WO2021212594A1 - Procédé et dispositif d'étalonnage de trajet optique pour instrument de pcr, et instrument de pcr - Google Patents

Procédé et dispositif d'étalonnage de trajet optique pour instrument de pcr, et instrument de pcr Download PDF

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Publication number
WO2021212594A1
WO2021212594A1 PCT/CN2020/092188 CN2020092188W WO2021212594A1 WO 2021212594 A1 WO2021212594 A1 WO 2021212594A1 CN 2020092188 W CN2020092188 W CN 2020092188W WO 2021212594 A1 WO2021212594 A1 WO 2021212594A1
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Prior art keywords
tube
dye
connected tube
calibration coefficient
holes
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PCT/CN2020/092188
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English (en)
Chinese (zh)
Inventor
曹进涛
李冬
贺贤汉
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杭州博日科技有限公司
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Publication of WO2021212594A1 publication Critical patent/WO2021212594A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation

Definitions

  • Real-time fluorescent quantitative polymerase chain reaction (PCR) technology can realize the quantitative analysis of DNA template, which is of great significance to molecular biology research and medical research.
  • Real-time polymerase chain reaction technology relies on accurate detection of fluorescence emission signals above the initial baseline.
  • the signal intensity of multiple wells of the PCR detector sample plate should be the same when detecting the same fluorescence signal.
  • the optical paths corresponding to different hole positions will be different, resulting in different hole positions. The value measured for the same signal is different, and signal distortion occurs, which leads to a decrease in the accuracy of the detection result.
  • the purpose of the present invention is to provide a method and a device for calibrating the optical path of a PCR machine, and a PCR machine to alleviate the technical problem that the accuracy of the detection result is reduced due to signal distortion.
  • the first multi-connected tube that is not doped with dye is used to measure the background signal values corresponding to the M ⁇ N holes; wherein, the first multi-connected tube is an M-connected tube or an N-connected tube;
  • the target calibration coefficients corresponding to the M ⁇ N hole positions are calculated.
  • the background signal values corresponding to the M ⁇ N hole positions are obtained through multiple measurements by using one of the first multi-connected tubes.
  • the step of measuring the signal value of the first dye corresponding to the M ⁇ N holes by using the first multi-tube doped with dye includes:
  • the dye-doped N-connected tubes are respectively placed on the M rows of the sample plate for M measurements to obtain the first dye signal values corresponding to the M ⁇ N holes.
  • the step of calculating the first calibration coefficients corresponding to the M ⁇ N hole positions according to the second dye signal value includes:
  • step of calculating the first calibration coefficient according to the first reference value and the second dye signal value includes:
  • Each value of the second dye signal value is divided by the first reference value to obtain the first calibration coefficient.
  • the step of obtaining the second calibration coefficient corresponding to each test tube in the first multi-connected tube includes:
  • the second multi-connected tube doped with dye is placed M times on the M rows of the sample plate for M measurements to obtain M characterizing the The measured value of each test tube in the M-connected tube;
  • the second multi-connected tube is the same as the first multi-connected tube, or the second multi-connected tube is not the same as the first multi-connected tube, and the second multi-connected tube
  • the number of test tubes is [1, M] or [1, N].
  • the step of calculating the target calibration coefficients corresponding to the M ⁇ N hole positions according to the first calibration coefficient and the second calibration coefficient includes:
  • the second calibration coefficient of each test tube in the M-connected tube is divided by the first calibration coefficient corresponding to each of the holes in the corresponding row. Coefficient, or divide the first calibration coefficient corresponding to each hole position in the corresponding row of each test tube in the M-connected tube by the second calibration coefficient of each test tube in the M-connected tube to obtain The target calibration coefficient;
  • the second calibration coefficient of each test tube in the N-connected tube is divided by all the corresponding holes in the corresponding row of each test tube.
  • the first calibration coefficient, or the first calibration coefficient corresponding to each hole in the corresponding row of each test tube in the N-connected tube is divided by the second calibration coefficient of each test tube in the N-connected tube. Calibration coefficient to obtain the target calibration coefficient.
  • an embodiment of the present invention provides a light path calibration device for a PCR machine.
  • the PCR machine includes a sample plate with M rows and N columns of holes. The holes are used for placing test tubes.
  • the device includes:
  • the first measuring module is used to measure the background signal value corresponding to the M ⁇ N holes by using the first multi-connected tube that is not doped with dye; wherein, the first multi-connected tube is an M-connected tube or an N-connected tube. Tube;
  • the first calculation module is configured to calculate the second dye signal value corresponding to the M ⁇ N hole positions according to the background signal value and the first dye signal value; wherein, the first dye signal value corresponding to each hole position
  • the second dye signal value is the difference between its corresponding first dye signal value and its corresponding background signal value;
  • the second calculation module is configured to calculate the first calibration coefficients corresponding to the M ⁇ N hole positions according to the second dye signal value
  • the third calculation module is configured to calculate the target calibration coefficients corresponding to the M ⁇ N hole positions according to the first calibration coefficient and the second calibration coefficient.
  • an embodiment of the present invention provides a PCR machine, including a sample plate with M rows and N columns of holes, the holes are used for placing test tubes, and each of the holes corresponds to a light path, where M, N is an integer, and M ⁇ 2, N ⁇ 2, and also includes the optical path calibration device of the PCR machine as described above.
  • a multi-connected tube with multiple test tubes is used to calibrate the light path corresponding to the multiple holes of the sample plate.
  • the use of the multi-connected tube can calibrate multiple holes at one time, improve the efficiency of calibration, and realize the control of the entire optical path.
  • Fast calibration; and the second calibration is performed on the influence of the difference between the different test tubes in the multi-connected tube, which reduces the impact of the difference between the different test tubes in the multi-connected tube, and further improves the accuracy of the calibration and avoids
  • the optical path calibration method of the PCR instrument in the embodiment of the present invention can take into account the calibration efficiency and accuracy, and achieve high calibration accuracy while achieving high-efficiency calibration.
  • FIG. 1 is a schematic diagram of a sample plate and a multi-connected tube of a PCR machine provided by an embodiment of the present invention
  • FIG. 2 is a flowchart of a method for calibrating the optical path of a PCR instrument provided by an embodiment of the present invention
  • Fig. 4 is a schematic diagram of an optical path calibration device of a PCR instrument provided by an embodiment of the present invention.
  • the light paths corresponding to the different hole positions will be caused. Differences occur, resulting in different hole positions for the same signal measurement value, signal distortion occurs, resulting in a decrease in the accuracy of the detection result.
  • the PCR instrument includes a sample plate with M rows and N columns of holes.
  • the holes are used for placing test tubes.
  • Each hole corresponds to an optical path.
  • the optical path usually includes optical fibers, lenses, and light sources.
  • M, N It is an integer, and M ⁇ 2, N ⁇ 2.
  • the sample plate 11 has 96 hole positions, and the arrangement is 8 rows and 12 columns. In addition, it can also have specifications such as 12 rows and 8 columns, 6 rows and 16 columns, 16 rows and 6 columns. It should be noted that the number of the above-mentioned holes is not limited, such as 12 holes, 24 holes, 48 holes, 96 holes, 192 holes or 384 holes and so on.
  • step S201 the first multi-tube that is not doped with dye is used to measure the background signal value corresponding to the M ⁇ N hole positions, for example, expressed as: A 1 , A 2 ... A M ⁇ N ;
  • the connecting pipes are M-connected pipes or N-connected pipes;
  • the first multi-tube that is not doped with dye can be an empty first multi-tube or the first multi-tube that contains a background solution that is not doped with dye, and it is placed on the M ⁇ N of the sample plate. The measurement is performed on each hole position, and the background signal values A 1 , A 2 ... A M ⁇ N corresponding to the above M ⁇ N hole positions are obtained.
  • the empty first multi-tube is doped with dye
  • step S201 when an empty test tube is used in step S201, the empty first multi-tube is doped with dye, and when step S201 is used as a test tube containing a background solution that is not doped with dye, the above background will be loaded
  • the first multi-tube of the liquid is doped with dyes.
  • the doped dyes can be FAM, SYBR GREEN, VIC, JOE, TAMRA, NED CY-3, Texas RED, CY-5, HEX, ROX or any other fluorescent dyes.
  • the first multi-tube doped with dye is placed in different rows or columns of the sample plate multiple times for measurement. Specifically, the dye is excited by the light in the light path corresponding to each hole. The fluorescent signal is detected by the detector, and the first dye signal value B 1 , B 2 ... B M ⁇ N of each hole position is obtained.
  • Step S204 Calculate the first calibration coefficients corresponding to the M ⁇ N hole positions according to the second dye signal value, for example, expressed as: K 1 , K 2 ... K M ⁇ N .
  • step S206 the target calibration coefficients corresponding to the M ⁇ N hole positions are calculated according to the first calibration coefficient and the second calibration coefficient, for example, expressed as: L 1 , L 2 ... L M ⁇ N .
  • the optical path calibration method of the PCR machine provided by the embodiment of the present invention, through the target calibration coefficients L 1 , L 2 ... L M ⁇ N corresponding to the M ⁇ N hole positions finally obtained, can be used to calibrate multiple holes of the PCR machine. Calibration is performed to reduce the difference between the corresponding optical paths of different holes, reduce background noise, and improve the accuracy of detection results.
  • a multi-connected tube with multiple test tubes is used to calibrate the light path corresponding to the multiple holes of the sample plate.
  • the use of the multi-connected tube can calibrate multiple holes at one time, improve the efficiency of calibration, and realize the control of the entire optical path.
  • Fast calibration; and the second calibration is performed on the influence of the difference between the different test tubes in the multi-connected tube, which reduces the impact of the difference between the different test tubes in the multi-connected tube, and further improves the accuracy of the calibration and avoids
  • the optical path calibration method of the PCR instrument in the embodiment of the present invention can take into account the calibration efficiency and accuracy, and achieve high calibration accuracy while achieving high-efficiency calibration.
  • a first multi-connected tube is used to obtain background signal values corresponding to M ⁇ N hole positions through multiple measurements.
  • step S202 can be implemented in the following manner: Place the dye-doped M-connected tubes on the N columns of the sample plate N times, and perform N measurements to obtain the first corresponding to the M ⁇ N holes. Dye signal value; or, place the dye-doped N-connected tube on the M row of the sample plate for M times to perform M measurements to obtain the first dye signal value corresponding to the M ⁇ N holes.
  • the specific implementation can be as follows: prepare a certain amount of dye solution of appropriate concentration, and place a dye solution with the same volume as the background solution in the first step in each test tube of the 8-tube; the concentration value of the dye solution is not specifically limited, but is It is possible to make the measured dye signal intensity greater than or equal to 10 times the background signal intensity as a limit.
  • the second dye signal value C 1 corresponding to each well position can be calculated according to the background signal value A 1 ... A 96 and the first dye signal value B 1 ... B 96 . ..C 96 , where C 1 ... C 12 is the second dye signal value corresponding to the first row of wells; and so on, C 85 ... C 96 is the second dye signal corresponding to the eighth row of wells value.
  • step S204 may include the following steps:
  • Step S301 Use any one of the second dye signal values or the average value thereof as the first reference value, for example, expressed as C ⁇ ;
  • Step S302 Calculate a first calibration coefficient according to the first reference value and the second dye signal value.
  • step S302 may include the following methods: divide the first reference value by each of the second dye signal values to obtain the first calibration coefficient; or, divide each of the second dye signal values by each value. With the first reference value, the first calibration coefficient is obtained.
  • the first reference value C ⁇ can be the maximum value, minimum value, average value, or any value among 96 second dye signal values.
  • the above measurement using 8-connected tubes in 12 rows of well positions achieved the first optical path calibration corresponding to the light paths of the 96-hole positions of the sample plate; however, each of the eight test tubes in the 8-connected tubes used in the above calibration process There may also be differences between them, which will also cause differences in the signal strength measured for the same signal when different test tubes are used for optical path calibration. Therefore, in order to eliminate the background influence that may be caused by the difference between the different test tubes in the 8-tube tube, next, on the basis of the first optical path calibration, perform the second optical path calibration, that is, the step S205 above obtains the first multi-connected tube.
  • the second calibration coefficient of each test tube in the tube specifically includes the following two embodiments.
  • step S205 may include the following steps:
  • Step 1) When the first multi-connected tube is an M-connected tube, the second multi-connected tube doped with dye is placed on the M row of the sample plate for M measurements in M times to obtain M characteristic M-connected tubes.
  • the measured value of each test tube for example, is expressed as: D 1 ′, D 2 ′...D M ′;
  • the second multi-connecting tube can also be an 8-connected tube, which will carry the dye 8
  • the connecting tube is placed on a certain row of the 8 rows of holes, for example, on the first row first, so that the 8 dye signal values D 1 ... D 8 of the 8 test tubes are measured.
  • the second reference value D ⁇ can be the maximum value, minimum value, average value or any value among the 8 values of D 1 ′...D 8 ′.
  • Step a) When an M-connected tube is used for the first multi-connected tube, the second calibration coefficient of each test tube in the M-connected tube is divided by the first calibration coefficient corresponding to each hole position in the corresponding row, or the M connected tube The first calibration coefficient corresponding to each hole position in the corresponding row of each test tube in each tube is divided by the second calibration coefficient of each test tube in the M connecting tube to obtain the target calibration coefficient;
  • Step b) When N-connected tubes are used for the first multi-connected tube, the second calibration coefficient of each test tube in the N-connected tube is divided by the first calibration coefficient corresponding to each hole in the corresponding row of each test tube, or The first calibration coefficient corresponding to each hole position in the corresponding row of each test tube in the N-connected tube is divided by the second calibration coefficient of each test tube in the N-connected tube to obtain the target calibration coefficient.
  • the light path corresponding to each hole position is calibrated according to the above target calibration coefficient, which can reduce the difference between the light paths corresponding to different hole positions, reduce background noise, and improve the accuracy of the detection result.
  • the first calculation module 43 is configured to calculate a second dye signal value according to the background signal value and the first dye signal value, wherein the second dye signal value corresponding to each hole position is its corresponding first dye The difference between the signal value and its corresponding background signal value;
  • the third calculation module 46 is configured to calculate the target calibration coefficients corresponding to the M ⁇ N holes according to the first calibration coefficients K 1 , K 2 ... K M ⁇ N and the second calibration coefficient.
  • the first multi-connected tube is used to measure the background signal values corresponding to the M ⁇ N hole positions; wherein, the first multi-connected tube is empty or holds a background solution that is not doped with dye. Specifically, it may include: using multiple first multi-connected tubes to obtain background signal values corresponding to M ⁇ N hole positions through one measurement; or, using one first multi-connected tube to obtain M ⁇ N hole positions corresponding to M ⁇ N hole positions through multiple measurements The value of the background signal.
  • the second calculation module 44 is also used to:
  • Calculating the first calibration coefficient based on the first reference value and the second dye signal value may specifically include: dividing the first reference value by each of the second dye signal values to obtain the first calibration coefficient; or Each of the two dye signal values is divided by the first reference value to obtain the first calibration coefficient.
  • the second multi-connected tube doped with dye is placed on the M row of the sample plate M times for M measurements, and each test tube in the M characteristic M-connected tube is obtained. Measured value;
  • the first multi-connected tube is an N-connected tube
  • the first multi-connected tube adopts M-connected tubes
  • the first calibration coefficient corresponding to each hole position in the corresponding row of the test tube is divided by the second calibration coefficient of each test tube in the M connecting tube to obtain the target calibration coefficient
  • the first multi-connected tube adopts N-connected tubes
  • the first calibration coefficient corresponding to each hole position in the corresponding row of each test tube in the tube is divided by the second calibration coefficient of each test tube in the N-connected tube to obtain the target calibration coefficient.
  • the multiple hole positions of the PCR instrument can be calibrated through the target calibration coefficients corresponding to the M ⁇ N hole positions finally obtained, thereby reducing the light path corresponding to different hole positions.
  • the difference between them reduces the background noise and improves the accuracy of the detection results.

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Abstract

L'invention concerne un procédé et un dispositif d'étalonnage de trajet optique pour un instrument de PCR, et instrument de PCR. L'instrument de PCR comprend une plaque d'échantillon pourvue de M rangées et de N colonnes de positions de trous, les positions de trous étant utilisées pour placer des tubes de test, et chaque position de trou correspondant à un trajet optique, M ≥ 2, N ≥ 2 et M et N étant des nombres entiers. Des secondes valeurs de signal de colorant correspondant à M × N positions de trou sont calculées en fonction de valeurs de signal d'arrière-plan acquises et de premières valeurs de signal de colorant mesurées ; des premiers coefficients d'étalonnage correspondant aux M × N positions de trou sont calculés en fonction des secondes valeurs de signal de colorant ; un second coefficient d'étalonnage correspondant à chaque tube de test dans un premier tube multiconnecté est acquis ; et des coefficients d'étalonnage cibles correspondant aux M × N positions de trou sont calculés en fonction des premiers coefficients d'étalonnage et des seconds coefficients d'étalonnage. Au moyen des coefficients d'étalonnage cibles, une pluralité de positions de trous de l'instrument de PCR peut être étalonnée de façon à réduire la différence entre les trajets optiques correspondant à différentes positions de trous, de sorte que différentes positions de trou maintiennent la même intensité de signal lors de la détection du même signal, ce qui améliore la précision du résultat de détection.
PCT/CN2020/092188 2020-04-21 2020-05-26 Procédé et dispositif d'étalonnage de trajet optique pour instrument de pcr, et instrument de pcr WO2021212594A1 (fr)

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CN202010319634.9A CN111707646B (zh) 2020-04-21 2020-04-21 Pcr仪的光路校准方法、装置及pcr仪

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