Method for rapid ultramicro nondestructive detection of mycoplasma pollution in cultured cells and application thereof

Abstract

The invention provides a method for rapid ultramicro nondestructive detection of mycoplasma pollution in cultured cells and application thereof, and aims to solve the problems of inaccurate detection precision, complex process, long time consumption and the like in the existing detection technology. According to the method, only 3 microliters of supernate in a cell culture dish is sucked through a pipette to serve as a sample, and the mycoplasma in the supernate is directly observed by adopting a freezing sample preparation technology and utilizing a freezing electron microscope under the condition of being amplified by 100 thousand times. According to the method, ultramicro sampling is realized, interference on cell growth is reduced, early detection of mycoplasma pollution is also realized, the lower limit of detection concentration is as low as 40 pieces / microliter, and the detection process is rapid and can be completed in only one hour. Experimental results show that the method can accurately detect mycoplasma contamination in cultured cells, and an efficient and nondestructive detection means is provided for cell culture quality control.

Description

Technical Field

[0001] The invention relates to the field of cell culture, and in particular to a method for rapid ultra-trace non-destructive detection of mycoplasma contamination in cultured cells and an application thereof. Background Art

[0002] Mycoplasma is a type of prokaryotic microorganism without cell wall and highly polymorphic. Its size is between that of bacteria and viruses, and it is one of the smallest and simplest prokaryotes known so far. Due to its tiny size, some mycoplasmas can penetrate the 0.2μm pore size filter membrane commonly used in laboratories and reproduce in artificial culture media, thus becoming a common source of contamination in cell culture. When mycoplasmas contaminate cells, they will significantly change the expression levels of DNA, RNA and proteins in the cells, resulting in a decrease in the authenticity and reliability of the experimental results, and will also have a serious impact on the normal growth and reproduction of cells.

[0003] However, the current methods for detecting mycoplasma contamination in cells have many shortcomings, such as the isolation and culture method is time-consuming and easily affected by environmental factors; although the DNA staining method, PCR method and TLR2 activation method are highly sensitive, they all require the collection of a certain number of cell samples for detection, which is destructive to cells and cannot achieve non-destructive detection; the scanning electron microscope detection method has the disadvantages of expensive equipment and complex operation. Therefore, the development of a rapid, ultra-micro and non-destructive detection method is of great significance to ensure the quality of cell culture and the reliability of experimental results.

[0004] In order to solve the above problems, the applicant proposes a method for rapid ultra-trace non-destructive detection of mycoplasma contamination in cultured cells and its application. Summary of the invention

[0005] The object of the present invention is to provide a method for rapid ultra-trace non-destructive detection of mycoplasma contamination in cultured cells and its application, so as to solve the problems in the prior art.

[0006] To achieve the above object, the present invention provides the following technical solutions: 1. A method for rapid ultra-trace non-destructive detection of mycoplasma contamination in cultured cells, characterized in that it comprises the following steps:

[0007] Sampling: Use a pipette to draw 3 μl of supernatant from the cell culture dish as the sample to be tested;

[0008] Sample preparation: Add 3 μl of the supernatant obtained in the sampling step to the front of the hydrophilic grid, then use filter paper to quickly adsorb it on the back of the grid until only a layer of water film remains, and then quickly put the grid into liquid ethane to complete the frozen sample preparation;

[0009] Detection: Place the frozen grid prepared in the sample preparation step into a cryo-electron microscope for observation, and check whether mycoplasma exists in the supernatant under a magnification of 100,000 times.

[0010] Optionally, in the detection step, the observation conditions of the cryo-electron microscope are optimized to achieve ultra-fast detection of mycoplasma contamination, with the detection time being as short as less than 1 hour.

[0011] Optionally, this method achieves ultra-trace non-destructive testing, with the sampling volume being only 3 microliters of cultured cell supernatant, and causing no damage to the cells.

[0012] Optionally, this method achieves early detection of mycoplasma contamination through frozen sample preparation and condition optimization, with the lower limit of detection concentration as low as 40 / μl, which can detect and control mycoplasma contamination as early as possible.

[0013] An application of rapid ultra-trace non-destructive detection of mycoplasma contamination in cultured cells, characterized in that the mycoplasma contamination in cultured cells is detected by the method described in any one of claims 1 to 4.

[0014] Beneficial effects: Ultra-trace non-destructive testing: The sampling volume is only 3 microliters of the supernatant of cultured cells, which does not damage the cells and realizes non-destructive testing in the true sense.

[0015] High sensitivity and accuracy: Through freezing sample preparation and cryo-electron microscopy observation, the presence of mycoplasma can be accurately identified under low-concentration contamination conditions, with the detection concentration limit as low as 40 per microliter. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The present invention selects 6 dishes of cells with different contamination levels, takes the supernatant and prepares samples for cryo-electron microscopy, and shows representative pictures;

[0017] Figure 2 This is a graph showing the results of the secondary PCR test in an embodiment of the present invention; DETAILED DESCRIPTION

[0018] The following describes the preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0019] Example 1

[0020] The core of the present invention is to provide a method for rapid ultra-trace non-destructive detection of mycoplasma contamination in cultured cells, which specifically comprises the following steps:

[0021] Sampling: Use a pipette to accurately draw 3 microliters of supernatant from the cell culture dish as the sample to be tested. The key to this step is the precise control of the sampling volume, which not only ensures the sensitivity of the test, but also avoids affecting the normal growth of cells.

[0022] Sample preparation: Add 3 microliters of the sampled supernatant to the front of the hydrophilic grid. The hydrophilic treatment helps to evenly distribute the supernatant on the grid and reduce the detection error caused by uneven samples. Subsequently, use filter paper to quickly adsorb on the back of the grid until only a layer of water film remains. The purpose of this step is to remove excess water and make the sample more concentrated for subsequent observation. Finally, quickly put the grid into liquid ethane to complete the frozen sample preparation. The ultra-low temperature of liquid ethane can quickly freeze the microbial morphology in the sample, maintain its original state, and provide clear and accurate images for subsequent observations.

[0023] Detection: Place the frozen sample carrier into a cryo-electron microscope for observation. Cryo-electron microscopes are known for their high resolution and cryo-imaging technology, which can clearly image microorganisms without destroying the sample structure. Check the presence of mycoplasma in the supernatant under a magnification of 100,000 times. The key to this step is the optimization of observation conditions, including the adjustment of the electron microscope's acceleration voltage, magnification, exposure time and other parameters to ensure the best imaging effect.

[0024] In order to verify the effectiveness and reliability of the present invention, we conducted the following experiments.

[0025] Experimental materials: 6 dishes of cells with different degrees of contamination were selected as experimental objects, including uncontaminated cells, lightly contaminated cells, moderately contaminated cells and heavily contaminated cells.

[0026] Experimental steps: Follow the above sampling, sample preparation and detection steps. During the sampling process, use a calibrated pipette to accurately draw 3 microliters of supernatant; during the sample preparation process, pay attention to controlling the speed and strength of filter paper adsorption to avoid sample loss; during the detection process, adjust the observation conditions according to the instruction manual of the cryo-electron microscope to ensure a clear image.

[0027] Experimental results: Through cryo-electron microscopy observation, we found that mycoplasma morphological structures existed in the supernatants of all contaminated cells, and the number and density of mycoplasmas increased accordingly as the degree of contamination increased. At the same time, we also found that even in the case of low-concentration contamination (such as less than 40 / μl), cryo-electron microscopy can clearly identify the presence of mycoplasmas. This result fully demonstrates the high sensitivity and accuracy of the present invention in the detection of mycoplasma contamination.

[0028] In order to further verify the reliability of the present invention, we also used PCR as a control experiment. The PCR method detects the presence of mycoplasma by amplifying specific gene fragments of mycoplasma. However, the experimental results show that the PCR method has missed detection in low-concentration contamination conditions (such as RRE-1 has not yet detected mycoplasma), while cryo-electron microscopy can accurately detect mycoplasma in all contaminated cells. This result further proves the superiority of the present invention in mycoplasma contamination detection.

[0029] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the attached claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention. Any figure mark in the claims should not be regarded as limiting the claims involved.

[0030] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for rapid ultra-trace non-destructive detection of mycoplasma contamination in cultured cells, characterized in that The following steps are involved: Sampling: Use a pipette to draw 3 μl of supernatant from the cell culture dish as the sample to be tested; Sample preparation: Add 3 μl of the supernatant obtained in the sampling step to the front of the hydrophilic grid, then use filter paper to quickly adsorb it on the back of the grid until only a layer of water film remains, and then quickly put the grid into liquid ethane to complete the frozen sample preparation; Detection: Place the frozen grid prepared in the sample preparation step into a cryo-electron microscope for observation, and check whether mycoplasma exists in the supernatant under a magnification of 100,000 times.

2. The method according to claim 1, characterized in that: In the detection step, the observation conditions of the cryo-electron microscope are optimized, which can achieve ultra-fast detection of mycoplasma contamination, and the detection time is as short as less than 1 hour.

3. The method according to claim 1 or 2, characterized in that: This method achieves ultra-trace non-destructive testing, with the sampling volume being only 3 microliters of cultured cell supernatant, and causing no damage to the cells.

4. The method according to any one of claims 1 to 3, characterized in that This method achieves early detection of mycoplasma contamination through frozen sample preparation and condition optimization. The lower limit of detection concentration is as low as 40 cells / μL, which can detect and control mycoplasma contamination as early as possible.

5. An application of rapid ultra-trace non-destructive detection of mycoplasma contamination in cultured cells, characterized in that: The method according to any one of claims 1 to 4 is used to detect mycoplasma contamination in cultured cells.

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