, and further can collect background noise and signal-to-noise ratio and simulate the whole amplification process.

Specifically, the method for simulating a gene amplification standard curve according to the present invention, wherein the control of the emission luminance of the standard light source is performed by correlation simulation based on the change of the fluorescent signal of the fluorescent group in the amplification reaction. In the amplification reaction process, the real-time fluorescent quantitative PCR instrument collects the fluorescent signal once in each cycle, and in a standard PCR amplification curve, the change of the fluorescent signal can be divided into four stages, namely a baseline stage, an exponential amplification stage, a linear amplification stage and a platform stage. The brightness change of the standard light source is subjected to correlation simulation on the fluorescent signal change in the four periods. In a baseline period, the PCR reaction is in an initial stage, the amplification product is few, and the generated fluorescence signal is very low, which belongs to the system background condition; considering that in the baseline period, the amplification product can also generate a lower fluorescence signal, although the fluorescence detection threshold of the fluorescence quantitative PCR is not reached, the fluorescence detection threshold still changes, and the standard light source can also simulate the signal-to-noise ratio process; in the exponential amplification period, the PCR product is exponentially multiplied after each cycle until a stable linear amplification stage of the linear amplification period is reached, the PCR product is increased in geometric multiple after each cycle, and the brightness of the standard light source successively simulates the change of the exponential and the geometric multiple according to the change of a set fluorescence signal; when the stage is reached, the fluorescence signal of the PCR product reaches the highest value, and the brightness of the standard light source also reaches 100%.

In the whole fluorescent quantitative PCR reaction process, the method for simulating the gene amplification standard curve realizes the simulation of a fluorescent acquisition background noise acquisition process, a fluorescent signal-to-noise ratio process, a fluorescent group exponential amplification process, a fluorescent group linear amplification process and a fluorescent group end point amplification process. Therefore, the method can not only trace the amplification process and related parameters of the fluorescence quantitative PCR instrument and judge whether the deviation of the amplification result is caused by a temperature control system or a light path system, but also analyze the influence relationship of the temperature and the fluorescence of the real-time fluorescence quantitative PCR instrument on the final quantitative result, the error amount, the error source and other specific numerical values.

The Ct value is a key parameter of the real-time fluorescence quantitative PCR instrument, and the number of amplification cycles which pass when the fluorescence signal of an amplification product reaches a fluorescence detection threshold value in the PCR amplification process is the Ct value; when the next cycle after the cycle of the baseline period exceeds the fluorescence detection threshold (for example, the fluorescence signal intensity corresponding to the setting of the luminous brightness of the standard light source to be 20% is the fluorescence detection threshold), the current standard cycle number, namely the standard Ct value, is compared and analyzed with the Ct value result generated by the real-time fluorescence quantitative PCR instrument, so that the accuracy and reliability of the optical path system of the real-time fluorescence quantitative PCR instrument can be analyzed.

The invention provides a new method for simulating a gene amplification standard curve, in particular to a physical method for calibrating a temperature field and a light path system of a real-time fluorescence quantitative PCR instrument, which can be used for comprehensively analyzing performance data and can comprise the following steps: ct value indicating error, Ct value uniformity, Ct value precision, channel peak height consistency and linear sensitivity coefficient, and the temperature analysis comprises temperature accuracy, inter-hole temperature difference, temperature overshoot, temperature rising and falling rate and the like.

The invention provides a new method for simulating a gene amplification standard curve, wherein the brightness change of a simulated fluorescent group amplification reaction is realized by controlling the luminous intensity of a standard light source; methods of controlling the luminous intensity may include voltage control, current control, duty cycle control. The standard device is provided with a system power supply for voltage division, the system power supply has small text wave and small interference, stable power output of the standard light source is ensured, accurate control of the brightness of the standard light source in the detection end can be realized, the voltage division control precision can reach 10 microvolts and can be accurate to 1 microvolts at most, and therefore the absolute brightness resolution ratio for controlling the brightness of the standard light source can reach one ten-thousandth of the absolute brightness.

The invention also provides a method for simulating the gene amplification standard curve, wherein the calibration test program setting comprises a baseline period, an exponential amplification period and a linear amplification period for simulating the amplification reaction of the standard samples with different concentrations, and the calibration test program setting can be stopped until the brightness for simulating the amplification reaction of the standard samples with different concentrations reaches 100%.

The method for simulating the gene amplification standard curve needs to select the arrangement of different standard devices containing a plurality of detection ends. The arrangement of the standard devices with different numbers of detection ends is selected according to different concentrations of simulated target standard samples, at least 5 concentration gradients are simulated, and each concentration gradient is simulated for 1 time or for multiple times. Each concentration gradient is simulated for 1 time, and the simulated hole positions can be arranged differently; each concentration gradient of the target standard sample is simulated for multiple times, and the arrangement of the standard device can be different due to different simulation times and different hole site arrangement. Each concentration gradient is simulated for multiple times, and the theoretical values of multiple detection data are consistent; the multiple detection data are deviated, and the performance deviation of the hole where the deviation exists is reflected. The arrangement of different standard devices comprises setting unknown samples with determined concentration or not setting unknown samples with determined concentration; the arrangement of different standard devices also comprises the arrangement of negative controls, or no negative controls. When negative control is set, the whole course of the calibration test program corresponding to the standard device of the hole has no change of simulated brightness, and the theoretical value is 0 or very low luminous brightness. We did not let it be 0 when simulating, and let it be 20% all the time since 0 would not be detected. There will typically be a low background light. Therefore, the method for simulating the gene amplification standard curve is more definite and targeted.

The method for simulating the gene amplification standard curve provided by the invention can replace the application of plasmid DNA standard substances, ribonucleic acid standard substances or dye standard substances and the like in calibration standards and industrial standards, and can also be used for detecting sample linearity and fluorescence linearity.

The method for simulating the gene amplification standard curve comprises the step of using the standard device, wherein the standard device comprises a detection end and a circuit board. The detection end comprises a standard light source, a temperature probe and a shell. One end of the detection end is a standard light source, and the other end is a temperature probe. The outer side of the standard light source is wrapped with a shell made of light-transmitting materials; the standard light source adopts an LED cold light source, and other light sources can also be adopted; the spectral range of the standard light source comprises the emission wavelengths of various fluorophores, such as FAM, SYBR GREEN, CY-3, JOE, VIC, TAMRA, ROX, TEXAS RED, CY-5 and the like. The emission light of the standard light source can be mixed light with a spectral range of 320-780nm, and can also be used with a filter with a fixed wavelength, so that the standard light with the fixed wavelength is emitted, and the emission wavelengths of different fluorescent groups are adapted, for example, when the fluorescent marker is FAM, the emission light of the standard light source selects the mixed light and is used with the filter with 520 nm; the standard light source can also select a special standard light source device of a certain fluorescent group; the standard light source can be traced to national optical standards in China, and has traceability; the light-transmitting material can be PE light-transmitting material, porous organic silicon material, acrylic and the like, and can enable the light source to emit light stably and uniformly. The temperature probe consists of a thermistor element and a metal shell wrapped outside, the detection range of the thermistor element is 0-120 ℃, and the precision can be +/-0.05 ℃ by a five-point six-section correction method; the thermistor element and the metal shell are fixed through heat-conducting sealant; the metal shell can be made of materials with relatively high heat conductivity coefficient, such as red copper gold plating, pure aluminum, pure copper, pure gold and the like, particularly metal materials with high heat conductivity coefficient, and the heat conductivity coefficient lambda is more than 90W/m.K; the shell of the transparent material wrapping the standard light source is fixedly connected with the metal shell wrapping the thermistor element through threads. The detection end is jointed with a PCR reaction hole of the real-time fluorescence quantitative PCR instrument so as to collect real temperature field data.

The detection end is fixedly connected with the circuit board through screws, a light transmission point which can enable light of the standard light source to pass through is arranged at the joint, the diameter of the light transmission point is 2-3mm, and the material of the light transmission point can be consistent with that of a light transmission material wrapped outside the standard light source, so that the light source can be uniformly and stably emitted and collected by a light path system of the detected real-time fluorescence quantitative PCR instrument.

The circuit board can be a PCB board or an FPCB board; a supporting bottom plate is further covered on the circuit board, and the circuit board is fixedly connected with the supporting bottom plate; the supporting bottom plate is preferably a carbon fiber bottom plate; the circuit board is also provided with a wireless communication module, and the wireless communication module is connected with the control circuit; the wireless communication module adopts a Bluetooth communication protocol, and can also adopt other wireless communication modes or wired communication modes, and is used for establishing communication connection with a PC end or an APP end; in the circuit design of the circuit board, the brightness change of the standard light source is controlled by voltage, and in order to realize the accurate brightness change and the linear satisfaction condition of the standard light source, namely the absolute brightness resolution of the standard light source is guaranteed to reach one ten-thousandth of the absolute brightness, the voltage control sets a system power supply to carry out voltage division control, so that the voltage division control precision at least reaches 10 mu V, and the setting of the system power supply realizes small voltage ripple, high stability and high output precision; the control method of the structural circuit to the standard light source brightness can also adopt control modes such as current, duty ratio and the like. Different control methods are set on the circuit board, and the aim of ensuring the absolute brightness resolution of the standard light source to reach one ten-thousandth precision is taken into consideration.

The detection end of the standard device can be one, the linearity of the standard light source is larger than 0.998 within the range of 10% -100% of brightness, the linearity can be corrected, and the linearity is larger than 0.999 through correction. When one detection end is arranged, the temperature field calibration and the linear analysis of the light path system can be realized in the single-hole amplification process of the real-time fluorescence quantitative PCR instrument; the standard device can be directly realized in a single-hole mode, and has higher accuracy and pertinence. The standard device also comprises a plurality of detection ends, the plurality of detection ends are fixed on a circuit board according to a certain arrangement to be used as a set of standard device, and the number of the plurality of detection ends can be 2, 3, 4, 5, 6 or 7 … …; the number of the detection ends does not exceed the maximum value of the plate hole number of the plate type matched with the real-time fluorescent quantitative PCR instrument at most; when a plurality of detection ends are used, the standard light source can simulate light emission according to the same control program and can also simulate light emission according to different control programs; the multiple detection ends can simulate the change of fluorescence of samples with different initial concentrations, simulate luminescence through different calibration test programs, and can quantitatively analyze the sample concentration of the initial template based on the linear relation with the amount of each cyclic amplification product in the qPCR program; when a plurality of detection ends are used, the linear regression coefficient of the drawn standard curve can be infinitely close to 1, so that a more ideal and accurate Ct value and standard curve can be obtained, and unnecessary deviation or error caused by samples, enzymes and various objective or artificial reasons in the traditional biochemical method is avoided; when a plurality of detection ends simulate light emission according to different control programs, different control programs can be designed according to different requirements and purposes, and the pertinence is more definite; when a plurality of detection ends simulate samples with the same concentration, the detection result can reflect the difference value of each corresponding hole, the average value is not calculated, and the detection hole target is more definite; the use mode of a plurality of detection ends is more flexible.

In addition, the standard device used by the method for simulating the gene amplification standard curve is a device for detecting and calibrating the real-time fluorescence quantitative PCR instrument by using the method, can be used as a small instrument for repeated use, saves the consumption of the traditional standard consumable reagent and the standard sample, has low requirement on the storage condition of the physical instrument, is convenient to use, and can overcome the inherent problems and defects of the biochemical method in the detection of the optical path system of the real-time fluorescence quantitative PCR instrument.

Drawings

FIG. 1 is a block diagram of an embodiment of a method for simulating a fluorophore-mediated amplification reaction according to the present invention;

FIG. 2 is a block diagram of an amplification reaction scheme according to the present invention with a simulated Ct value of 19;

FIG. 3 is a block diagram of an amplification reaction scheme according to the present invention with a simulated Ct value of 23;

FIG. 4 is a schematic diagram of a single detection end of a standard device;

FIG. 5 is a schematic view of the overall structure of the housing of the light-transmitting material wrapped outside the standard light source at a single detection end of the standard device;

FIG. 6 is a schematic diagram of an appearance structure of a standard device with 15 detection terminals and a matched external power box;

FIG. 7 is an amplification curve of the real-time fluorescence quantitative PCR instrument according to the process of FIG. 2;

FIG. 8 is a standard curve calibrated and drawn by a gene mutation standard substance for a real-time fluorescent quantitative PCR instrument;

FIG. 9 is an ideal result graph of a standard curve drawn by a standard device with 7 detection ends simulating 6 concentration arrangements;

FIG. 10 is a standard curve plotted against African swine fever pseudovirus standard substance;

FIG. 11 is a standard curve of a standard device simulating a African swine fever pseudovirus standard substance.

Detailed Description

The inventor of the application creatively discovers a method for simulating a gene amplification standard curve through a large amount of basic research, and is particularly suitable for simultaneously detecting and calibrating a temperature field and an optical system of a real-time fluorescence quantitative PCR instrument. Based on this, preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements. Furthermore, in the following detailed description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the disclosed embodiments of the invention. It may be evident, however, that one or more embodiments may be practiced without these specific details. In other instances, well-known structures and devices are shown in schematic form in order to simplify the drawing.

The invention provides a method for simulating a gene amplification standard curve and a standard device used by the method for monitoring a temperature field and a light path in the reaction process of a real-time fluorescence quantitative PCR instrument.

According to the general concept of the invention, a method for simulating a gene amplification standard curve is provided, the method also relates to a standard device in the using process, the standard device simulates the brightness change of a fluorescent group in an amplification reaction by using a standard light source in the standard device, the standard device can collect the temperature in a porous metal heating module hole of a real-time fluorescence quantitative PCR instrument by introducing a thermistor element and the standard light source, can circularly control the brightness change of the standard light source according to the temperature, so as to simulate the brightness change of the fluorescent group amplification reaction, the change is optically detected by the real-time fluorescence quantitative PCR instrument in real time, and finally, the amplification curve and the standard curve can be drawn by combining the temperature data collected in the module hole and the change data of the brightness of the standard light source through the fluorescence detection data generated by the real-time fluorescence quantitative PCR instrument, the accuracy and sensitivity of the temperature field and the light path system of the real-time fluorescence quantitative PCR instrument can be specifically analyzed through data comparison and the drawn curve.

FIG. 1 shows a flow chart of an embodiment of a method for simulating a fluorophore amplification reaction according to the present invention. In the figure, a real-time fluorescent quantitative PCR instrument is abbreviated as a qPCR instrument, a software system matched with the qPCR instrument is abbreviated as qPCR instrument software, and an operation program corresponding to the qPCR instrument software is abbreviated as a qPCR program; the software matched with and installed in the standard device is standard device software, and the running program corresponding to the standard device software is a calibration test program which comprises a temperature program and an optical program. Firstly, opening a qPCR instrument, and starting qPCR instrument software matched with the qPCR instrument; setting a qPCR program on an operation interface of qPCR instrument software according to actual conditions; the setting of the qPCR program comprises a temperature setting program and an optical setting program, and particularly, the data such as the type of the detected fluorescent marker and the like are required to be set, wherein common fluorescent markers include FAM, SYBR GREEN, CY-3, CY-5, JOE, VIC, TAMRA, ROX, TEXAS RED and the like, for example, SYBR GREEN can be selected; connecting a standard device and matched standard device software through a USB adapter; connecting a standard device and matched standard device software, and activating the standard device; and (3) putting the standard device constructed by the method into a qPCR instrument to be tested, attaching the detection end and the bottom end of a PCR reaction hole of the qPCR instrument, opening the software of the standard device, and starting a calibration test program.

The specific operation flow of the calibration test program is as follows: clicking on qPCR instrument software matched with the qPCR instrument to start, and running a preset qPCR program; when the qPCR instrument is started to operate, a detection end starts to acquire the temperature in a hole of a heating module (block) of the qPCR instrument, and standard device software completes the test and acquisition of the temperature of the heating module of the qPCR instrument through the detection end; meanwhile, the standard light source in the detection end adjusts and controls the light emitting brightness of the standard light source through the standard device software according to the light emitting proportion set by the standard device software corresponding to the temperature cycle data in the preset qPCR program, the qPCR instrument acquires the light change data provided by the standard device to generate a detection result, the detection result is analyzed and compared with the data result recorded by the calibration test program used in the standard device software, and the performance data of the temperature field and the light path system of the qPCR instrument can be obtained through comparison and analysis.

Data recorded by a calibration test program used in standard device software matched with the standard device and data acquired by qPCR instrument software matched with the qPCR instrument can be automatically analyzed through respective software systems, and can also be respectively exported for comparative analysis. The acquisition result of the analog brightness change of the standard device can be analyzed on qPCR instrument software matched with the qPCR instrument to obtain a fluorescence signal value, a curve, a Ct value and the like acquired in the whole amplification process; and inputting the data derived from the qPCR instrument into the matched software of the standard device for comparison and analysis, and generating performance evaluation results such as temperature accuracy, inter-hole temperature difference, temperature overshoot, temperature rise and fall rate, Ct value indicating error, Ct value uniformity, Ct value precision, channel peak height consistency, linear sensitivity coefficient and the like.

It should be noted that: the data collected by the qPCR instrument software and the standard device software can be automatically analyzed through the qPCR instrument software and the standard device software, and can also be respectively exported, and then manually sorted and contrastively analyzed after exporting. Whether the system analysis or the manual analysis is adopted, the related evaluation results such as the accuracy, the analysis error, the error source and the like of the temperature field and the light path of the real-time fluorescence quantitative PCR instrument can be finally obtained through analysis.

The specific implementation scheme of the method for simulating a fluorophore-mediated amplification reaction in a specific amplification reaction procedure is shown in FIG. 2. In fig. 2, a real-time fluorescence quantitative PCR instrument is abbreviated as a qPCR instrument, a real-time fluorescence quantitative PCR is abbreviated as a qPCR, the qPCR instrument is turned on, the standard device constructed by the method and a matched USB adapter are put into the qPCR instrument to be tested, the qPCR software matched with the qPCR instrument is turned on, meanwhile, the standard device software matched with the standard device is also turned on, and a temperature reaction program of the qPCR instrument to be tested is set according to a preset control temperature program; setting the types of the qPCR reaction sample and the fluorescent marker, and the like; the standard device uses temperature as a control signal, in the embodiment, the standard device starts to work by presetting a temperature t1 (such as 30 ℃) as a starting signal through standard device software, and presetting a temperature t2 (such as 85 ℃) and a temperature t3 (such as 60 ℃) as light emitting control signals.

In fig. 2, the pre-simulation phase of the standard apparatus: firstly, when the acquisition temperature of the standard device reaches the starting temperature t1=30 ℃, the standard device receives a starting signal and is started to enter a starting state, the standard device is started, and the corresponding PCR cycle number n =0 at the moment; and the real-time fluorescence quantitative PCR instrument is used for heating or cooling control according to a preset program. Secondly, a t2 is identified, and a t3 counts a cycle is identified, namely when the standard device collects t2=85 ℃ and t3=60 ℃ every time, the real-time fluorescence quantitative PCR instrument completes 1 cycle, the PCR cycle number n = n +1, and the first 3 cycles do not emit light. When n is less than 3, the corresponding PCR cycle number (n + 1) < 4, namely the first 3 PCR cycles, the standard device enters a pre-simulation stage, the relative luminous intensity I =0%, namely the standard light source does not emit light, and the standard device mainly detects the temperature field of the real-time fluorescence quantitative PCR instrument.

In fig. 2, the background noise acquisition process of the standard apparatus: at this stage, PCR amplification is in a baseline stage, although amplification products are amplified exponentially, the generated fluorescence signal is very low and belongs to the system background condition, and a standard device constantly gives a fixed brightness of 20%, so that the process of collecting background noise by fluorescence is simulated. Namely, when the PCR cycle number n is more than or equal to 3 and n is less than or equal to 10 and 3 is less than or equal to n is less than or equal to 10, the PCR cycle number (n + 1) is more than or equal to 4 and less than or equal to 11, namely, when the 4 th to 11 th PCR cycles are carried out, the relative luminous intensity is a fixed value, namely, the relative luminous intensity I = 20%; starting from a simulated 20% brightness, a standard amplification curve for the fluorophores simulated by the method can be plotted until the brightness increases to 100%.

In FIG. 2, the simulated SNR process for the Standard deviceThe PCR amplification is in an exponential amplification stage of the amplification reaction, although the amplification product is amplified exponentially, the fluorescence signal is changed in exponential growth, but the fluorescence signal does not reach the fluorescence detection threshold of the fluorescence quantitative PCR, so that the standard device performs small-amplitude increment on the brightness of each cycle in the cycle (12 th cycle to 18 th cycle), namely, the brightness of each cycle is increased by 0.11%, thereby simulating the signal-to-noise ratio process of the stage. Namely, when the number of PCR cycles n is more than 10, n is less than or equal to 18, 10 is less than n and less than 18, 11 is less than the number of PCR cycles (n + 1) is less than 19, namely, when the 12 th to 18 th PCR cycles begin to simulate the background signal-to-noise ratio part, the relative luminous intensity of each cycle is increased by 0.11 percent, namely, the relative luminous intensity I is I_(n+1)=I_n+0.11%。

In fig. 2, the simulated standard Ct value of the standard device: the cycle is the later stage of the amplification reaction index amplification period, namely the relative luminous intensity given by the standard device is increased by 0.2 percent on the basis of the brightness of the previous cycle, and the fluorescent detection threshold value is reached, I_(n+1)=I_n+0.2%, namely when the PCR cycle number n is more than or equal to 18 and the PCR cycle number (n + 1) =19, namely the PCR cycle number reaches the standard cycle number, namely the Ct value = n +1=19, the relative luminous intensity I = I +0.2%, and at the moment, the relative luminous intensity given by the standard device is compared with the Ct value result generated by the real-time fluorescence quantitative PCR instrument, so that the accuracy and reliability of the optical path system can be analyzed.

In FIG. 2, the simulated fluorophore linearized amplification procedure of the standard set-up: and (3) the next cycle of the standard Ct value is a linear amplification period of the PCR reaction, under an ideal condition, the PCR product is increased in a geometric multiple after each cycle, but the increase of the PCR product has no clear mathematical relation, the brightness of the standard light source simulates the index and the geometric multiple change according to a preset light-emitting ratio until the brightness I =100% is reached, and the PCR amplification reaction enters a plateau period. Namely, when the PCR cycle number n is more than or equal to 19, n is less than 42, n is less than or equal to 19, n is less than or equal to 42, 20 is more than or equal to the PCR cycle number (n + 1) < 43, namely, when the PCR cycles are from 20 to 42, the relative luminous intensity is simulated to be linearly increased according to a set program until I =100%, when the PCR cycle number =42 enters a standard device to simulate the fluorescent group plateau phase amplification, the brightness of the standard light source is kept at I =100%, the PCR product is hardly increased any more, and the fluorescent signal is hardly increased.

In fig. 2, the simulation of the standard device is turned off: after the 42 th PCR cycle is completed, the standard device software matched with the standard device starts a closing process, the standard device is closed, and the brightness I =0% of the standard light source.

The flow of the method for simulating a fluorophore-mediated amplification reaction in another specific amplification reaction procedure is shown in FIG. 3. The starting process and the preset related procedures are similar to those described in fig. 2, and will not be described in detail here.

In fig. 3, the standard apparatus first enters a pre-simulation phase: firstly, when the acquisition temperature of the standard device reaches a starting temperature t1, the standard device is started to enter a starting state after receiving a starting signal, the standard device is started, and the corresponding PCR cycle number n =0 at the moment; and the real-time fluorescence quantitative PCR instrument is used for heating or cooling control according to a preset program. Secondly, a t2 is identified, and a t3 counts a cycle is identified, namely when the standard device collects t2=85 ℃ and t3=60 ℃ every time, the real-time fluorescence quantitative PCR instrument completes 1 cycle, the PCR cycle number n = n +1, and the first 3 cycles do not emit light. The number of PCR cycles (n + 1) < 4, namely the first 3 PCR cycles, the standard device enters a pre-simulation stage, the relative luminous intensity I =0%, namely the standard light source does not emit light, and the standard device mainly detects the temperature field of the real-time fluorescence quantitative PCR instrument.

In fig. 3, the background noise acquisition process of the standard apparatus: at this stage the PCR amplification was in the baseline phase and the standard device was constant giving a fixed brightness of 20% simulating the fluorescent acquisition background noise process. When n is equal to or greater than 3 and equal to or less than 10, namely 4 and equal to or less than the number of PCR cycles (n + 1) and equal to or less than 11, and the 4 th to 11 th PCR cycles, the relative luminous intensity is a fixed value, namely the relative luminous intensity I = 20%; starting from a simulated 20% brightness, a standard amplification curve for the fluorophores simulated by the method can be plotted until the brightness increases to 100%.

In FIG. 3, the simulated SNR process of the standard device is shown, in which the PCR amplification is in the exponential amplification stage of the amplification reaction, and although the amplification product is amplified exponentially and the fluorescence signal is also changed exponentially, the fluorescence signal does not reach the fluorescence detection threshold of the quantitative fluorescence PCR, so thatThe standard device simulated the snr process at this stage in small increments of brightness per cycle (0.11% per cycle) during the cycles of this cycle (12 th-22 th cycles). When 10 < n < 22, 11 < PCR cycle number (n + 1) < 23 is satisfied, i.e., when 12 th to 22 th PCR cycles, the background signal-to-noise ratio portion begins to be simulated, and the relative luminous intensity per cycle increases by 0.11%, i.e., the relative luminous intensity I_(n+1)=I_n+0.11%。

Standard apparatus simulation standard Ct value: the cycle is the later stage of the amplification reaction index amplification period, namely the relative luminous intensity given by the standard device is increased by 0.2 percent on the basis of the brightness of the previous cycle, and the fluorescent detection threshold value is reached, I_(n+1)=I_n+0.2%, when n is larger than or equal to 22 and the PCR cycle number (n + 1) =23 is met at the same time, namely the PCR cycle number reaches the standard cycle number, namely the Ct value = n +1=23, the relative luminous intensity I = I +0.2%, at the moment, the relative luminous intensity given by the standard device is compared with the Ct value result generated by the real-time fluorescence quantitative PCR instrument, and the accuracy and reliability of the optical path system can be analyzed.

The standard device simulates a fluorophore linearization amplification process: and (3) the next cycle of the standard Ct value is a linear amplification period of the PCR reaction, under an ideal condition, the PCR product is increased in a geometric multiple after each cycle, but the increase of the PCR product has no clear mathematical relation, the brightness of the standard light source simulates the index and the geometric multiple change according to a preset light-emitting ratio until the brightness I =100% is reached, and the PCR amplification reaction enters a plateau period. When 23 ≦ n < 42, 24 ≦ PCR cycle number (n + 1) < 43, i.e., when PCR cycles 24 to 42 were performed, the relative luminescence intensity was increased linearly in a simulated manner according to the established program until I =100%, and when PCR cycle number =42 was entered into the standard device to simulate the fluorescent group plateau amplification, the brightness of the standard light source remained I =100%, the PCR product was hardly increased any more, and the fluorescence signal was hardly increased.

It should be noted that: the method for simulating the fluorophore-mediated amplification reaction is described above with reference to fig. 2 and fig. 3, respectively, in the specific implementation process of the specific amplification procedure, the temperature, the Ct value, and the cycle number set in the process are only a specific application example, and do not limit the present simulation method. The control temperature and the cycle number preset in the simulation method can be adjusted according to the practical application example. In the actual simulation process, the modified settings of the specific application data can be performed according to the specific conditions of the type of the fluorescent marker used in the specific PCR reaction, the denaturation or annealing temperature of the sample, the PCR cycle number and the like.

FIG. 4 is a schematic cross-sectional view of a single detection end of a preferred standard device constructed according to the method of the present invention for simulating a fluorophore amplification reaction. Fig. 4 shows that the detection end comprises a standard light source 3, a temperature probe and a housing 2. One end of the detection end is provided with a temperature probe, and the other end is provided with a standard light source 3. And the outer side of the standard light source is wrapped by a shell 2 made of a light-transmitting material. The temperature probe consists of a thermistor element 1 and a metal shell wrapped outside, the detection range of the thermistor element is 0-120 ℃, and the precision can be +/-0.05 ℃ by a five-point six-section correction method; the diameter of the thermistor element is 0.5 mm; the thermistor element and the metal shell are fixed through a heat-conducting sealant 7; the metal shell can be made of high-thermal conductivity coefficient materials such as red copper gold plating, pure aluminum, pure copper and pure gold. The standard light source 3 adopts an LED cold light source, and can also adopt other light sources; the emission light of the standard light source 3 is mixed light, the spectral range is (320-; the standard light source 3 can also select a special light source of a certain fluorescent group; the standard light source 3 can be traced to the national optical standard of China, and has traceability. The shell 2 is made of a light-transmitting material, and the light-transmitting material can be a PE light-transmitting material, and can also be a light-transmitting material which can enable a light source to stably and uniformly emit light, such as a porous organic silicon material, an acrylic material and the like; the shell 2 of the detection end is jointed with a PCR reaction hole of the real-time fluorescence quantitative PCR instrument so as to collect real temperature field data; the height of the single detection end is 21.5 mm. In addition, the lower part of the detection end is also provided with a light transmission point 8 which can enable the light of the standard light source to pass through, the diameter of the light transmission point 8 can be 2-3mm, and the material of the light transmission point can be the same as that of the light transmission material used by the shell 2 of the standard light source, so that the light source can be ensured to be uniformly and stably emitted and collected by a light path system of the detected real-time fluorescence quantitative PCR instrument.

Furthermore, the housing 2 made of the light-transmitting material is connected to the metal housing by means of a thread 4.

FIG. 5 is a schematic view of the overall structure of the housing of the light-transmitting material wrapped outside the standard light source in the single detection end of FIG. 4, which is a schematic view of the three-dimensional structure of the light-transmitting material used in the housing of the single detection end of a preferred standard device constructed by the method for simulating the fluorophore amplification reaction according to the present invention. Fig. 5 is a whole shell structure of the standard device, and the transparent material of the shell 2 for wrapping the standard light source 3 is shown in the figure, and in fig. 5, the upper end of the shell can be seen to comprise a thread 4 structure, and the metal shell for wrapping the thermistor element in the temperature probe at the detection end is connected with the shell made of the transparent material of fig. 5 through the thread 4 structure to form a single detection end.

Fig. 6 is an appearance structure diagram of a standard device with 15 detection terminals and a matched external power box. The simulation method of the present invention requires the use of standard devices, but the standard devices used are arranged in various ways according to implementation purposes. In practical use, the standard device may have one detection end or a plurality of detection ends, and the number of the plurality of detection ends may be 2, 3, 4, 5, 6, and 7 … …. the method for simulating a fluorophore amplification reaction according to the present invention may also be used to simulate a standard gene amplification curve, so when used to simulate a standard gene amplification curve, the standard gene amplification curve simulates a minimum number of 5 detection ends in the standard device because the standard requires at least 5 concentrations of the standard substance.

FIG. 6 shows that the plate contains 15 detection ends, and the number of the detection ends does not exceed the maximum number of the plate wells of the real-time PCR instrument in practical application. For example, if the PCR apparatus is adapted to be a 96-well plate, the number of the detection terminals can be 96 at most, and if the PCR apparatus is adapted to be a 384-well plate, the number of the detection terminals can be 384 at most, and a plurality of detection terminals are fixed on a circuit board in a certain arrangement to be used as a set of apparatus. When a plurality of detection ends are used, the standard light source can simulate light emission according to the same control program and can also simulate light emission according to different control programs; when the plurality of detection ends simulate light emission according to different control programs, each detection end can simulate the change of the fluorescent group in the sample with different initial concentrations, so that analysis is performed through the obtained standard curve.

Fig. 6 shows an arrangement of a standard device with 15 test terminals, from which the thermistor element 1, the housing 2 made of a light-transmitting material, the circuit board 5 and the support base 6 of the standard device are still visible. The detection end shown in fig. 4 is fixedly connected with the circuit board 5 through a screw, and the circuit board 5 is a PCB circuit board. The circuit board 5 is provided with a wireless communication module, the wireless communication module adopts a Bluetooth communication protocol, and can also adopt other wireless communication modes or wired communication modes, and the wireless communication module is used for establishing communication connection with a PC end or an APP end; in the circuit design of the circuit board 5, the brightness of the standard light source 3 is controlled by voltage, in order to realize the accurate brightness change and the linear satisfaction condition of the standard light source 3, namely, the absolute brightness resolution of the standard light source 3 is guaranteed to reach one ten thousandth of the absolute brightness, the voltage control resolution is required to reach 10 μ V, the ripple of the power voltage is required to be realized to be as small as possible, the stability is high, and the output precision is high, and for the purpose, a system power supply is further arranged on the circuit board 5 and used for realizing voltage division. The circuit board 5 shown in fig. 6 is provided with a system power supply for voltage division, wherein the system power supply is a branch power supply installed on the circuit board of the standard device and is used for realizing accurate control of the brightness of the detection end after voltage division. The system has small power supply text wave and small interference, ensures stable power supply output to the standard light source, and can realize accurate control of the brightness of the detection end, the control precision of the voltage division can reach 10 microvolts and can be accurate to 1 microvolts at most, thereby controlling the absolute brightness resolution of the standard light source brightness to one ten thousandth of the absolute brightness. The realization of the precision control of the analog optical equipment by the partial pressure method is the creative invention achievement of the inventor after a great deal of basic research.

The system power supply comprises a single chip microcomputer, an ADC, a DAC and an operational amplifier. The single chip microcomputer is used for controlling the whole system, controlling the ADC, controlling the DAC and controlling the storage. The operational amplifier is used for controlling and driving the LED, and the LED brightness can be accurately controlled due to the low output impedance of the operational amplifier. The function of the ADC is to collect temperature. The DAC is used for controlling the brightness of the LED, and the DAC can achieve accurate analog quantity output. The system power supply has three paths: the power supply of the single chip microcomputer, the power supply of the ADC and the DAC and the power supply of the operational amplifier. In fig. 6, there are 16 channels for 2 DACs, which correspond to 15 light source points (corresponding to 15 detection terminals), and there are 1 remaining DAC. The DAC inputs adjustable analog signals to the operational amplifier, and the operational amplifier gives different voltage changes according to the input signals to control and detect the brightness of the standard light source at the end.

The circuit board 5 may also be provided with a memory, a firmware area, and an analog switch, among other components.

The control method of the circuit board 5 for the brightness of the standard light source 3 can also adopt control modes such as current, duty ratio and the like.

The circuit board 5 is further covered with a supporting bottom plate 6, the circuit board 5 is fixedly connected with the supporting bottom plate 6, and the supporting bottom plate 6 is preferably a carbon fiber bottom plate.

The standard device shown in fig. 6 also shows an appearance structure diagram of a matched external power supply box. In practical application, the standard device in the figure is placed in a real-time fluorescence quantitative PCR instrument, the standard device is connected with a matched external power supply box shown in the figure through a flat cable 11, a power supply 12 is arranged in a main body of the matched external power supply box, and a switch 13 is also arranged on the external power supply box and is mainly used for controlling the on and off of the power supply.

In actual use, the external power supply of the standard device can be supplied with power by a battery, and can also be directly connected with a computer for power supply through a USB interface, the battery can be supplied with power from different power sources, including dry batteries, lead storage batteries or lithium batteries, and also can be different models, including No. 1, No. 2, No. 3, No. 5, No. 7 and the like, for example, the external power supply can be set into No. 3 batteries, No. 7 batteries, No. 2 batteries, and also can be set into button batteries, columnar batteries, square batteries and the like.

FIG. 7 is an amplification curve of the real-time fluorescence quantitative PCR apparatus according to the flow chart of FIG. 3. The amplification curve is a standard amplification curve generated from the standard device simulation data according to the procedure of FIG. 3. In the figure, the abscissa represents the cycle number, and the ordinate represents the fluorescence intensity detected by the real-time fluorescence quantitative PCR instrument corresponding to the standard light source brightness. The standard curve in the figure corresponds to a simulation of the standard light source luminance from I =20% to I =100% for the standard device in the embodiment of fig. 3. The abscissa in the figure represents the number of PCR cycles and the ordinate represents the fluorescence intensity, and it can be seen from the figure that when the number of PCR cycles reaches 23, the abscissa corresponding to the intersection of the standard amplification curve and the dashed line is the standard number of cycles, i.e. Ct value 9, and Ct value = 23. The ordinate corresponding to the dotted line in the figure is the fluorescence detection threshold 10, and the fluorescence detection threshold 10 in the figure is the corresponding fluorescence intensity variation when the standard light source brightness changes from 21% to 21.2%.

Example 1: use of a real-time fluorescent quantitative PCR instrument calibrated with a standard substance.

Experimental materials: EGFR-1 (18-; 19-, 20-; 21-) gene mutation standard substance from Chinese metrological scientific research institute is national grade standard substance, and the standard substance number is GBW (E) 090640. Details are shown in table 1 below.

Table 1: basic information of gene mutation standard substance for real-time fluorescent quantitative PCR instrument

Firstly, preparing a standard substance;

preparation of a calibration Standard for real-time fluorescent quantitative PCR Instrument with a calibrated precision balance (0.00000)1g) Serial preparations of standard substances for real-time fluorescent quantitative PCR instrument calibration are carried out, wherein the copy number concentration of national standard substance (GBW (E)090640) is 1.28X 10¹⁴copy/uL, the concentration is too high compared with the fluorescent quantitative PCR instrument, and the sample concentration of the real-time quantitative PCR instrument is 1.0 multiplied by 10²copy/μL-1.0×10⁸copy/uL. Thus, the standard was formulated to give a marker concentration of 1.28X 10¹⁴copy/uL was used as the initial standard and then it was subjected to gradient dilution to obtain the required various concentration gradients, where S1-S7 are the concentrations of plasmid DNA standard for PCR calibration, U1 and U2 are unknown samples of defined concentrations, and NTC is a negative control, as shown in Table 2 below.

Table 2: standard substance configuration table of different concentration gradients of real-time fluorescence quantitative PCR instrument

Secondly, preparing a PCR reaction system;

comprises a reagent of sterilization double distilled water ddH₂O, 10 XPCR buffer, 25mmol/LMgCl₂dNTPs, 10. mu. mol/L probe, 10. mu. mol/

L primer

1, 10. mu. mol/L primer 2, 5U/. mu.L Taq enzyme, DNA.

Thirdly, arranging and preparing a calibration plate;

the prepared standard substance real-time fluorescent quantitative PCR reaction system for calibration is arranged according to a 96-well plate type, and is respectively added into 96-well PCR reaction tubes, and the specific arrangement is shown in the following table 3.

Table 3: arrangement of standard substance calibration plate for calibrating real-time fluorescent quantitative PCR instrument

Fourthly, carrying out PCR reaction;

the temperature control program for this PCR amplification was set according to the requirements of the national calibration standards, as shown in Table 4 below.

Table 4: temperature control program setting of real-time fluorescence quantitative PCR instrument

And a fifth step of calculating an average value of actual detection values of 6 wells of the same concentration as a result of completion of the PCR reaction, as detailed in Table 5 below.

Table 5: table for summarizing and calculating average value of Ct value of actually measured calibration result of standard substance

The logarithm of the standard substance concentration is used as the abscissa, and the Ct value corresponding to each standard substance is used as the ordinate, to draw a standard curve of the plasmid DNA standard substance gradient dilution, as shown in detail in FIG. 8.

From FIG. 8, R can be obtained²=0.9936, thereby calculating a linear regression coefficient R = 0.9967.

Example 2: application of standard device calibration for real-time fluorescent quantitative PCR instrument

In the first step, different numbers of standard devices are arranged, and since the "calibration specification for polymerase chain reaction analyzer (PCR)", JJF1527-2015, requires at least 5 concentration gradients, it is at least necessary to arrange a standard device having 5 detection ends, and for comparison with the calibration result of the standard substance, the concentration set by different detection ends for simulating the concentration gradients is shown in table 6 below.

TABLE 6 configuration table of standard devices with different concentration gradients of real-time fluorescence quantitative PCR instrument

In addition, in order to fully demonstrate the flexible application of the standard device in the detection standard curve, the following tables 7 to 11 specifically list the arrangement of different numbers of detection ends in the calibration of the real-time fluorescence quantitative PCR reaction body, and the arrangement is exemplified by a 96-well plate type.

TABLE 7 Standard device with 5 detection ends to simulate 5 concentrations of arrangement

Table 8 standard device with 7 detection ends simulates arrangement of 7 concentrations

TABLE 9 Standard device with 7 detection ends to simulate 6 concentration arrangement

TABLE 10 Standard device with 15 detection ports to simulate 7 concentration configurations

TABLE 11 Standard device with 96 detection ports to simulate 7 concentration configurations

In addition, it should be emphasized that, in practical use, the standard apparatus including a plurality of detection terminals can be flexibly adjusted in arrangement according to implementation purposes.

Secondly, carrying out PCR reaction;

since this example is mainly compared with the results of the calibration curve of the standard substance, the temperature control procedure for PCR amplification was set as in Table 4. Correspondingly, standard device software matched with the standard device is set, and the brightness of the standard light source is controlled in a voltage control mode in the PCR reaction process according to the temperature and the corresponding cycle number acquired by the standard device.

It should be noted that the calibration using the standard apparatus is more specific, for example, before a specific amplification experiment is performed, different arrangement types are selected and specific simulated temperatures are set according to the type and purpose of the sample DNA amplified by the actual experiment. Similarly, the control program can be correspondingly adjusted, and different temperature control points can be set, so that the calibration result is more targeted, and the corresponding accuracy is higher.

This example is intended to compare with the standard curve of gene amplification simulated by the standard substance, and therefore the temperature control program for PCR amplification used is the same as that in Table 4. In the practical application process, different amplification programs are set according to the use purposes, for example, the temperature control program for the amplification of the real-time fluorescence quantitative PCR instrument can be set according to the temperature program for controlling the brightness set by the standard device, and the operation is more flexible compared with different use purposes.

And thirdly, drawing a standard curve of the simulated gradient of the standard device by taking the logarithmic values of different concentrations of the standard substance simulated by the standard device as abscissa and the Ct values corresponding to different concentrations of the standard substance simulated by the standard device as ordinate as a result of the PCR reaction. FIG. 9 is a graph showing the ideal results of a standard curve drawn by simulating the arrangement of 6 concentrations according to the standard device having 7 detection ends shown in Table 8, where the results are an ideal value for the simulation, provided that both the temperature field and the optical system of the real-time fluorescence quantitative PCR apparatus are accurate and non-biased.

The result graph of the standard curve is simulated by using a standard device, can be drawn manually according to the actual measurement result of the detected Ct value, and can also be automatically generated by a system of a real-time fluorescence quantitative PCR instrument according to the actual measurement Ct value.

By comparing the ideal result and the actual measurement result of the simulated standard curve, if the ideal result is different from the actual measurement result, the difference can be presumed to be caused by the equipment performance error of the real-time fluorescence quantitative PCR instrument.

From a comparison of example 1 and example 2, the linear theoretical value of the plotted standard curve for the standard set-up should be 1. Of course, if the linearity of the standard curve generated by the final detection of the real-time fluorescence quantitative PCR instrument is not equal to 1, the linearity is inevitably caused by the real-time fluorescence quantitative PCR instrument, possibly caused by the inaccuracy of a light path system, the edge effect and the like, and the linearity can be used for analyzing the performance problem of the instrument. Furthermore, if linearity ≠ 1 is found, it can also be used to evaluate the uniformity of individual detection wells of a quantitative fluorescence PCR machine.

The method has clear requirements in the specification for the analysis process of the fluorescence signal value, the amplification curve, the Ct value, the temperature accuracy, the inter-pore temperature difference, the temperature overshoot, the temperature rising and falling rate, the Ct value indicating error, the Ct value uniformity, the Ct value precision, the channel peak height consistency, the linear sensitivity coefficient and other results and performance evaluation measured by the real-time fluorescence quantitative PCR instrument. The method for simulating the fluorescent group amplification reaction and the method for simulating the gene amplification standard curve are used for analyzing the specific parameter performance, and the specific parameter performance can be analyzed according to the existing specifications.

Because the standard has a definite template of 'calibration original record (reference) format', 'calibration certificate result page (reference) format' and 'uncertainty evaluation example of temperature and Ct value measurement result', the result of physical mode simulation of the standard device is compared and analyzed with the calibration result of the standard substance by the arrangement of the standard device with 7 detection ends similar to table 8, and because of space limitation of the format of the standard analysis result, table 12 below only lists the record and comparison results of 'Ct value indication error, Ct value uniformity and Ct value precision'.

Table 12: ct value indicating error, Ct value uniformity and Ct value precision comparison calibration result

Example 3: the application of the real-time fluorescence quantitative PCR instrument for calibrating the African swine fever pseudovirus standard substance.

Firstly, preparing a standard substance;

the configuration is performed according to the description of the usage instruction of the African swine fever pseudovirus standard sample, and the configuration process is not repeated herein. A total of 5 concentration gradients were set up as detailed in table 13 below.

Table 13: concentration gradient configuration table of African swine fever pseudovirus standard sample

Secondly, preparing a PCR reaction system;

thirdly, arranging and preparing a calibration plate;

the prepared standard sample real-time fluorescent quantitative PCR reaction system for calibration is arranged according to a 96-well plate type, and is respectively added into 96-well PCR reaction tubes, and the specific arrangement is shown in the following table 14.

Table 14: arrangement of calibration plate for African swine fever pseudovirus standard sample

Fourthly, carrying out PCR reaction;

description of the drawings: the PCR reaction system of the second step and the temperature control program of the PCR reaction of the step are specifically set with reference to the instruction book;

in the fifth step, as a result of the completion of the PCR reaction, the average value of the actual detection values of 16 wells having the same concentration is calculated, as detailed in Table 15 below.

Table 15: ct average value of calibration result of African swine fever pseudovirus standard sample

The logarithm of the concentration of the standard sample is used as the abscissa, and the Ct value corresponding to each standard sample is used as the ordinate, to draw a standard curve of the plasmid DNA standard sample gradient dilution, as shown in fig. 10.

From FIG. 10, R can be obtained²=0.9991, thereby calculating a linear regression coefficient R = 0.9995.

Example 4: application of standard device in simulation of African swine fever pseudovirus standard sample

Firstly, arranging standard devices;

contains 5 concentration gradients, and the simulated concentrations of the different concentration gradients are detailed in table 13 above. And the arrangement of the standard devices in the upper table 5 is selected.

In practical use, the standard device with a plurality of detection ends can be flexibly adjusted according to implementation purposes in arrangement and arrangement.

Secondly, carrying out PCR reaction;

it should be noted that, in this example, the purpose is to compare with the application of calibration of the African swine fever pseudovirus standard sample, and the temperature control program of the PCR reaction is specifically set up by referring to the standard sample specification as in example 3, therefore, it is not described in detail.

And step three, obtaining the result after the PCR reaction is finished.

The corresponding Ct values obtained by simulating the African swine fever pseudovirus standard sample by the standard device are shown in the following table 16:

table 16: ct value of standard device for simulating African swine fever pseudovirus standard sample

Finally, the logarithmic values of the different concentrations of the standard sample simulated by the standard device are used as the abscissa, the Ct values corresponding to the different concentrations of the standard sample simulated by the standard device are used as the ordinate, the standard curve of the simulation gradient of the standard device is drawn, and fig. 11 is a standard curve diagram of the standard device for simulating 5 concentrations of the african swine fever pseudovirus standard sample according to the standard device in table 16.

As can be seen from the comparison between example 3 and example 4, the linearity of the standard curve of the calibration of the standard sample is not equal to 1, which indicates that certain errors exist in the uniformity of each detection well of the quantitative fluorescence PCR instrument.

In the actual detection of the African swine fever, the method of example 4 can be used for simulating a standard curve of an African swine fever plasmid DNA standard sample in advance, calibrating and quality control detection is carried out on a real-time fluorescence quantitative PCR instrument, and then the real-time fluorescence quantitative PCR instrument which is calibrated and quality controlled is used for detecting the African swine fever sample. Therefore, the standard curve is drawn by using the standard device more pertinently, and the practical application purpose is more clear.

The invention provides a method for simulating a gene amplification standard curve, which simulates the change of a fluorescent group in an amplification reaction by a standard light source, and provides a standard device.

The method for simulating the gene amplification standard curve and the constructed standard device can detect whether the light path system of the real-time fluorescence quantitative PCR instrument is accurate and reliable. Standard gene amplification procedures can be detected. Meanwhile, the brightness change control of the standard light source in the standard device is controlled by the temperature signal provided by the real-time fluorescence quantitative PCR instrument temperature field, and the deviation of the measurement result can be analyzed by analyzing the detection result, whether the deviation is caused by the real-time fluorescence quantitative PCR instrument temperature field or the light path system, and the correlation is given. The standard light source can be traced to the national optical standard of China, and has traceability. The physical device can be used as a standard device for repeated use, and the consumption of reagents and standard samples is saved.

The invention also provides a method for simulating the gene amplification standard curve, which can be used for replacing the existing standard that plasmid DNA standard substances, ribonucleic acid standard substances or dye standard substances are used for carrying out sample linearity and fluorescence linearity detection; the standard curve of the standard sample gradient dilution of various DNA samples can be simulated, and the linear detection or quality control can be carried out on the fluorescent quantitative PCR instrument; the method can simulate the standard curves of the standard substances of various DNA samples and carry out calibration test on the equipment in a targeted manner; the standard device can be used as instrument equipment for repeated use, the requirement on storage conditions is low, and the use is convenient, so that the consumption of reagents and standard samples is saved, and the cost is reduced; in addition, the method for simulating the gene amplification standard curve by using the standard device is directly realized by a single-hole mode, is not an average value, and has more accuracy and pertinence.

While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and variations of the specific details may be made in light of the overall teachings of the disclosure, and such variations are within the scope of the invention. The scope of the invention is to be determined primarily by the following claims and their equivalents.

Claims

1. A method of simulating a gene amplification standard curve for calibrating a real-time fluorescent quantitative PCR instrument, the method comprising:

constructing a standard device which comprises a circuit board (5) and a plurality of detection ends, wherein the detection ends are used for collecting temperature and simulating fluorescence group luminous brightness, and each detection end comprises a standard light source (3);

starting the real-time fluorescent quantitative PCR instrument and the standard device, selecting the arrangement of the standard device, setting a qPCR program matched with the real-time fluorescent quantitative PCR instrument, setting a calibration test program matched with the standard device, and putting the standard device into the real-time fluorescent quantitative PCR instrument;

operating the qPCR program, collecting the temperature data of a heating module of the real-time fluorescent quantitative PCR instrument by a detection end, and transmitting the data to standard device software;

the standard device controls the brightness of the emitted light according to a preset light proportion of temperature data acquired by a calibration test program, so that the brightness change of the fluorescent group amplification reaction is simulated;

a calibration test program for simulating the brightness change of the fluorescent group amplification reaction is operated until the brightness of the standard light source is 100%, and the simulation is closed;

the luminous brightness of the standard light source (3) is detected by a real-time fluorescence quantitative PCR instrument, and simulated fluorescence detection data is generated;

and drawing a standard curve according to the detection data output by the real-time fluorescence quantitative PCR instrument.

2. The method of modeling a gene amplification standard curve according to claim 1, wherein: the standard device is arranged, wherein the number of the plurality of detection ends is in a range of at least 5, and at most, the number of the detection ends does not exceed the plate hole number of the maximum configuration plate type of the real-time fluorescence quantitative PCR instrument.

3. The method of modeling a gene amplification standard curve according to claim 1, wherein: the arrangement of the selection standard device is selected according to different concentrations of simulated target standard samples, at least 5 concentration gradients are simulated, and each concentration gradient is simulated for 1 time or multiple times.

4. The method of modeling a gene amplification standard curve according to claim 3, wherein: when each concentration gradient of the target standard sample is simulated for 1 time, the arrangement of the standard device is provided with a plurality of types; each concentration gradient of the target standard sample is simulated for multiple times, and the arrangement of the standard device is also multiple.

5. The method of modeling a gene amplification standard curve according to claim 3, wherein: and when each concentration gradient of the target standard sample is simulated for multiple times, detecting the deviation of data for multiple times, and reflecting the performance deviation of the hole in which the deviation data is positioned.

6. The method of modeling a gene amplification standard curve according to claim 1, wherein: the qPCR program is set according to the sample type of the simulation standard sample, and the calibration test program is set according to the sample type of the simulation standard sample and the arrangement setting of the selection standard device.

7. The method of modeling a gene amplification standard curve according to claim 1, wherein: the calibration test program realizes the simulation of the gene amplification standard curve of the standard samples with different concentrations by controlling the change of the luminous brightness of the standard light source (3) according to the change of the fluorescence signal intensity of the amplification reaction of the standard samples with different concentrations.

8. The method of modeling a gene amplification standard curve according to claim 7, wherein: the arrangement of the standard device comprises setting an unknown sample with determined concentration or not setting the unknown sample with determined concentration.

9. The method of modeling a gene amplification standard curve according to claim 1, wherein: the arrangement of the standard device comprises the arrangement of a negative control or no negative control.

10. The method of modeling a gene amplification standard curve according to claim 9, wherein: when the negative control is set, the whole course of the calibration test program corresponding to the standard device of the hole has no change in simulated brightness, and the whole course is always 20%.