Cell preparation mycoplasma detection method and kit thereof

Abstract

The invention provides a cell preparation mycoplasma detection method and a kit thereof. The cell preparation mycoplasma detection method comprises the following steps: carrying out solid-liquid separation on a to-be-detected cell preparation to obtain cell precipitate; a cell lysis solution is added into the cell precipitate, cells are suspended in the lysis solution, then incubation is conducted for 15 min to 25 min at the room temperature, and the cell lysis solution comprises thiourea with the molar concentration of 0.8 mol / L to 1.8 mol / L; and carrying out nucleic acid extraction on the lysate. The extracted nucleic acid reacts with a qPCR reagent, then a fluorescent signal is collected through a fluorescent quantitative PCR instrument, and the PCR reaction process is monitored according to the change of the fluorescent signal. After the reaction is finished, testing to obtain a circulation threshold value, and judging whether the mycoplasma exists in the cell preparation or not according to the circulation threshold value. According to the method, the detection accuracy of the mycoplasma of the cell preparation can be effectively improved through thiourea-assisted testing.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a method and kit for detecting mycoplasma in cell preparations. Background Technology

[0002] Cellular preparations are increasingly used in biomedical research and clinical treatment, such as in cell transplantation, gene therapy, and regenerative medicine. The quality of cellular preparations directly affects the reliability of research results and the safety of clinical treatment. Mycoplasma contamination is one of the major quality problems faced by cellular preparations. Mycoplasma is a type of prokaryotic microorganism without a cell wall, highly pleomorphic, capable of passing through bacterial filters, and able to grow and reproduce in lifeless culture media. When mycoplasma contaminates cells, it competes with host cells for nutrients, secretes harmful substances, and affects cell growth, metabolism, morphology, and function, leading to biased experimental results and even posing serious health risks to patients in clinical applications.

[0003] Currently, commonly used methods for mycoplasma detection mainly include culture methods, DNA staining methods, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR). Culture methods are the "gold standard" for mycoplasma detection, but their detection cycle is long, usually requiring 2-4 weeks, and their sensitivity is low, making them unsuitable for rapid detection. DNA staining is simple to operate, but its specificity is poor and it is easily affected by contamination from other microorganisms. ELISA, based on antigen-antibody reactions, has a certain degree of specificity and sensitivity, but cross-reactivity is possible, and the detection steps are relatively cumbersome.

[0004] PCR (Polymerase Chain Reaction) has been widely used in mycoplasma detection due to its high sensitivity, specificity, and speed. Quantitative real-time PCR (qPCR), in particular, allows for real-time monitoring of PCR products, enabling quantitative detection of mycoplasma and further improving accuracy and efficiency. However, in actual testing, complex components in cell preparations, such as proteins, polysaccharides, and lipids, can inhibit nucleic acid extraction and the PCR reaction, leading to reduced sensitivity, increased false negatives, and affecting detection accuracy.

[0005] To improve the accuracy of PCR detection, researchers have explored various methods to optimize the detection process, such as optimizing nucleic acid extraction methods and improving the PCR reaction system. Cell lysis is a crucial step in nucleic acid extraction; effective cell lysis releases more nucleic acids and reduces interference from impurities. Existing cell lysis buffers typically contain detergents and proteases, which, while capable of lysing cells, are not ideal for mycoplasma lysis and struggle to effectively remove inhibitory substances released during cell lysis. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing cell preparation mycoplasma detection methods, which suffer from low detection accuracy, and to provide a cell preparation mycoplasma detection method and kit. This method, by adding a specific concentration of thiourea to the cell lysis buffer, can effectively improve the lysis efficiency of cells and mycoplasma, reduce the inhibitory effect of impurities on nucleic acid extraction and PCR reactions, thereby improving detection accuracy.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for detecting mycoplasma in cell preparations includes the following steps:

[0009] S101: Solid-liquid separation: The cell preparation to be tested is separated into solid and liquid components to obtain cell precipitate;

[0010] S102: Cell lysis: Add cell lysis buffer to the cell pellet and suspend the cells in the lysis buffer, then incubate at room temperature for 15 min to 25 min, wherein the cell lysis buffer comprises thiourea with a molar concentration of 0.8 mol / L to 1.8 mol / L;

[0011] S103: Nucleic acid extraction: Nucleic acid is extracted from the lysis buffer to obtain nucleic acid;

[0012] S104: qPCR reaction: The nucleic acid is reacted with qPCR reagent, and then the fluorescence signal is collected by a real-time PCR instrument. The progress of the PCR reaction is monitored based on the change of the fluorescence signal.

[0013] S105: Result judgment: After the reaction is completed, the cycle threshold is obtained by testing, and the presence of mycoplasma in the cell preparation is determined based on the cycle threshold.

[0014] Optionally, in step S101, the solid-liquid separation method is centrifugation, with a centrifugation speed of 800 r / min to 1500 r / min and a centrifugation time of 5 min to 10 min.

[0015] Optionally, in step S102, the cell lysis buffer further includes sodium dodecyl sulfate at a mass percentage of 1% to 3%, glycerol at a volume percentage of 5% to 15%, and Tris-HCl at a molar concentration of 10 mmol / L to 30 mmol / L, and the pH of the cell lysis buffer is 8.0 to 8.5.

[0016] Optionally, in step S102, the molar concentration of the thiourea is 1.0 mol / L to 1.5 mol / L.

[0017] Optionally, in step S102, the room temperature incubation time is 18 min to 22 min.

[0018] Optionally, in step S103, the nucleic acid extraction is performed using magnetic bead extraction or column extraction.

[0019] Optionally, in step S104, the qPCR reagent includes primers, probes, Taq DNA polymerase, dNTPs, and Mg. 2+ And a buffer solution, wherein the primers are designed for the mycoplasma 16S rRNA gene.

[0020] Optionally, the primer sequences are: upstream primer: 5'-GGTGTTAATGATGAAGAAAC-3'; downstream primer: 5'-CTTAAGCCAACATCCTCTGC-3'; and the probe sequence is: 5'-FAM-CAGCAGTAGGGAATCTTCCACA-TAMRA-3'.

[0021] Optionally, in step S104, the qPCR reaction procedure is as follows: 40-50 cycles, each cycle consisting of 95℃ pre-denaturation for 3-5 minutes; 95℃ denaturation for 10-15 seconds; and 60℃ annealing extension for 30-40 seconds.

[0022] A kit for the above-mentioned cell preparation mycoplasma detection method includes a cell lysis buffer and a qPCR reagent. The cell lysis buffer comprises thiourea at a molar concentration of 0.8 mol / L to 1.8 mol / L, sodium dodecyl sulfate at a mass percentage of 1% to 3%, glycerol at a volume percentage of 5% to 15%, and Tris-HCl at a molar concentration of 10 mmol / L to 30 mmol / L. The pH of the cell lysis buffer is 8.0 to 8.5. The qPCR reagent comprises primers, probes, Taq DNA polymerase, dNTPs, and Mg2+. 2+ And buffer solution.

[0023] Beneficial effects

[0024] This invention adds a specific concentration of thiourea to the cell lysis buffer. Thiourea, as a strong denaturant, can effectively disrupt the cell membrane structure of cells and mycoplasma, promote the full lysis of cells and mycoplasma, and release more nucleic acids. At the same time, thiourea can also denature impurities such as proteins, reducing their inhibitory effect on subsequent nucleic acid extraction and PCR reaction, thereby improving the accuracy of detection. Detailed Implementation

[0025] One embodiment of the present invention provides a method for detecting mycoplasma in cell preparations, comprising the following steps:

[0026] S101: Solid-liquid separation: The cell preparation to be tested is separated into solid and liquid components to obtain cell precipitate;

[0027] S102: Cell lysis: Add cell lysis buffer to the cell pellet and suspend the cells in the lysis buffer, then incubate at room temperature for 15 min to 25 min. The cell lysis buffer contains thiourea with a molar concentration of 0.8 mol / L to 1.8 mol / L.

[0028] S103: Nucleic acid extraction: Nucleic acid is extracted from the lysis buffer to obtain nucleic acid;

[0029] S104: qPCR reaction: Nucleic acid is reacted with qPCR reagents, and then the fluorescence signal is collected by a real-time PCR instrument. The progress of the PCR reaction is monitored based on the changes in the fluorescence signal.

[0030] S105: Result Judgment: After the reaction is completed, the cycle threshold is obtained by testing, and the presence of mycoplasma in the cell preparation is determined based on the cycle threshold.

[0031] In the above embodiments, a specific concentration of thiourea was added to the cell lysis buffer. Thiourea, as a strong denaturant, effectively disrupts the cell membrane structure of cells and mycoplasma, promoting complete lysis and releasing more nucleic acids. Simultaneously, thiourea denatures proteins and other impurities, reducing their inhibitory effect on subsequent nucleic acid extraction and PCR reactions, thereby improving detection accuracy. Furthermore, in the above embodiments, controlling the incubation time effectively improves the lysis efficiency of cells and mycoplasma, reduces interference from impurities, and thus improves detection accuracy, laying a solid foundation for subsequent nucleic acid extraction and qPCR reactions.

[0032] In some embodiments, in step S101, the solid-liquid separation is performed by centrifugation at a speed of 800 r / min to 1500 r / min for a time of 5 min to 10 min. Centrifugation effectively separates cell precipitate and supernatant. Specific centrifugation speed and time ensure sufficient collection of cell precipitate, reduce the impact of impurities in the supernatant on subsequent detection steps, and guarantee the accuracy and stability of the detection.

[0033] In some embodiments, in step S102, the cell lysis buffer further includes 1%–3% sodium dodecyl sulfate (w / w), 5%–15% glycerol (v / v), and Tris-HCl (w / v) at a molar concentration of 10 mmol / L–30 mmol / L, with a pH of 8.0–8.5. In addition to thiourea, sodium dodecyl sulfate, glycerol, and Tris-HCl are added to the cell lysis buffer. Sodium dodecyl sulfate acts as a detergent, further disrupting the cell membrane structure and enhancing the lysis effect. Glycerol stabilizes the nucleic acid structure, preventing degradation during extraction. Tris-HCl maintains the pH stability of the lysis buffer, providing a suitable environment for cell lysis and nucleic acid stability. The synergistic effect of these components improves the lysis efficiency of cells and mycoplasma, reduces nucleic acid degradation, and provides high-quality samples for nucleic acid extraction.

[0034] In some embodiments, in step S102, the molar concentration of thiourea is 1.0 mol / L to 1.5 mol / L. This concentration range of thiourea can ensure good lysis while avoiding the inhibition of subsequent PCR reactions by high concentrations of thiourea, further improving the sensitivity and accuracy of detection.

[0035] In some embodiments, in step S102, the room temperature incubation time is 18 min to 22 min. This room temperature incubation time ensures sufficient lysis of cells and mycoplasma, while avoiding excessive incubation time that could lead to nucleic acid degradation, thus improving the quality and yield of nucleic acid extraction.

[0036] In some embodiments, in step S103, nucleic acid extraction is performed using either magnetic bead extraction or column extraction. Magnetic bead extraction and column extraction have the advantages of high extraction efficiency and high purity, effectively removing impurities and obtaining high-quality nucleic acids, providing excellent templates for qPCR reactions and ensuring the reliability of detection.

[0037] In some embodiments, in step S104, the qPCR reagent includes primers, probes, Taq DNA polymerase, dNTPs, and Mg. 2+ The buffer solution contains primers designed for the Mycoplasma 16S rRNA gene. Primers designed for the Mycoplasma 16S rRNA gene are highly specific, capable of specifically amplifying Mycoplasma nucleic acid, reducing non-specific amplification, decreasing false negative and false positive results, and improving the specificity and accuracy of detection.

[0038] In some embodiments, the primer sequences are: upstream primer: 5'-GGTGTTAATGATGAAGAAAC-3'; downstream primer: 5'-CTTAAGCCAACATCCTCTGC-3'; and the probe sequence: 5'-FAM-CAGCAGTAGGGAATCTTCCACA-TAMRA-3'. These sequences exhibit high specificity and sensitivity to mycoplasma, accurately identifying and amplifying the target nucleic acid, further ensuring the accuracy of the detection results.

[0039] In some embodiments, in step S104, the qPCR reaction program is as follows: 40-50 cycles, each cycle consisting of 95°C pre-denaturation for 3-5 minutes, 95°C denaturation for 10-15 seconds, and 60°C annealing extension for 30-40 seconds. In this qPCR reaction program, the pre-denaturation step fully activates enzyme activity, and the denaturation and annealing extension parameters ensure the specificity and efficiency of the PCR reaction, improving amplification efficiency and detection sensitivity, and ensuring accurate detection even at low concentrations of mycoplasma. For example, 95°C pre-denaturation for 3-5 minutes can fully activate Taq DNA polymerase activity, and the cycle parameters of 95°C denaturation for 10-15 seconds and 60°C annealing extension for 30-40 seconds can ensure the specificity and efficiency of the PCR reaction and improve detection sensitivity.

[0040] Another embodiment of the present invention provides a kit for the above-mentioned cell preparation mycoplasma detection method, comprising cell lysis buffer and qPCR reagent. The cell lysis buffer comprises thiourea at a molar concentration of 0.8 mol / L to 1.8 mol / L, sodium dodecyl sulfate at a mass percentage of 1% to 3%, glycerol at a volume percentage of 5% to 15%, and Tris-HCl at a molar concentration of 10 mmol / L to 30 mmol / L. The pH of the cell lysis buffer is 8.0 to 8.5. The qPCR reagent comprises primers, probes, Taq DNA polymerase, dNTPs, and Mg... 2+ The kit contains the key reagents required for the assay. The reagents are stable and optimized, easy to use, and ensure the repeatability and reliability of the assay method. This facilitates its widespread application in practical testing and meets the testing needs of various scenarios.

[0041] Examples 1-3, Comparative Examples 1-3

[0042] The components of the cell lysis buffers in the examples and comparative examples are shown in Table 1.

[0043] Table 1

[0044]

[0045] The cell preparations for mycoplasma detection in Examples 1-3 and Comparative Examples 1-3 include the following steps:

[0046] S101: Solid-liquid separation: Take 1 mL of the cell preparation to be tested, place it in a 1.5 mL centrifuge tube, centrifuge at 1200 r / min for 8 min, discard the supernatant, and obtain the cell pellet.

[0047] S102: Cell lysis: Add 200 μL of each cell lysis buffer from Table 1 to the cell pellet, gently pipette to suspend the cells in the lysis buffer, and then incubate at room temperature for 20 min.

[0048] S103: Nucleic acid extraction: Nucleic acid was extracted using the magnetic bead method to obtain a nucleic acid solution, which was then stored at -20℃ for later use.

[0049] S104: qPCR reaction: The qPCR reaction system is 25 μL, including 12.5 μL of 2×qPCRMix (containing Taq DNA polymerase, dNTPs, Mg). 2+ The following reagents were prepared: buffer solution, upstream primer (5'-GGTGTTAATGATGAAGAAAC-3', 10 μmol / L) 0.5 μL, downstream primer (5'-CTTAAGCCAACATCCTCTGC-3', 10 μmol / L) 0.5 μL, probe (5'-FAM-CAGCAGTAGGGAATCTTCCACA-TAMRA-3', 10 μmol / L) 0.5 μL, nucleic acid template 5 μL, and ddH2O to bring the total volume to 25 μL.

[0050] The qPCR reaction program was as follows: 95℃ pre-denaturation for 4 min; 95℃ denaturation for 12 s; 60℃ annealing and extension for 35 s, for a total of 40 cycles. Fluorescence signals were collected during the annealing and extension phase of each cycle.

[0051] S105: Result Interpretation: After the reaction, the cycle threshold (Ct value) was obtained by analysis using a real-time PCR instrument. When the Ct value is ≤35, it is judged as positive, that is, mycoplasma is present in the cell preparation; when the Ct value is >35, it is judged as negative, that is, mycoplasma is not present in the cell preparation.

[0052] Test case

[0053] (1) Ct value: The Ct values ​​of each example and comparative example were directly read using a real-time PCR instrument. The smaller the Ct value, the more mycoplasma nucleic acid was detected, and the higher the detection sensitivity. The test results are shown in Table 2.

[0054] Table 2

[0055]

[0056] As shown in Table 2, the Ct values ​​of Examples 1-3 are all lower than those of Comparative Examples 1-3, indicating that the detection method of the present invention can detect mycoplasma in cell preparations more sensitively. Among them, Example 2 has the smallest Ct value and the highest detection sensitivity.

[0057] (2) Detection sensitivity: The Mycoplasma pneumoniae standard was serially diluted (10⁻⁶ mcg / mcg) 0 ~10 -6 The samples (copies / μL) were detected using the detection methods of Examples 2 and Comparative Examples 1-3, respectively. The lowest detectable concentration of mycoplasma, i.e., the detection sensitivity, was recorded. The test results are shown in Table 3.

[0058] Table 3

[0059] Group Mycoplasma pneumoniae Reproductive tract mycoplasma Mycoplasma hominis Example 2 <![CDATA[10 -4 ]]> <![CDATA[10 -4 ]]> <![CDATA[10 -3 ]]> Comparative Example 1 <![CDATA[10 -2 ]]> <![CDATA[10 -2 ]]> <![CDATA[10 -2 ]]> Comparative Example 2 <![CDATA[10 -3 ]]> <![CDATA[10 -3 ]]> <![CDATA[10 -2 ]]> Comparative Example 3 <![CDATA[10 -3 ]]> <![CDATA[10 -3 ]]> <![CDATA[10 -2 ]]>

[0060] As shown in Table 3, the detection sensitivity of Example 2 is significantly higher than that of Comparative Examples 1-3, and it can detect lower concentrations of mycoplasma. Comparative Example 1 has the lowest detection sensitivity because its cell lysis buffer does not contain thiourea, resulting in poor lysis. The thiourea concentrations of Comparative Examples 2 and 3 are not within the range specified in this invention, and their detection sensitivity is also lower than that of Example 2.

[0061] (3) Specificity test: Using Mycoplasma pneumoniae, Mycoplasma genitalium, and Mycoplasma hominis standards, as well as the nucleic acids of non-mycoplasma microorganisms such as Escherichia coli, Staphylococcus aureus, and fungi, as templates, the detection method of Example 2 was used for detection. The presence of specific amplification was observed to evaluate the specificity of the detection method. The test results are shown in Table 4.

[0062] Table 4

[0063] template Whether amplification occurred (Ct value) Mycoplasma pneumoniae Yes (25.6) Reproductive tract mycoplasma Yes (26.3) Mycoplasma hominis Yes (27.1) E. coli No (>40) Staphylococcus aureus No (>40) fungi No (>40)

[0064] As shown in Table 4, the detection method of Example 2 can specifically amplify Mycoplasma pneumoniae, Mycoplasma genitalium, and Mycoplasma hominis, but does not amplify non-mycoplasma microorganisms such as Escherichia coli, Staphylococcus aureus, and fungi, indicating that the detection method of the present invention has high specificity.

[0065] (4) Repeatability test: For the same positive cell preparation sample, the detection method of Example 2 was repeated 6 times, and the coefficient of variation (CV) of the Ct value was calculated to evaluate the repeatability of the detection method. The test results are shown in Table 5.

[0066] Table 5

[0067]

[0068] As shown in Table 5, the coefficient of variation of Ct values ​​after six repeated tests using the detection method of Example 2 is 0.68%, which is less than 5%, indicating that the detection method of the present invention has good repeatability.

[0069] Comparative Example 4

[0070] The cell lysis buffer from Example 2 was used, and the cells were incubated at room temperature for 20 min. However, conventional PCR methods were employed for detection, with primers and reaction conditions identical to those described above. The PCR products were detected by agarose gel electrophoresis. The detection results of Comparative Example 4 required observation via agarose gel electrophoresis, making quantitative analysis impossible, and the detection sensitivity was low. When the mycoplasma concentration was low (e.g., 10...),... -4 In Example 4, no amplification product was detected (copies / μL), while in Example 2, it was accurately detected by Ct value. Furthermore, conventional PCR methods are relatively cumbersome and time-consuming, while qPCR methods allow for real-time monitoring of the reaction process and rapid detection results.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0072] For those skilled in the art, based on the ideas of this invention, there will be changes in the specific implementation methods and application scope. Therefore, the content of this specification should not be construed as a limitation of this invention.

Claims

1. A method for detecting mycoplasma in cell preparations, characterized in that, Includes the following steps: S101: Solid-liquid separation: The cell preparation to be tested is subjected to solid-liquid separation to obtain cell precipitate; S102: Cell lysis: Add cell lysis buffer to the cell pellet and suspend the cells in the lysis buffer, then incubate at room temperature for 15 min to 25 min, wherein the cell lysis buffer comprises thiourea with a molar concentration of 0.8 mol / L to 1.8 mol / L; S103: Nucleic acid extraction: Nucleic acid is extracted from the lysis buffer to obtain nucleic acid; S104: qPCR reaction: The nucleic acid is reacted with qPCR reagent, and then the fluorescence signal is collected by a real-time PCR instrument. The progress of the PCR reaction is monitored based on the changes in the fluorescence signal. S105: Result judgment: After the reaction is completed, the cycle threshold is obtained by testing, and the presence of mycoplasma in the cell preparation is determined based on the cycle threshold.

2. The method for detecting mycoplasma in cell preparations as described in claim 1, characterized in that, In step S101, the solid-liquid separation method is centrifugation, with a centrifugation speed of 800 r / min to 1500 r / min and a centrifugation time of 5 min to 10 min.

3. The method for detecting mycoplasma in cell preparations as described in claim 1, characterized in that, In step S102, the cell lysis buffer further includes sodium dodecyl sulfate at a mass percentage of 1% to 3%, glycerol at a volume percentage of 5% to 15%, and Tris-HCl at a molar concentration of 10 mmol / L to 30 mmol / L, and the pH of the cell lysis buffer is 8.0 to 8.

5.

4. The method for detecting mycoplasma in cell preparations as described in claim 1, characterized in that, In step S102, the molar concentration of the thiourea is 1.0 mol / L to 1.5 mol / L.

5. The method for detecting mycoplasma in cell preparations as described in claim 1, characterized in that, In step S102, the incubation time at room temperature is 18 min to 22 min.

6. The method for detecting mycoplasma in cell preparations as described in claim 1, characterized in that, In step S103, the nucleic acid extraction is performed using either magnetic bead extraction or column extraction.

7. The method for detecting mycoplasma in cell preparations according to any one of claims 1-6, characterized in that, In step S104, the qPCR reagent includes primers, probes, Taq DNA polymerase, dNTPs, and Mg. 2+ And a buffer solution, wherein the primers are designed for the mycoplasma 16S rRNA gene.

8. The method for detecting mycoplasma in cell preparations as described in claim 7, characterized in that, The primer sequences are: upstream primer: 5'-GGTGTTAATGATGAAGAAAC-3'; downstream primer: 5'-CTTAAGCCAACATCCTCTGC-3'; the probe sequence is: 5'-FAM-CAGCAGTAGGGAATCTTCCACA-TAMRA-3'.

9. The method for detecting mycoplasma in cell preparations as described in any one of claims 1-6, characterized in that, In step S104, the qPCR reaction procedure is as follows: 40-50 cycles are performed, with each cycle consisting of 95℃ pre-denaturation for 3-5 minutes, 95℃ denaturation for 10-15 seconds, and 60℃ annealing extension for 30-40 seconds.

10. A kit for the mycoplasma detection method of cell preparations according to any one of claims 1-9, characterized in that, The reagent includes cell lysis buffer and qPCR reagents. The cell lysis buffer comprises thiourea at a molar concentration of 0.8 mol / L to 1.8 mol / L, sodium dodecyl sulfate at a mass percentage of 1% to 3%, glycerol at a volume percentage of 5% to 15%, and Tris-HCl at a molar concentration of 10 mmol / L to 30 mmol / L. The pH of the cell lysis buffer is 8.0 to 8.

5. The qPCR reagents include primers, probes, Taq DNA polymerase, dNTPs, and Mg2+. 2+ And buffer solution.

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