Method for determining fluorescent calibration template and fluorescent calibration method of nucleic acid detector

Abstract

A method for determining a fluorescent calibration template and a fluorescent calibration method of a nucleic acid detector, the determining method comprising: preparing a first fluorescent reagent with a first fluorescent value; detecting the fluorescent value and waveband of the first fluorescent reagent by using a to-be-calibrated instrument, and calibrating the fluorescent value of the to-be-calibrated instrument as the first fluorescent value, the to-be-calibrated instrument comprising a plurality of detection holes; selecting a plurality of fluorescent plates corresponding to the waveband of the first fluorescent reagent detected by the to-be-calibrated instrument; processing the plurality of fluorescent plates so that each fluorescent plate comprises a plurality of fluorescent detection parts for cooperating with the detection holes; detecting the plurality of fluorescent detection parts of the same fluorescent plate by using the same detection hole of the to-be-calibrated instrument; and selecting the fluorescent plate whose difference between the fluorescent value of all the fluorescent detection parts and the first fluorescent value is within a preset range as the fluorescent calibration template. The determining method of the present application can calibrate multiple detection holes at the same time, improve the calibration efficiency, and improve the accuracy of fluorescent calibration.

Description

Technical Field

[0001] The present application relates to the field of fluorescence detection technology, and in particular to a method for determining a fluorescence calibration template and a fluorescence calibration method for a nucleic acid detector. Background Art

[0002] Polymerase Chain Reaction (PCR) is a molecular biology technique used to amplify specific DNA fragments. It can be seen as a special DNA replication outside of an organism. The greatest feature of PCR is its ability to significantly increase trace amounts of DNA.

[0003] Real-time fluorescence quantitative PCR technology involves adding a fluorescent group to the PCR reaction system, using the accumulated fluorescence signal to monitor the entire PCR process in real time, and finally using a standard curve to quantitatively analyze unknown templates. Real-time fluorescence quantitative PCR effectively overcomes the limitation of traditional quantitative methods, which are limited to endpoint detection. By measuring the intensity of the fluorescence signal once per cycle and recording it in the software, quantitative results are obtained by calculating the Ct value for each sample based on the standard curve. The Ct value (cycle threshold) refers to the number of cycles required for the fluorescence signal in each reaction tube to reach the set threshold.

[0004] Long-term use of a PCR nucleic acid detector may reduce test accuracy and cause experimental misdiagnosis. To avoid returning the instrument to the factory for repair, users can use a fluorescence calibration device to calibrate the PCR nucleic acid detector.

[0005] In the prior art, when the prepared fluorescence calibration standard template paper is used to calibrate a PCR nucleic acid detector with a multi-hole tube arrangement, if a single standard template paper is used, only single-hole detection can be performed, resulting in low calibration efficiency; or if multiple standard template paper sheets are used for separate detection, the fluorescence values ​​between the multiple standard template paper sheets differ greatly, resulting in low accuracy of fluorescence calibration. Summary of the Invention

[0006] The purpose of this application is to provide a method for determining a fluorescence calibration template and a fluorescence calibration method for a nucleic acid detector, so as to solve the problem of low calibration efficiency of the manufactured fluorescence calibration template.

[0007] To achieve the purpose of this application, this application provides the following technical solutions:

[0008] In a first aspect, the present application provides a method for determining a fluorescence calibration template, wherein the fluorescence calibration template is used to perform fluorescence calibration on an instrument to be calibrated, the determination method comprising:

[0009] preparing a first fluorescent reagent with a first fluorescence value;

[0010] detecting a fluorescence value and a waveband of the first fluorescence reagent by using a to-be-calibrated instrument, and calibrating the fluorescence value of the to-be-calibrated instrument as the first fluorescence value, wherein the to-be-calibrated instrument comprises a plurality of detection holes;

[0011] selecting a plurality of fluorescence plates corresponding to the waveband according to the waveband of the first fluorescence reagent measured by the to-be-calibrated instrument;

[0012] processing the plurality of fluorescence plates so that each of the fluorescence plates comprises a plurality of fluorescence detection parts for cooperating with the detection holes;

[0013] detecting a plurality of fluorescence detection parts of the same fluorescence plate by using the same detection hole of the to-be-calibrated instrument;

[0014] selecting a fluorescence plate whose difference between the fluorescence value of all the fluorescence detection parts and the first fluorescence value is within a preset range as a fluorescence calibration template.

[0015] In an embodiment, the first fluorescence reagent with the first fluorescence value comprises:

[0016] providing a second fluorescence reagent with a second fluorescence value;

[0017] diluting the second fluorescence reagent according to a detection range of the to-be-calibrated instrument to prepare the first fluorescence reagent with the first fluorescence value.

[0018] In an embodiment, the first fluorescence reagent with the first fluorescence value further comprises:

[0019] dispensing the first fluorescence reagent with the first fluorescence value into n continuous tubes, wherein the number of each row of detection holes of the to-be-calibrated instrument is m, and n≤m.

[0020] In an embodiment, the calibration of the fluorescence value of the to-be-calibrated instrument as the first fluorescence value comprises:

[0021] calibrating the fluorescence value of the detection hole of the to-be-calibrated instrument in which the n continuous tubes are placed as the first fluorescence value.

[0022] In an embodiment, the providing of the second fluorescence reagent with the second fluorescence value comprises:

[0023] The second fluorescence reagent comprises one of FAM, HEX, Texas Red, Cy5 and Quasar 705.

[0024] In an embodiment, the selecting of the plurality of fluorescence plates corresponding to the waveband according to the waveband of the first fluorescence reagent measured by the to-be-calibrated instrument comprises:

[0025] The fluorescence plate is an acrylic plate.

[0026] In one embodiment, the plurality of fluorescent plates are processed such that each of the fluorescent plates comprises a plurality of fluorescent detection portions for cooperating with the detection holes.

[0027] Each of the fluorescent plates comprises m / 2 fluorescent detection portions.

[0028] In one embodiment, the plurality of fluorescent detection portions of the same fluorescent plate are detected by the same detection hole of the instrument to be calibrated.

[0029] The plurality of detection holes comprises a first detection hole, a second detection hole, a third detection hole and a fourth detection hole, and the plurality of fluorescent detection portions comprises a first fluorescent detection portion and a second fluorescent detection portion.

[0030] The first fluorescent detection portion and the second fluorescent detection portion are inserted into the first detection hole and the second detection hole, respectively, and the fluorescent value of the second fluorescent detection portion is detected by the second detection hole.

[0031] The fluorescent plate is moved, and the first fluorescent detection portion and the second fluorescent detection portion are inserted into the second detection hole and the third detection hole, respectively, and the fluorescent value of the first fluorescent detection portion is detected by the second detection hole.

[0032] In one embodiment, the fluorescent plate selected from among the fluorescent plates whose difference between the fluorescent value of the fluorescent detection portion and the first fluorescent value is within the preset range comprises:

[0033] The difference between the fluorescent value of the fluorescent detection portion of the selected fluorescent plate and the first fluorescent value divided by the first fluorescent value is less than 0.01.

[0034] In a second aspect, the present application also provides a fluorescent calibration method for a nucleic acid detection instrument, comprising:

[0035] The plurality of fluorescent plates are inserted into the detection holes of the nucleic acid detection instrument, and the fluorescent values of each of the detection holes of the nucleic acid detection instrument are calibrated by the fluorescent plates.

[0036] The method for determining the fluorescence calibration template provided by the embodiment of the present application can detect the wave band of the first fluorescence reagent through the to-be-calibrated instrument, then select the fluorescence plate corresponding to the wave band of the first fluorescence reagent, and manufacture the fluorescence plate into a plate including multiple fluorescence detection parts, so that the multiple detection holes of the to-be-calibrated instrument can be calibrated at the same time, and the calibration efficiency is improved; and the multiple fluorescence detection parts of the same fluorescence plate are detected through the same detection hole, and the fluorescence plate with the difference between the fluorescence values of all the fluorescence detection parts and the first fluorescence value within the preset range is selected as the fluorescence calibration template, so that the fluorescence calibration template with the fluorescence values of the multiple fluorescence detection parts consistent is obtained, and the accuracy of the fluorescence calibration is improved. BRIEF DESCRIPTION OF DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative effort.

[0038] Figure 1 A perspective view of the fluorescence calibration template according to an embodiment of the present application;

[0039] Figure 2 A perspective view of the test tube table and eight connecting tubes according to an embodiment of the present application;

[0040] Figure 3 A flowchart of the method for determining the fluorescence calibration template according to an embodiment of the present application;

[0041] Figure 4 A specific flowchart of step S100 according to an embodiment of the present application;

[0042] Figure 5 Waveform diagrams corresponding to various second fluorescence reagents according to an embodiment of the present application;

[0043] Figure 6 A specific flowchart of step S500 according to an embodiment of the present application;

[0044] Figure 7 A perspective view of the test tube table and fluorescence plate when detecting the fluorescence value of the fourth fluorescence detection part according to the second embodiment of the present application;

[0045] Figure 8 A perspective view of the test tube table and fluorescence plate when detecting the fluorescence value of the third fluorescence detection part according to the second embodiment of the present application;

[0046] Figure 9 A perspective view of the test tube table and fluorescence plate when detecting the fluorescence value of the second fluorescence detection part according to the second embodiment of the present application;

[0047] Figure 10 Figure 6 is a perspective view of a test tube platform and a fluorescence plate for detecting the first fluorescence detection part of the second embodiment of the present application.

[0048] Figure 11 Figure 7 is a schematic view of a fluorescence calibration structure of the nucleic acid detector of one embodiment of the present application.

[0049] Explanation of Reference Signs:

[0050] 100, fluorescence plate; 110, first fluorescence detection part; 120, second fluorescence detection part; 130, third fluorescence detection part; 140, fourth fluorescence detection part; 200, test tube platform; 210, first detection hole; 220, second detection hole; 230, third detection hole; 240, fourth detection hole; 250, fifth detection hole; 260, sixth detection hole; 270, seventh detection hole; 280, eighth detection hole; 300, eight connecting tube; 310, tube body. DETAILED DESCRIPTION

[0051] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0052] It should be noted that when a component is referred to as being "fixed" to another component, it can be directly on the other component or there can be an intervening component. When a component is referred to as being "connected" to another component, it can be directly connected to the other component or there can be an intervening component.

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The use of the terms "and / or" includes a combination of one or more of the associated listed items.

[0054] Some embodiments of the present application will be described in detail below with reference to the drawings. The following embodiments and features of the embodiments can be combined with each other in the case of no conflict.

[0055] PCR nucleic acid detector is an instrument for amplifying specific DNA through PCR (polymerase chain reaction) technology. In simple terms, when the content of the collected sample is too low, the PCR nucleic acid detector can amplify the sample several times, which plays a role in signal amplification, facilitating subsequent detection.

[0056] Real-time fluorescent quantitative PCR technology is a method of adding a fluorescent group to the PCR reaction system, using fluorescent signal accumulation to monitor the entire PCR process in real time, and finally quantitatively analyzing the unknown template through the standard curve. Real-time fluorescent quantitative PCR technology effectively solves the limitations of traditional quantitative endpoint detection, realizes the detection of the intensity of the fluorescent signal once every cycle, and records it in the software. Through the calculation of the Ct value of each sample, the quantitative result is obtained according to the standard curve. Among them, the Ct value (Cycle threshold, cycle threshold) refers to the number of cycles experienced by the fluorescent signal in each reaction tube to reach the set threshold. The PCR detector can record the Ct value of the fluorescent signal. As long as a new DNA sequence is added, the Ct value will increase a little. Therefore, if the sample contains the new coronavirus, the final displayed Ct value is an upward "S" curve, which is diagnosed as a nucleic acid detection positive patient. Conversely, there is no rising curve, which is negative.

[0057] The PCR nucleic acid detector may cause test precision to decrease and cause experimental misjudgment after long-term use. In order to avoid factory repair, the user can use the fluorescent calibration device to calibrate the PCR nucleic acid detector.

[0058] In the prior art, the fluorescent calibration standard template sheet is applied to the calibration of the multi-hole row pipe PCR nucleic acid detector. If a single standard template sheet is used, only single-hole detection can be performed, and the calibration efficiency is low. Or multiple standard template sheets are used for detection, and the fluorescent values of the multiple standard template sheets are greatly different, resulting in low accuracy of fluorescent calibration.

[0059] With reference to Figure 1 , Figure 2 and Figure 3 , the application provides a fluorescent calibration template determination method. The fluorescent calibration template is used for fluorescent calibration of a to-be-calibrated instrument, which can be a PCR nucleic acid detector, a fluorescent probe instrument, etc. The to-be-calibrated instrument includes a test tube table 200 and a fluorescent detection device. The test tube table 200 can be heated during the DNA amplification process. The test tube table 200 is provided with multiple detection holes for inserting test tubes. The fluorescent detection device can detect the fluorescent value of the object in the detection hole and display the fluorescent value of the object in each detection hole.

[0060] In some embodiments of the application, the determination method comprises:

[0061] S100, a first fluorescent reagent with a first fluorescent value is prepared;

[0062] S200, a fluorescence value and a waveband of the first fluorescent reagent are detected by using a to-be-calibrated instrument, and the fluorescence value of the to-be-calibrated instrument is calibrated as the first fluorescent value, wherein the to-be-calibrated instrument comprises a plurality of detection holes;

[0063] S300, a plurality of fluorescent plates 100 corresponding to the waveband of the first fluorescent reagent are selected according to the waveband of the first fluorescent reagent detected by the to-be-calibrated instrument;

[0064] S400, the plurality of fluorescent plates are processed so that each fluorescent plate 100 comprises a plurality of fluorescent detection parts for cooperating with the detection holes;

[0065] S500, a plurality of fluorescent detection parts of the same fluorescent plate 100 are detected by using the same detection hole of the to-be-calibrated instrument;

[0066] S600, a fluorescent plate 100 is selected, wherein the difference between the fluorescence value of each fluorescent detection part of the fluorescent plate 100 and the first fluorescent value is within a preset range.

[0067] In the process of making the fluorescent calibration template, the first fluorescent reagent with the first fluorescent value is prepared in step S100, and a test tube containing the first fluorescent reagent is inserted into the detection hole of the to-be-calibrated instrument. The first fluorescent reagent can be detected by the fluorescence detection device of the to-be-calibrated instrument in step S200. Since the fluorescence value detected by the to-be-calibrated instrument is not accurate, the fluorescence value corresponding to the detection hole into which the test tube is inserted is calibrated as the first fluorescent value. A plurality of fluorescent plates 100 corresponding to the waveband of the first fluorescent reagent are selected in step S300, wherein the fluorescent plates 100 are colored, and the wavebands corresponding to the fluorescent plates 100 of different colors are different. In step S400, the fluorescent plates 100 can be processed by cutting or the like to obtain fluorescent plates 100 comprising a plurality of fluorescent detection parts, wherein the fluorescent detection parts are used for being inserted into the detection holes of the to-be-calibrated instrument. Since the plurality of fluorescent detection parts of the same fluorescent plate 100 will have deviations in the fluorescence value in the process of making the fluorescent plate 100, the plurality of fluorescent detection parts of the same fluorescent plate 100 are detected by using the same detection hole in step S500, and the fluorescent plate 100 is selected in step S600, wherein the difference between the fluorescence value of each fluorescent detection part of the fluorescent plate 100 and the first fluorescent value is within a preset range, so as to improve the consistency of the fluorescence values between the plurality of fluorescent detection parts in the same fluorescent plate 100.

[0068] The fluorescent calibration template made by the determination method of the present application is selected by the instrument to be calibrated, does not need to be detected by other instruments, and is not limited by the absence of standard instruments. The plurality of fluorescent detection parts of the determined fluorescent standard template can simultaneously calibrate the plurality of detection holes of the instrument to be calibrated, thereby improving the calibration efficiency; and the plurality of fluorescent detection parts of the same fluorescent plate 100 are detected by the same detection hole, and the fluorescent plate 100 whose difference between the fluorescent value of all fluorescent detection parts and the first fluorescent value is within the preset range is selected as the fluorescent calibration template, so that the fluorescent plate 100 with relatively consistent fluorescent values of the plurality of fluorescent detection parts is obtained, thereby improving the accuracy of the fluorescent calibration.

[0069] Reference Figure 3 And Figure 4 , step S100 includes: step S110, providing a second fluorescent reagent with a second fluorescent value; and step S120, diluting the second fluorescent reagent to prepare a first fluorescent reagent with a first fluorescent value according to the detection range of the instrument to be calibrated. Specifically, the second fluorescent reagent is a standard fluorescent reagent that can be purchased on the market, so the second fluorescent value is a known standard fluorescent value. Because the second fluorescent value of the second fluorescent reagent usually exceeds the detection range of the instrument to be calibrated, the second fluorescent reagent needs to be diluted to obtain the first fluorescent reagent with the first fluorescent value. During the dilution of the second fluorescent reagent, it needs to be mixed uniformly so that the fluorescent value of the obtained first fluorescent reagent is close.

[0070] In an embodiment, step S100 further includes: step S130, dispensing the first fluorescent reagent with the first fluorescent value into n tubes, and the number of detection holes of each row of the instrument to be calibrated is m, and n≤m. Wherein, the instrument to be calibrated can include multiple rows of detection holes, and the number of detection holes of each row is m. This embodiment takes a single row of eight detection holes and eight-tube 300 test tubes as an example, that is, n and m are both eight, which is not limited in other embodiments. In this embodiment, the eight-tube 300 test tube includes eight tube bodies 310 for containing the first fluorescent reagent. Because the second fluorescent reagent is diluted, the fluorescent values of the first fluorescent reagents contained in the eight tube bodies 310 will deviate, and the first fluorescent reagent is used to preliminarily calibrate all detection holes of the instrument to be calibrated at this time.

[0071] Reference Figure 5The second fluorescent reagent of the present application includes one of FAM (carboxyfluorescein), HEX (hexachlorofluorescein), Texas Red (streptavidin conjugate), Cy5 (sulphorhodamine 5) and Quasar 705 (indocarbocyanine). Among them, the corresponding wave band of FAM (carboxyfluorescein) is 450-490 nm, and the reflected color is blue; the corresponding wave band of HEX (hexachlorofluorescein) is 515-535 nm, and the reflected color is green; the corresponding wave band of Texas Red (streptavidin conjugate) is 560-590 nm, and the reflected color is yellow; the corresponding wave band of Cy5 (sulphorhodamine 5) is 620-650 nm, and the reflected color is orange; and the corresponding wave band of Quasar 705 (indocarbocyanine) is 672-684 nm, and the reflected color is red. One second fluorescent reagent corresponds to one color of fluorescent plate 100, and the determination method of the present application can determine a plurality of fluorescent calibration templates, so that a plurality of instruments to be calibrated can be calibrated.

[0072] In step S200, the fluorescent value of the instrument to be calibrated is calibrated as the first fluorescent value, including: the fluorescent value corresponding to the detection hole in which the n connecting pipe is placed in the instrument to be calibrated is calibrated as the first fluorescent value. Specifically, the instrument to be calibrated can detect the fluorescent value after the first fluorescent reagent is placed in the detection hole, and at this time the fluorescent value corresponding to the detection hole is calibrated as the first fluorescent value, so that the fluorescent values corresponding to the n detection holes of the instrument to be calibrated are preliminarily calibrated.

[0073] In step S300, the fluorescent plate 100 can be an acrylic plate, colored glass, etc. The fluorescent plate 100 of the present application adopts an acrylic plate, which is not limited in other embodiments. The acrylic plate is cut into the shape of a reagent tube, which can be stored for a long time and is not easy to damage, and the calibration ability is stable. Alternatively, the acrylic plate is colored, and the colored acrylic plate is processed and cut into the shape of a reagent tube to make a fluorescent plate 100. The thickness of the fluorescent plate 100 has no fixed requirement, only needs to be able to be placed in the detection hole of the test tube table 200, and the thickness of the acrylic plate can be 5 mm. The fluorescent plate 100 can be processed into a complete reagent tube shape.

[0074] In step S400, a plurality of fluorescent plates 100 are processed, so that each fluorescent plate 100 includes m / 2 fluorescent detection parts. That is, for one instrument to be calibrated, each row of detection holes can complete the calibration of the fluorescent value corresponding to the detection hole through two fluorescent plates 100, so as to improve the calibration efficiency.

[0075] In an implementation, the plurality of detection holes include a first detection hole 210, a second detection hole 220, a third detection hole 230, and a fourth detection hole 240, the plurality of fluorescence detection parts include a first fluorescence detection part 110 and a second fluorescence detection part 120, the interval between two adjacent detection holes is equal to the interval between two adjacent fluorescence detection parts, so that the plurality of fluorescence detection parts can cooperate with the plurality of detection holes.

[0076] Reference Figure 3 And Figure 6 The step S500 includes: a step S510, inserting the first fluorescence detection part 110 and the second fluorescence detection part 120 into the first detection hole 210 and the second detection hole 220 respectively, and detecting the fluorescence value of the second fluorescence detection part 120 through the second detection hole 220.

[0077] The step S520, moving the fluorescence plate 100, inserts the first fluorescence detection part 110 and the second fluorescence detection part 120 into the second detection hole 220 and the third detection hole 230 respectively, and detects the fluorescence value of the first fluorescence detection part 110 through the second detection hole 220.

[0078] Specifically, the plurality of fluorescence detection parts of the fluorescence plate 100 are detected through one of the detection holes after preliminary calibration, so that the deviation of the fluorescence values of the plurality of fluorescence detection parts of the same fluorescence plate 100 caused by the difference in fluorescence detection between the plurality of detection holes can be avoided, and the fluorescence plate 100 with relatively consistent fluorescence values of the plurality of fluorescence detection parts is obtained.

[0079] Embodiment one

[0080] First, insert the first fluorescence detection part 110 and the second fluorescence detection part 120 of the fluorescence plate 100 into the first detection hole 210 and the second detection hole 220 respectively, read the fluorescence value of the second detection hole 220, which corresponds to the fluorescence value of the second fluorescence detection part 120 at this time; second, move the fluorescence plate 100, insert the first fluorescence detection part 110 and the second fluorescence detection part 120 of the fluorescence plate 100 into the second detection hole 220 and the third detection hole 230 respectively, read the fluorescence value of the second detection hole 220, which corresponds to the fluorescence value of the first fluorescence detection part 110 at this time. Thus, the fluorescence plate 100 including two fluorescence detection parts with relatively consistent fluorescence values is obtained.

[0081] Embodiment two

[0082] The difference between this embodiment and embodiment one is that for the single-row eight-detection-hole instrument to be calibrated, the detection holes further include a fifth detection hole 250, a sixth detection hole 260, a seventh detection hole 270, and an eighth detection hole 280, and the fluorescence detection parts further include a third fluorescence detection part 130 and a fourth fluorescence detection part 140.

[0083] Reference Figure 7, Figure 8 , Figure 9 and Figure 10 , the first step, first fluorescent plate 100 of the first fluorescent detection part 110, the second fluorescent detection part 120, the third fluorescent detection part 130 and the fourth fluorescent detection part 140 are inserted into the first detection hole 210, the second detection hole 220, the third detection hole 230 and the fourth detection hole 240 respectively, read the fourth detection hole 240 of the fluorescence value, at this time corresponding to the fourth fluorescent detection part 140 of the fluorescence value; the second step, the first fluorescent plate 100 of the first fluorescent detection part 110, the second fluorescent detection part 120, the third fluorescent detection part 130 and the fourth fluorescent detection part 140 are inserted into the second detection hole 220, the third detection hole 230, the fourth detection hole 240 and the fifth detection hole 250 respectively, read the fourth detection hole 240 of the fluorescence value, at this time corresponding to the third fluorescent detection part 130 of the fluorescence value; the third step, again, the first fluorescent plate 100 of the first fluorescent detection part 110, the second fluorescent detection part 120, the third fluorescent detection part 130 and the fourth fluorescent detection part 140 are inserted into the third detection hole 230, the fourth detection hole 240, the fifth detection hole 250 and the sixth detection hole 260 respectively, read the fourth detection hole 240 of the fluorescence value, at this time corresponding to the second fluorescent detection part 120 of the fluorescence value; the fourth step, finally, the first fluorescent plate 100 of the first fluorescent detection part 110, the second fluorescent detection part 120, the third fluorescent detection part 130 and the fourth fluorescent detection part 140 are inserted into the fourth detection hole 240, the fifth detection hole 250, the sixth detection hole 260 and the seventh detection hole 270 respectively, read the fourth detection hole 240 of the fluorescence value, at this time corresponding to the first fluorescent detection part 110 of the fluorescence value. Thus, through three times of movement, the fluorescence value of the four fluorescent detection parts of the fluorescent plate 100 can be read through one detection hole, and the consistency of the fluorescence values of the multiple fluorescent detection parts on the fluorescent plate 100 is improved.

[0084] In step S600, the fluorescent plate 100 whose difference between the fluorescence value of all fluorescent detection parts and the first fluorescence value is within the preset range is selected, the difference preset range is that the difference between the fluorescence value of all fluorescent detection parts and the first fluorescence value is less than 0.01, and the consistency of the fluorescence values of the multiple fluorescent detection parts is better.

[0085] Reference Figure 11The application also provides a fluorescence calibration method of a nucleic acid detector, comprising: inserting a plurality of fluorescence plates 100 into detection holes of the nucleic acid detector, and calibrating fluorescence values of each detection hole in the nucleic acid detector by the fluorescence plates 100. Specifically, the fluorescence plates 100 manufactured by the calibration method of the application only need to be inserted into a test tube table 200 of the nucleic acid detector by two fluorescence plates 100, so that the nucleic acid detector can be calibrated, the fluorescence values corresponding to each detection hole of the nucleic acid detector are calibrated as first fluorescence values, and the fluorescence calibration is performed, so that the fluorescence calibration efficiency is high.

[0086] In the description of the embodiments of the application, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer" and the like refer to the orientation or positional relationship based on the drawings described, and are only intended to facilitate the description of the application and simplify the description, and do not indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the application.

[0087] The above only discloses a preferred embodiment of the application, and of course cannot limit the scope of the application. Those skilled in the art can understand that all or part of the above-mentioned processes can be implemented, and equivalent changes made according to the claims of the application still belong to the scope of the application.

Claims

1. A method for determining a fluorescence calibration template, wherein the fluorescence calibration template is used to perform fluorescence calibration on an instrument to be calibrated, characterized in that: The determination method includes: preparing a first fluorescent reagent with a first fluorescence value; Using an instrument to be calibrated to detect the fluorescence value and wavelength of the first fluorescent reagent, and calibrating the fluorescence value of the instrument to be calibrated to the first fluorescence value, wherein the instrument to be calibrated includes a plurality of detection holes; Selecting a plurality of fluorescent plates of corresponding wavelengths according to the wavelength of the first fluorescent reagent measured by the instrument to be calibrated; Processing a plurality of the fluorescent plates so that each of the fluorescent plates includes a plurality of fluorescent detection parts for cooperating with the detection holes; Using the same detection hole of the instrument to be calibrated to detect multiple fluorescence detection parts of the same fluorescent plate; A fluorescent plate in which the differences between the fluorescence values ​​of all the fluorescence detection parts and the first fluorescence value are within a preset range is selected as a fluorescence calibration template.

2. The determination method according to claim 1, characterized in that The first fluorescent reagent for preparing the first fluorescent value comprises: a second fluorescent reagent providing a second fluorescent value; The second fluorescent reagent is diluted according to the detection range of the instrument to be calibrated to prepare the first fluorescent reagent with the first fluorescence value.

3. The determination method according to claim 2, characterized in that: The first fluorescent reagent for preparing the first fluorescent value further comprises: The first fluorescent reagent having the first fluorescent value is divided into n connected tubes, and the number of detection wells in each row of the instrument to be calibrated is m, where n≤m.

4. The determination method according to claim 3, characterized in that: The step of calibrating the fluorescence value of the instrument to be calibrated to the first fluorescence value includes: The fluorescence value corresponding to the detection hole where the n-connected tube is placed in the instrument to be calibrated is calibrated as the first fluorescence value.

5. The determination method according to claim 2, characterized in that: The second fluorescent reagent providing a second fluorescent value comprises: The second fluorescent reagent includes one of FAM, HEX, Texas Red, Cy5, and Quasar 705.

6. The determination method according to claim 1, characterized in that: The step of selecting a plurality of fluorescent plates of corresponding wavelengths according to the wavelength of the first fluorescent reagent measured by the instrument to be calibrated comprises: The fluorescent plate is an acrylic plate.

7. The determination method according to claim 3, characterized in that: The processing of the plurality of fluorescent plates so that each of the fluorescent plates includes a plurality of fluorescence detection parts for cooperating with the detection holes comprises: Each of the fluorescent plates includes m / 2 fluorescent detection sections.

8. The determination method according to claim 7, characterized in that: The method of using the same detection hole of the instrument to be calibrated to detect the multiple fluorescence detection parts of the same fluorescent plate includes: The plurality of detection holes include a first detection hole, a second detection hole, a third detection hole, and a fourth detection hole, and the plurality of fluorescence detection parts include a first fluorescence detection part and a second fluorescence detection part; inserting the first fluorescence detection unit and the second fluorescence detection unit into the first detection hole and the second detection hole respectively, and detecting the fluorescence value of the second fluorescence detection unit through the second detection hole; The fluorescent plate is moved, and the first fluorescence detection part and the second fluorescence detection part are respectively inserted into the second detection hole and the third detection hole, and the fluorescence value of the first fluorescence detection part is detected through the second detection hole.

9. The determination method according to claim 1, characterized in that: The step of selecting a fluorescent plate whose differences between the fluorescence values ​​of all the fluorescence detection parts and the first fluorescence value are within a preset range as a fluorescence calibration template comprises: The difference between the fluorescence value of the fluorescence detection part of all the selected fluorescent plates and the first fluorescence value divided by the first fluorescence value is less than 0.

01.

10. A fluorescence calibration method for a nucleic acid detector, characterized in that: include: Use multiple fluorescent plates determined by the determination method as described in any one of items 1-9, insert the multiple fluorescent plates into the detection holes of the nucleic acid detector, and calibrate the fluorescence value of each detection hole in the nucleic acid detector using the fluorescent plates.

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