A method for detecting carryover rate of a lysing instrument

Abstract

The application discloses a kind of detection methods of carry pollution rate of cleaving instrument, it is related to cleaving instrument carrying pollution detection technical field.This method first takes 3 test tubes and is filled into marker solution, 3 test tubes are filled into equal amount of purified water;The test tube is sequentially placed on the sample holder of cleaving instrument, places 6 empty flow tubes on sampling tray, completes sampling;Liquid in 6 the flow tubes is directly (or after dilution) added to counting plate, and the counting under microscope is respectively i1, i2, i3;Take the liquid of proper amount in the flow tube filled with purified water and add to counting plate, and the counting under microscope is respectively j1, j2, j3;The above sampling and counting process is repeated several times;According to formula [(j3-j1) / (i3-j3)]×100%, the carry pollution rate of cleaving instrument is calculated in each repeated time, take maximum value, obtain the carry pollution rate of cleaving instrument.The test result of this method is little different from existing detection method, and the reliability is very high.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of detection of carryover contamination of lysing instrument, and particularly relates to a detection method of carryover contamination rate of lysing instrument. BACKGROUND

[0002] The lysing instrument is a core component of a medical examination front-end processing product lysing instrument for full automation, miniaturization and intelligentization of flow cytometry technology, mainly including three blocks of mechanical block, liquid path block and circuit board block. The mechanical block refers to a mechanical operation organization framework, mainly including a sample rack feeding end, a reagent shaft, a horizontal shaft and a turntable mixing device. The liquid path block refers to a liquid path flow-through part, mainly including a peristaltic pump liquid inlet end, a sample needle and a reagent needle cleaning device, a plunger pump sample adding end and an electromagnetic valve control system. The circuit board block is controlled by the main control board to control the movement of each interface board block and the on-off of the liquid path block. The current market lysing instrument has the same idea for carryover contamination detection, mainly in that the detection methods are different. There are currently three main detection methods: biochemical detection method, chemiluminescence method and flow counting method.

[0003] The biochemical detection method and the chemiluminescence method have similar basic principles. The biochemical detection method uses a full-automatic biochemical analyzer to detect the absorbance of a suitable amount of orange human serum solution (stock solution), and calculates the carryover contamination rate by measuring the absorbance values of the stock solution and deionized water. The chemiluminescence method also uses a full-automatic chemiluminescence immunoassay analyzer to detect the absorbance values of high-concentration samples and zero-concentration samples of a clinical test item, and then calculates the carryover contamination rate by calculating the absorbance ratio of the zero-concentration sample and the high-concentration sample.

[0004] The flow counting method uses microspheres as a representative detection object, uses a flow cytometer to test standard microsphere solution and blank solution, and calculates the carryover contamination rate by calculating the ratio of the number of microspheres in the blank solution to the number of microspheres in the standard microsphere solution.

[0005] The three methods for detecting carryover contamination on the market all have certain drawbacks: (1) special professional instrument equipment is required for testing carryover contamination, which has high cost; (2) the operation process of the above-mentioned equipment is complex, the training period is long, and the requirement for professional knowledge is high; (3) the above-mentioned equipment has certain requirements for the sample sampling amount when testing carryover contamination, and it is not easy to test the sample needle with extremely small sampling amount; for example, the sampling amount of the flow cytometer cannot be less than 100 μL; (4) the three detection methods cannot be interrupted during the detection process, and the test results are obtained by the equipment analysis. If the equipment fails during the process, the used samples will be completely wasted, and therefore the reliability is affected. SUMMARY

[0006] In view of the above deficiencies of the prior art, the present application provides a detection method for carry-over contamination rate of a lysing instrument, which specifically realizes the method through the following techniques.

[0007] A detection method for carry-over contamination rate of a lysing instrument, comprising the following steps:

[0008] S1, three test tubes are filled with a marker solution, and three test tubes are filled with an equal amount of purified water; the test tubes filled with the marker solution and the purified water are sequentially placed on a sample holder of the lysing instrument, six empty flow tubes are placed on a sampling tray, a sampling amount is set, and a sampling program is run; first, samples are sequentially taken from the three test tubes filled with the marker solution, and then samples are sequentially taken from the three test tubes filled with the equal amount of purified water until the sampling of the six flow tubes is completed; the marker is used to observe and record the number under a microscope;

[0009] S2, six flow tubes are taken out, an appropriate amount of liquid is taken from the flow tubes filled with the marker solution, or an appropriate amount of liquid is taken after dilution with purified water (if the observed marker is too much when the liquid is directly taken, appropriate dilution is required), and the liquid is added to a counting plate; the number of markers is observed and recorded under a microscope, and is recorded as i1, i2 and i3 respectively;

[0010] An appropriate amount of liquid is taken from the flow tubes filled with the purified water and added to the counting plate; the number of markers is observed and recorded under a microscope, and is recorded as j1, j2 and j3 respectively;

[0011] S3, the carry-over contamination rate of the lysing instrument in each repetition is calculated according to the formula [(j3-j1) / (i3-j3)]x100%, and the maximum value is taken;

[0012] The steps S1-S3 are repeated several times.

[0013] The sampling process and principle of the detection method provided by the present application are as follows: (1) the sampling needle of the lysing instrument first sequentially takes samples from the three test tubes filled with the marker solution, and then sequentially takes samples from the three test tubes filled with the equal amount of purified water; (2) the appropriate amount of liquid taken is added to the corresponding six flow tubes one by one; after each test tube is added, the liquid path plate of the lysing instrument will clean the sampling needle once, until the six flow tubes are all added. It should be noted that the sampling must be performed in the three test tubes filled with the marker solution first, and then in the three test tubes filled with the equal amount of purified water.

[0014] Preferably, in step S1, the amount of the marker solution or the purified water added to the test tube is 0.8-1.0 mL.

[0015] Preferably, in step S2, the flow tubes filled with the marker solution are diluted 100-200 times with purified water, and then an appropriate amount of liquid is taken and added to the counting plate.

[0016] Preferably, the amount of liquid added to the counting plate in step S2 is no more than 10 μL.

[0017] Preferably, the number of times of repeating steps S1-S3 is no less than 3.

[0018] More preferably, the marker is one that facilitates observation under a microscope and can be counted (by the naked eye or an instrument), and thus any high concentration of biological or non-biological material that can serve the above purpose can be used as a marker in the present application.

[0019] Further preferably, the marker is a microsphere, or a cell, or a microorganism. Cells and microorganisms (e.g., bacteria) are in a dispersed state that facilitates counting. If conditions permit, cells and microorganisms can also be stained with a dye for the purpose of facilitating observation and counting; for example, Gram staining can be used to stain Gram-positive bacteria.

[0020] Compared with the prior art, the present application has the following advantages:

[0021] 1. The detection method of the present application uses a microscope counting method that has not been used by anyone in the industry to detect the carryover contamination rate. The final test results are similar to those obtained by three detection methods (represented by flow cytometry) currently available on the market, and the method has high reliability and can perfectly replace flow cytometry for carryover contamination rate detection.

[0022] 2. The entire method is simpler to operate and requires less expertise from the tester. If an error occurs during testing, the test can be repeated, greatly improving the error tolerance of the detection.

[0023] 3. The detection instrument is mainly a microscope, and the consumables and reagents (counting plates, markers, etc.) are all commercially available products, and the testing of finished products is less expensive. Since there are no special requirements for the amount of sample, the testing application range is also more extensive. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Observation of the microsphere solution diluted 100 times in the three flow cytometry tubes of the first group of examples under a microscope.

[0025] Figure 2 Observation of the liquid in the three flow cytometry tubes containing purified water of the first group of examples under a microscope. DETAILED DESCRIPTION

[0026] The technical solutions of the present application will be described clearly and completely below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0027] The marker solution used in the following examples and comparative examples is a microsphere solution, and the microsphere material is a commercially available product that meets the requirements of YY / T 0588-2017: Flow Cytometer.

[0028] Embodiment

[0029] The detection method of the carryover rate of the lysing instrument provided in the embodiment has the following steps:

[0030] S1, take 3 test tubes and fill them with 1.0 mL of standard microsphere solution, and take 3 test tubes and fill them with 1.0 mL of purified water; place the test tubes containing the microsphere solution and the purified water on the sample holder of the lysing instrument in turn, and place 6 empty flow tubes on the sampling tray, set the sampling amount, and run the sampling program until the 6 flow tubes are sampled;

[0031] S2, take out the 6 flow tubes, dilute the flow tubes containing the microsphere solution with purified water by 100 times, take 8 μL of the liquid, and add it to the counting plate, and observe and record the number of microspheres under a microscope; record i1, i2, and i3 respectively;

[0032] Take an appropriate amount of liquid from the flow tubes containing the purified water and add it to the counting plate, and observe and record the number of microspheres under a microscope; record j1, j2, and j3 respectively; the above steps are repeated 3 times;

[0033] S3, calculate the carryover rate of the lysing instrument for each repetition according to the formula [(j3-j1) / (i3-j3)]x100%, and take the maximum value.

[0034] The above steps S1-S3 are repeated 3 times (referred to as groups 1-3); the detection results are shown in Table 1 below.

[0035] Table 1: Carryover rate test results of Example 1

[0036]

[0037] Comparative Example

[0038] The detection method of the lysing instrument carrying pollution rate provided by the present comparative example has the following steps: using a standard microsphere solution to test in a flow cytometer, repeating the test for 3 times, calculating the number of microspheres in the calibration area, recording the number of microspheres as H1, H2 and H3 respectively, then testing the number of empty samples, repeating the test for 3 times (groups 1-3), calculating the number of microspheres in the calibration area, recording the number of microspheres as L1, L2 and L3 respectively, repeating the test for 3 times according to the method, then calculating the carrying pollution rate result according to the formula [(L1-L3) / (H3-L3)]x100%, and taking the maximum value.

[0039] Table 2 is the detection result of the carrying pollution rate of comparative example 1

[0040]

[0041] As shown in Tables 1, 2 and the accompanying Figure 1 , 2 , Figure 1 , 2 The results of microspheres observed under a microscope of the first group of examples are shown in Tables 1, 2 and the accompanying

[0042] The above specific embodiments describe the implementation of the present application in detail, but the present application is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concepts of the present application, the technical solutions of the present application can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present application.

Claims

1. A method for detecting carryover rate of a lysing instrument, characterized in that, The method comprises the following steps: S1, three test tubes are filled with a marker solution, and three test tubes are filled with purified water; the test tubes filled with the marker solution and the purified water are sequentially placed on a sample rack of a lysing instrument, six empty flow tubes are placed on a sampling tray, a sampling amount is set, and a sampling program is run; the sampling is performed from the three test tubes filled with the marker solution, and then the sampling is performed from the three test tubes filled with the purified water; and the sampling of the six flow tubes is completed; the marker is used for observing and recording the quantity under a microscope; S2, six flow tubes are taken out, an appropriate amount of liquid in the flow tubes filled with the marker solution is directly taken, or the flow tubes are diluted with purified water, and then an appropriate amount of liquid is taken and added to a counting plate; the quantity of the marker is observed and recorded under a microscope, and the quantity is recorded as i1, i2 and i3 respectively; an appropriate amount of liquid in the flow tubes filled with the purified water is taken and added to the counting plate; the quantity of the marker is observed and recorded under the microscope, and the quantity is recorded as j1, j2 and j3 respectively; S3, the carry-over rate of the lysing instrument in each repetition is calculated according to the formula [(j3-j1) / (i3-j3)]x100%, and the maximum value is taken; The steps S1-S3 are repeated for several times.

2. The method according to claim 1, wherein, In the step S1, the added amount of the marker solution or the purified water in the test tube is 0.8-1.0 mL.

3. The method of claim 1, wherein the carryover is detected by the lysing instrument. In the step S2, the flow tubes filled with the marker solution are diluted 100-200 times with purified water, and then an appropriate amount of liquid is taken and added to the counting plate.

4. The method of claim 1, wherein the carryover is detected by the lysing instrument. In the step S2, the amount of the liquid added to the counting plate is not more than 10 μL.

5. The method of claim 1, wherein the carryover is detected by the lysing instrument. The repetition times of the steps S1-S3 are not less than 3 times.

6. The method according to any one of claims 1 to 5, wherein the method is for detecting carryover of the lysing instrument. The marker refers to biological or non-biological material that can be directly observed and counted under a microscope.

7. The method of claim 6, wherein the method is used to detect carryover contamination in a lysing instrument. The marker is a microsphere, a cell or a microorganism.

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