10 CFU mycoplasma standard strain stationary liquid applicable to sensitivity verification of mycoplasma rapid detection method

Abstract

The invention belongs to the technical field of microorganism preservation, and particularly relates to 10 CFU mycoplasma standard strain stationary liquid applicable to sensitivity verification of a mycoplasma rapid detection method. The stationary liquid comprises a basal culture medium, a low-temperature protective agent and additive components, wherein the additive components comprise ethanolamine, insulin, transferrin, human serum albumin, vitamin C, a cell membrane stabilizer and a nuclease inhibitor. By using the stationary liquid provided by the invention, 10 CFU mycoplasma standard strains can be preserved for 15 months at-20 DEG C, the integrity of the mycoplasma cell structure can be basically maintained, and the mycoplasma nucleic acid can be stably preserved; in addition, the mycoplasma standard strain can tolerate thermal change and freeze-thaw change at 37 DEG C, and has good thermal stability and freeze-thaw stability, so that the technical problems of harsh transportation and storage conditions, short preservation time and poor stability of the 10 CFU mycoplasma standard strain are solved.

Description

Technical Field

[0001] This invention belongs to the field of microbial preservation technology, specifically relating to a 10 CFU mycoplasma standard strain fixative suitable for sensitivity verification of rapid mycoplasma detection methods. Background Technology

[0002] Mycoplasma contamination poses a potential safety risk in the development and production of cell therapy and biopharmaceutical products. To mitigate this risk, the Chinese Pharmacopoeia (ChP), European Pharmacopoeia (EP), United States Pharmacopoeia (USP), and Japanese Pharmacopoeia (JP) all stipulate relevant regulations for mycoplasma detection methods, with microbiological detection methods considered the "gold standard." However, these procedures are time-consuming (at least 28 days) and unsuitable for products with short shelf lives requiring rapid release. Nucleic acid amplification (NAT) technology offers advantages such as speed and high throughput, and is increasingly being used as an alternative to traditional microbiological methods. The application of NAT requires validation that it achieves a limit of detection (LOD) comparable to or better than culture methods. According to USP and EP requirements, NAT methods should undergo sensitivity validation, with a sensitivity of 10 CFU / mL being sufficient to replace culture methods. Therefore, the preparation and preservation of mycoplasma standard strains for assessing the reliability of the LOD of NAT detection methods is crucial.

[0003] Mycoplasma are the smallest known prokaryotes, characterized by the lack of a cell wall, making them highly sensitive to osmotic pressure, drying, chemical reagents, and mechanical shear forces. During NAT validation, 10 CFU of mycoplasma standard strains are mixed with the test sample matrix to simulate mycoplasma contamination. Due to the large sample volume or the inhibitory effect of the sample matrix, the simulated sample needs to be centrifuged to remove the sample matrix and enrich the mycoplasma. Maintaining the integrity of the mycoplasma structure ensures that this operation does not result in significant cell loss. 10 CFU of mycoplasma standard strains are trace DNA samples with low nucleic acid concentrations, making them difficult to preserve. If cell disruption releases nucleases or if additional matrix or consumables introduce nuclease contamination, there is a risk of degradation, leading to increased Ct values ​​or even negative results in PCR detection. This is ineffective for assessing the sensitivity of the detection method. Therefore, the 10 CFU mycoplasma standard strain fixative should maintain cell membrane integrity for a certain period to ensure centrifugation collection, while simultaneously maintaining the integrity of the nucleic acid structure to exhibit stable Ct values.

[0004] Traditional methods for preserving mycoplasma include freeze-drying and cryopreservation. Cryopreservation is further divided into liquid nitrogen gas phase preservation (-150°C to -196°C), -80°C freezing, and -20°C freezing. All these methods require suitable cryoprotectants. Freeze-drying offers the longest shelf life and excellent stability. Commonly used cryoprotectant formulations include 10% skim milk or 10% horse serum + 5% trehalose or 5% sucrose, or the addition of 1% monosodium glutamate. This method requires vacuum freeze-drying equipment and optimized freeze-drying procedures, making the operation relatively cumbersome. Liquid nitrogen preservation offers good stability, second only to freeze-drying. Commonly used cryoprotectant formulations include culture medium + 10-15% glycerol or 5-10% DMSO + 10-20% horse serum or fetal bovine serum. This method requires periodic replenishment of liquid nitrogen, resulting in high maintenance costs; it is inconvenient to handle, requiring protective equipment; and transportation is relatively difficult. The -80℃ preservation method offers good preservation results, the equipment is widely available, and the operation is relatively simple, facilitating access and management. The commonly used cryoprotectant formulation is similar to that of the liquid nitrogen method. The -20℃ preservation method is the most commonly used method in laboratories. It is extremely simple, requires no special equipment, and has the lowest cost. However, due to temperature fluctuations caused by frequent door opening and closing, it is prone to repeated freeze-thaw cycles. The cryoprotectant formulation is similar to that of the liquid nitrogen method, but this method causes greater ice crystal damage and does not achieve the same preservation effect as the liquid nitrogen and -80℃ methods. It is suitable for short-term preservation of high-concentration mycoplasma bacterial cultures.

[0005] For 10 CFU mycoplasma standard strains, the low bacterial concentration and low nucleic acid content make it difficult to balance preservation performance and ease of use with the above preservation methods. Minerva Biolabs' 10 CFU™ Sensitivity Standards (catalog number: 102-0002), in unopened lyophilized powder form, can be stored for 2 years at 2-8°C. After reconstitution, the solution should be stored at -18°C or below; however, no information was found regarding the storage period. Huzhou Shenke Biotechnology Co., Ltd.'s 10 CFU Mycoplasma oralis verification strain (catalog number: 1501501) and 10 CFU Mycoplasma pneumoniae verification strain (catalog number: 1501503), in inactivated bacterial solution form, have a shelf life of 6 months when stored at -65°C or below. Therefore, it is essential to develop a fixative solution suitable for 10 CFU mycoplasma standard strains that can be stably stored for a long time under non-lyophilized conditions and at relatively high freezing temperatures. Summary of the Invention

[0006] Based on the above background, this invention provides an innovative fixative that can not only stably preserve low concentrations of mycoplasma cells, but also effectively stabilize low concentrations of mycoplasma DNA. This fixative employs a unique composite formula of "culture medium + cryoprotectant + cell membrane stabilizing component + nuclease inhibitor," with the following specific mechanisms of action: 1) The culture medium provides a suitable survival environment, including maintaining appropriate osmotic pressure, pH value, and providing basic nutrients, ensuring the cells are in a relatively stable state; 2) The cryoprotectant and cell membrane stabilizing component work synergistically to effectively combat mechanical damage from ice crystal formation, osmotic shock, damage caused by cell membrane phase transitions, and oxidative stress damage, thereby significantly reducing cell membrane rupture and maintaining the integrity of the cell structure; 3) The nuclease inhibitor EDTA-2Na effectively inhibits nuclease activity by strongly chelating divalent metal ions, preventing nucleic acid degradation and ensuring that the integrity and quantity of nucleic acids are not compromised. For samples with a low absolute molecular weight, such as 10 CFU mycoplasma standard strains, the fixative should be used in conjunction with packaging materials that are low in adsorption, DNAase-free, and have good sealing properties to minimize losses caused by physical adsorption and nuclease degradation.

[0007] The specific performance characteristics of this fixative are as follows: This invention designs a fixative for preserving 10 CFU of mycoplasma standard strains without lyophilization, in the form of an inactivated bacterial solution. 1) It can be stably preserved for 7 days at 37℃; 2) It can be stably stored for at least 15 months at -20℃; 3) It can withstand 7 freeze-thaw cycles when switching between -20℃ and room temperature. The 10 CFU of mycoplasma standard strains stored using this fixative have a significantly extended shelf life, excellent thermal stability and freeze-thaw stability, demonstrating advantages such as simple preservation process, convenient access, and easy transportation. This makes it easy to transport and preserve under normal laboratory conditions, greatly improving the preservation quality and ease of use of the samples.

[0008] The specific technical solution of this application is as follows: A 10 CFU mycoplasma standard fixative for sensitivity validation of rapid mycoplasma detection methods includes basal culture medium, cryoprotectant and additives. The basal medium is IMDM medium; The cryoprotectant is 10% (vol / vol) DMSO; The added ingredients are ethanolamine 0.5 mM, insulin 10 mg / L, transferrin 50 mg / L, human serum albumin 2 g / L, vitamin C 50 mg / L, Kolliphor P 188 1 g / L, and EDTA-2Na 10 mM.

[0009] Preferably, the insulin is recombinant human insulin.

[0010] Preferably, the transferrin is recombinant human transferrin.

[0011] Preferably, the human serum albumin is recombinant human serum albumin.

[0012] Beneficial Effects: This fixative comprises a basal culture medium, a cryoprotectant, and additives, including ethanolamine, insulin, transferrin, human serum albumin, vitamin C, a cell membrane stabilizer, and a nuclease inhibitor. This invention relates to a fixative that reduces mycoplasma cell membrane damage, essentially maintaining cell structural integrity, while also exhibiting nuclease inhibition, slowing nucleic acid degradation, and stably preserving trace amounts of nucleic acid without affecting subsequent DNA extraction and amplification. Using the fixative described in this invention, 10 CFU of mycoplasma standard strains can be stably stored for at least 15 months at -20°C without lyophilization. Furthermore, it can withstand thermal changes up to 37°C and freeze-thaw cycles, exhibiting good thermal and freeze-thaw stability, thus solving the technical problems of harsh transportation and storage conditions, short storage time, and poor stability of 10 CFU of mycoplasma standard strains. Detailed Implementation

[0013] The technical solutions of the present invention will be further described below with reference to specific embodiments. The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0014] Terminology Explanation: qPCR: Quantitative Real-time Olymerase Chain Reaction; Ct value: Cycle threshold; GC / CFU: Genomic Copy / Colony Forming Unit; PC: Positive Control; NTC: No Template Control. NCS: Negative Control Sample; CG: Control Group.

[0015] A 10 CFU mycoplasma standard strain fixative for sensitivity validation of rapid mycoplasma detection methods includes a basal culture medium, a cryoprotectant, and additives, wherein the additives are ethanolamine, insulin, transferrin, human serum albumin, vitamin C, cell membrane stabilizer, and nuclease inhibitor.

[0016] The basal culture medium used was phenol red-free IMDM medium (manufacturer: Wuhan Pronosai, catalog number: PM150515). The added components were ethanolamine 0.5 mM, insulin 10 mg / L, transferrin 50 mg / L, human serum albumin 2 g / L, vitamin C 50 mg / L, cell membrane stabilizer 1 g / L, and nuclease inhibitor 10 mM.

[0017] Preferably, the insulin is recombinant human insulin. The transferrin is recombinant human transferrin. The human serum albumin is recombinant human serum albumin. The cell membrane stabilizer is Kolliphor P 188. The nuclease inhibitor is EDTA-2Na. The cryoprotectant is 10% (vol / vol) DMSO.

[0018] The preparation method of fixative for 10 CFU of mycoplasma standard strains includes the following steps: A high-concentration stock solution containing ethanolamine, insulin, transferrin, human serum albumin, vitamin C, Kolliphor P 188, and EDTA-2Na is prepared using a suitable solvent and sterilized by filtration through a 0.22 μm filter membrane. The required volume of each additive stock solution and DMSO is measured, and the volume is brought up to the required level with 1MDM medium. The solution is then sterilized by filtration through a 0.22 μm filter membrane to obtain the fixative. The fixative should be stored at 2–8°C, protected from light.

[0019] The method for using the 10 CFU mycoplasma standard fixative includes the following steps: The fixative can be used as a dilution buffer for mycoplasma bacterial suspension. Mycoplasma cultured to the early logarithmic growth stage is serially diluted with the fixative to obtain a 10 CFU mycoplasma standard. During the serial dilution process, the fixative gradually replaces the original solution. After dilution, it can be directly aliquoted into cryovials for storage, eliminating the need for mixing the fixative with the mycoplasma standard. Low-adsorption, DNase-free cryovials with good sealing properties should be used, and storage should be at -20°C or below.

[0020] Example

[0021] 1. Thermal accelerated stability test at 37℃ Test objective: 1) Simulate deviations in non-cold chain transportation or short-term storage to assess stability after short-term exposure to high-temperature conditions. For example, ice pack failure or temporary refrigerator malfunction during transportation.

[0022] 2) Predicting long-term stability: Based on the kinetic principle of accelerated degradation at high temperatures, the shelf life at the recommended storage temperature (-20℃) can be estimated in a short period of time.

[0023] Test method: The samples prepared in this invention were stored in a 37°C incubator at different time points and compared with the control samples that were always stored at the recommended storage temperature (-20°C). All samples were simultaneously subjected to DNA extraction and qPCR detection, and the ΔCt of the target nucleic acid was calculated as Ct(sample stored at 37°C) - Ct(sample stored at -20°C).

[0024] Sample: See Table 1.

[0025] Table 1. Information on 10 CFU Mycoplasma pneumoniae standard strains preserved in fixative according to this invention.

[0026] Extraction and purification of mycoplasma DNA (Reagent kit: EcoSy Biotech, Catalog No.: CRB00-0032): 10 CFU of Mycoplasma pneumoniae standard strain were supplemented with 0.9 mL of sterile 1×PBS solution. DNA extraction was then performed using the following two methods: 0.5 mL direct extraction method: Take 0.5 mL for DNA extraction and purification; 0.5 mL centrifugation method: Take 0.5 mL, centrifuge at 16,000 × 10⁻⁶. g Centrifuge for 20 min, remove 400 µL of supernatant, add 400 µL of sterile 1×PBS solution, resuspend thoroughly, and then extract and purify DNA.

[0027] qPCR detection (Reagent kit: EcoSy Biotech, Catalog No.: CRB00-1012): The total nucleic acid content was detected by the 0.5 mL direct extraction method, indicating that the fixative maintained the stability of mycoplasma nucleic acid; the nucleic acid content of the centrifuged bacterial cells was detected by the 0.5 mL centrifugation method, indicating that the fixative maintained the integrity of the mycoplasma cell structure.

[0028] Test results: qPCR results showed that, using two DNA extraction methods, samples stored at 37℃ for 3, 5, and 7 days under accelerated thermal conditions did not show a decreasing trend in the detection signal of the target nucleic acid compared to the control group (stored at -20℃), demonstrating good thermal stability. The results are shown in Tables 2 and 3.

[0029] Table 2. Results of qPCR detection using direct extraction from 0.5 mL samples stored at 37℃.

[0030] In Table 2, VIC is the detection channel for the internal reference gene (Recovery Control), used to monitor the extraction and purification process of mycoplasma DNA. ΔCt (sample-PC) *1 The difference between the PC (sample detection) and the Ct (sample CG) value is represented; a value ≤ 1 indicates that the DNA extraction process was effective. FAM is the detection channel for the target nucleic acid (mycoplasma target gene), ΔCt(sample - CG). *2 The difference in Ct values ​​represents the difference between samples stored at 37°C and the control group (stored at -20°C). A smaller ΔCt value indicates a smaller difference, meaning a better fixative effect on maintaining the stability of mycoplasma nucleic acids. No Ct *3 This indicates that after the qPCR reaction procedure set according to the CRB00-1012 instruction manual was completed, the fluorescence signal did not reach the set fluorescence threshold. This result is judged as the target nucleic acid in the sample not being detected (negative).

[0031] Table 3. Results of qPCR detection by centrifugation of samples stored at 37℃ (0.5 mL).

[0032] In Table 3, △Ct (sample - CG) *4 The difference in Ct values ​​between the sample stored at 37°C and the control group (stored at -20°C) is represented by ΔCt. The smaller the ΔCt value, the smaller the difference, indicating that the fixative is more effective in maintaining the structural integrity of mycoplasma cells.

[0033] 2. Real-time long-term storage test at -20℃ Test objective: To simulate the actual recommended storage temperature (-20℃) and evaluate the stability under long-term storage conditions.

[0034] Test method: The samples prepared in this invention were stored at -20°C for different time points and compared with the control sample at the initial zero point. After DNA extraction and qPCR detection, it was determined whether the detection signal of the target nucleic acid could be stably reported.

[0035] Sample: See Table 4.

[0036] Table 4 Information on 10 CFU Mycoplasma standard strains preserved in fixative according to the present invention.

[0037] Extraction and purification of mycoplasma DNA (Reagent kit: EcoSy Biotech, Catalog No.: CRB00-0032): Add 0.9 mL of sterile 1×PBS solution to the 10 CFU mycoplasma standard strain. Centrifuge at 16,000 × 10⁻⁵ mL: gCentrifuge for 20 min, remove 400 µL of supernatant, add 400 µL of sterile 1×PBS solution, resuspend thoroughly, and then extract and purify DNA.

[0038] qPCR detection (Reagent kit: EcoSai Biotechnology, catalog number: CRB00-1012).

[0039] Test Results: qPCR results showed that, using the fixative solution described in this invention, the two 10 CFU Mycoplasma standard strains, when stored at -20℃ for 15 months and 17 months respectively, exhibited stable Ct values ​​for the target nucleic acids compared to the initial zero-temperature control samples, with no significant degradation observed. This indicates that they can achieve long-term stable storage at -20℃ for at least 15 months. Data from the 10 CFU Mycoplasma pneumoniae standard strains further suggest that their stable storage period could potentially be extended to 17 months. Results are shown in Tables 5 and 6.

[0040] Table 5. Results of qPCR detection of 10 CFU oral mycoplasma standard strains stored at -20℃ for 15 months.

[0041] Note: PC *5 and PC *6 The batch numbers are different. According to the CRB00-1012 instructions, for the VIC channel, only the ΔCt (sample-PC) needs to be monitored (the Recovery Control added to the sample should be from the same batch number as the PC used for qPCR testing). A value ≤1 indicates that the DNA extraction process of the sample is effective. For the FAM channel, a PC Ct ≤25 and a valid "S"-shaped amplification curve are sufficient, indicating that the sensitivity of the qPCR test kit is qualified.

[0042] Table 6. Results of qPCR detection of 10 CFU Mycoplasma pneumoniae standard strains stored at -20℃ for 17 months

[0043] Note: PC *7 and PC *8 Different batch numbers. Explanation as in the notes of Table 5.

[0044] 3. Freeze-thaw stability test Test objective: To simulate real-world freeze-thaw cycles and evaluate stability during repeated freezing and thawing processes, such as transportation from the production site to the point of use.

[0045] Test method: The samples prepared in this invention were subjected to "-20℃" test times at different rates. Freeze-thaw cycles at room temperature were performed and compared with control samples that were always stored at the recommended storage temperature (-20°C). All samples were simultaneously subjected to DNA extraction and qPCR detection, and the target nucleic acid ΔCt was calculated as Ct(freeze-thawed sample) - Ct(sample stored at -20°C).

[0046] Sample: See Table 1.

[0047] Extraction and purification of mycoplasma DNA: Same as in Example 1.

[0048] qPCR detection: Same as in Example 1.

[0049] Test results: qPCR results showed that two DNA extraction methods were used at -20℃. Compared with the unfrozen control group (stored at -20℃ throughout), samples subjected to 3, 5, and 7 freeze-thaw cycles at room temperature did not show a decreasing trend in the detection signal of the target nucleic acid, demonstrating good freeze-thaw stability. The results are shown in Tables 7 and 8.

[0050] Table 7 Results of qPCR detection using direct extraction from 0.5 mL of frozen-thawed samples

[0051] In Table 7, △Ct (sample - CG) *9 Represents -20℃ The difference in Ct values ​​between the room temperature freeze-thawed samples and the unfrozen control group (always stored at -20℃) is considered. The smaller the ΔCt value, the smaller the difference, indicating that the fixative solution is more effective in maintaining the stability of mycoplasma nucleic acid.

[0052] Table 8 Results of qPCR detection using 0.5 mL centrifugation method for frozen-thawed samples

[0053] In Table 8, △Ct (sample - CG) *10 Represents -20℃ The difference in Ct values ​​between the room temperature freeze-thawed samples and the unfrozen control group (always stored at -20℃) is considered. The smaller the ΔCt value, the smaller the difference, indicating that the fixative is more effective in maintaining the structural integrity of mycoplasma cells.

[0054] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A 10 CFU mycoplasma standard strain fixative for sensitivity verification of rapid mycoplasma detection methods, characterized in that, Includes basal culture medium, cryoprotectant, and additives; The basal medium is IMDM medium; The cryoprotectant is 10% (vol / vol) DMSO; The added ingredients are ethanolamine 0.5 mM, insulin 10 mg / L, transferrin 50 mg / L, human serum albumin 2 g / L, vitamin C 50 mg / L, Kolliphor P 188 1 g / L, and EDTA-2Na 10 mM.

2. The 10 CFU mycoplasma standard fixative solution for sensitivity verification of a rapid mycoplasma detection method as described in claim 1, characterized in that, The insulin in question is recombinant human insulin.

3. The 10 CFU mycoplasma standard fixative solution for sensitivity verification of a rapid mycoplasma detection method as described in claim 1, characterized in that... The transferrin in question is recombinant human transferrin.

4. The 10 CFU mycoplasma standard fixative solution for sensitivity verification of a rapid mycoplasma detection method as described in claim 1, characterized in that, The human serum albumin mentioned is recombinant human serum albumin.

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