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发光法支原体检测试剂盒
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为什么选择此级别
BioReagent,无菌,用于化学发光,用于细胞培养 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
避光,-20°C储存。超低温运输 。请查阅批次 COA 获取详细规格。
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SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
发光法支原体检测试剂盒利用支原体特有激酶的活性并通过 ATP 依赖的荧光素酶催化的发光反应进行检测，通过比较检测试剂加入前后 ATP 含量的变化来确定样品是否有支原体污染。支原体被裂解后，会释放特有激酶与底物相互反应，可催化ADP转换为ATP。
整个实验操作分为两步，第一步是在样品中加入支原体检测试剂 A 检测样品中原有 ATP 的含量；第二步是加入支原体检测试剂B，如果样品有支原体污染，其特有的激酶可以催化 ADP 转换为 ATP，此时检测的就是原有的本底 ATP 含量和由支原体特有酶催化新生成的 ATP 含量的总和。通过计算发光读值 B 与 A 的比值就可以判断是否有支原体污染。如果比值大于1.2 表示有支原体污染，比值越大说明污染程度越高，如果比值小于 0.9 说明没有支原体污染，如果比值介于 0.9 和 1.2 之间，建议原细胞（包含原培养液）继续培养 24-48 h 之后再测试一次。
适用样本：细胞
产品组分表
| L1506760 | Component | 20T | 200T | Storage |
| L1506760A | Mycoplasma Assay Buffer | 1 mL | 10 mL | -20℃. Store in the dark. |
| L1506760B | Luciferase | 1 μL | 10 μL | -20℃ |
| L1506760C | Mycoplasma Substrate A | 1 EA | 1 EA | -20℃. Store in the dark. |
| L1506760D | Mycoplasma Reagent B | 1 mL | 10 mL | -20℃ |
| L1506760E | Postive Control | 200 μL | 1 mL | -20℃ |
自备仪器和试剂
1、无菌离心管、可调节式移液枪及枪头
2、离心机、96 孔全黑酶标板或全白酶标板
3、灭菌水、PBS 或新鲜培养基
4、发光检测仪或多功能酶标仪
实验流程
1、试剂准备
| 试剂名称 | 试剂准备 | 注意事项 |
| Mycoplasma Assay Buffer | 即用型；使用前平衡到室温 | -20℃避光保存 |
| Luciferase | 即用型 | -20℃保存 |
| Mycoplasma Reagent A | 临用前配制；用 Mycoplasma Assay Buffer 溶解 Mycoplasma Substrate A，然后将溶解后的Mycoplasma Substrate A 和 Luciferase 加入到 Mycoplasma Assay Buffer 瓶中，充分混匀后得到 Mycoplasma Reagent A | 按使用需求分装并避光保存于-20℃；使用前，平衡到室温 |
| Mycoplasma Reagent B | 即用型；使用前平衡到室温 | 按使用需求分装；-20℃保存 |
| Postive Control | 即用型；使用前平衡到室温 | 按使用需求分装；-20℃保存 |
注意：所有试剂避免反复冻融，建议分装使用，分装的容器不能有 ATP 污染。
2、样本制备
2.1 贴壁细胞
细胞消化前取 0.2-1 mL 细胞培养上清，1,500 rpm 离心 5 min，取上清液用于检测。
2.2 悬浮细胞
细胞传代时取培养液 0.2-1 mL 细胞培养液，1,500 rpm 离心 5 min，取上清液用于检测。
2.3 复苏细胞
冻存细胞解冻后添加进新鲜的完全培养基，培养 1-2 h 后取 0.2-1 mL 细胞培养液，1,500 rpm离心5 min，取上清液用于检测。
注意：
①、细胞传代后或消化后，支原体检测信号将会降低。如在细胞传代或消化后取样检测，需在传代或消化完毕的24 h 后取样。
②、上清样品最好收集后立即检测，也可以在 4℃放置并当天检测，或者置于-80℃保存半年内检测。低温保存的样品检测的时候需平衡至室温后才可用于检测。
3、实验步骤
3.1 在 96 孔全黑或全白酶标板中分别加入 50 μL 的待检样品、Postive Control、阴性对照（如灭菌水、PBS、新鲜培养基）。
3.2 每孔加入 50 μL Mycoplasma Reagent A，混匀后室温静置 5 min，然后用多功能酶标仪测定化学发光值RLUA
3.3 每孔加入 50 μL Mycoplasma Reagent B，混匀后室温静置 10 min，然后用多功能酶标仪测定化学发光值RLUB。
3.4 计算比值（Ratio）=RLUB/RLUA。参考表1，比值大于 1.2 说明有支原体污染，比值小于 0.9 表示没有支原体污染，比值在0.9-1.2之间可以将原样品继续培养 24-48 h 后重新检测。
表1. 发光法支原体检测试剂盒结果分析
| Ratio | 结果分析 | 处理方法 |
| ＜0.9 | 阴性 | 无需处理 |
| 0.9-1.2 | 可疑 | 继续隔离培养培养 24-48 h 后再进行测试 |
| ＞1.2 | 阳性 | 细胞灭菌处理后丢弃或隔离后用专用的支原体预防或去除试剂处理 |
注意：
①、多功能酶标仪每孔的读数时间设置为 1,000 ms，需严格按照加入 Mycoplasma Reagent B 10min后进行检测，切勿提前或延后，否则可能会影响比值在临界值附近样品的结果判断。
②、本方法的最佳实验温度是 20-25℃，在使用前须确保试剂自然回复到室温，请勿以水浴加热或其他方式加热试剂，如需长期存放试剂， 推荐根据每次实验用量进行分装，并避免反复冻融。
③、皮肤表面含有大量 ATP，处理样品和进行实验时需佩戴手套防止样品和试剂污染而造成假阴性或假阳性。
④、有些样品如添加了特殊药物或使用某些特殊培养液，可能含有抑制或增强反应的成分而导致假阴性或假阳性，此时可以将样品稀释 10 倍后再进行检测，并进一步根据稀释后测定出来的 Ratio 来判定是否有支原体污染。 ⑤、同一样本使用不同批次试剂盒及不同仪器比值（Ratio）会有差异，但不影响定性判断。
4、结果展示
常见问题及解答
| 问题 | 解答 |
| 请问该产品对发光检测仪器的灵敏度有要求吗？ | 推荐使用高灵敏度仪器，如几次检测的比率一直约等于1，可能与检测仪器的灵敏度有关，可将读数时间从 1 s 调整到 1-10 s 的区间。 |
| 配制好的 Mycoplasma Reagent A 溶液可以保存多久？ | -20℃避光保存，推荐 3 个月内使用，-80℃避光保存，12 个月内有效。建议分装，避免反复冻融。 |
| 发光法相较于 PCR 检测法有何不同？ | 化学发光法检测更加迅速，可随时且大批量检测细胞培养上清有无支原体污染。 |
| 是否可以延长反应时间，增大读数方便计算？ | 不能，需严格按照操作步骤，不能提前或延后，否则会影响在临界值附近比值的判断。 |
| 是否可以改变样本用量，增加检测结果的准确性？ | 对于 96 孔板，推荐使用 50 μL 样本，可以增加样本量，同时按照比例增加Mycoplasma Reagent A 和 Mycoplasma Reagent B，样本量不能超过100μL；不建议减少样本、Mycoplasma Reagent A 和 Mycoplasma Reagent B，检测值在背景附近可能会影响比值及结果判断，可以稀释样本检测。 |
注意事项
本产品仅供科学研究使用，不适用于临床诊断。
This luminescence-based mycoplasma detection kit utilizes the activity of mycoplasma-specific kinases and an ATP-dependent luciferase-catalyzed luminescence reaction. It determines mycoplasma contamination by comparing the change in ATP levels before and after adding the detection reagents. Upon lysis, mycoplasma release specific kinases that interact with substrates, catalyzing the conversion of ADP to ATP.
The assay involves two steps. First, Mycoplasma Reagent A is added to the sample to measure the intrinsic ATP level. Second, Mycoplasma Reagent B is added. If mycoplasma contamination is present, their specific kinases catalyze the conversion of ADP to ATP. The luminescence measured in this step represents the sum of the intrinsic ATP and the newly generated ATP produced by the mycoplasma-specific enzymes. The presence of mycoplasma contamination is determined by calculating the ratio of Luminescence Reading B to Reading A. A ratio greater than 1.2 indicates mycoplasma contamination, with higher ratios suggesting greater contamination levels. A ratio less than 0.9 indicates no mycoplasma contamination. If the ratio falls between 0.9 and 1.2, it is recommended to continue culturing the original cells (including the original culture medium) for 24-48 hours and retest.
Applicable Sample: Cells
| L1506760 | Component | 20T | 200T | Storage |
| L1506760A | Mycoplasma Assay Buffer | 1 mL | 10 mL | -20℃. Store in the dark. |
| L1506760B | Luciferase | 1 μL | 10 μL | -20℃ |
| L1506760C | Mycoplasma Substrate A | 1 EA | 1 EA | -20℃. Store in the dark. |
| L1506760D | Mycoplasma Reagent B | 1 mL | 10 mL | -20℃ |
| L1506760E | Postive Control | 200 μL | 1 mL | -20℃ |
Materials Required but Not Provided
Sterile centrifuge tubes, adjustable pipettes and tips
Centrifuge, 96-well solid black or solid white microplate
Sterile water, PBS, or fresh culture medium
Luminometer or multifunctional microplate reader
Experimental Procedure
1. Reagent Preparation
| Reagent Name | Preparation | Notes |
| Mycoplasma Assay Buffer | Ready-to-use; equilibrate to room temperature before use. | Store at -20°C protected from light. |
| Luciferase | Ready-to-use | Store at -20°C. |
| Mycoplasma Reagent A | Prepare immediately before use. Dissolve Mycoplasma Substrate A in Mycoplasma Assay Buffer. Then add the dissolved Mycoplasma Substrate A and Luciferase to the Mycoplasma Assay Buffer bottle. Mix thoroughly to obtain Mycoplasma Reagent A. | Aliquot according to usage needs and store at -20°C protected from light. Equilibrate to room temperature before use. |
| Mycoplasma Reagent B | Ready-to-use; equilibrate to room temperature before use. | Aliquot according to usage needs; store at -20°C. |
| Postive Control | Ready-to-use; equilibrate to room temperature before use. | Aliquot according to usage needs; store at -20°C. |
Note: Avoid repeated freeze-thaw cycles for all reagents. It is recommended to aliquot them. The aliquot containers must be free of ATP contamination.
2. Sample Preparation
2.1 Adherent Cells
Collect 0.2-1 mL of cell culture supernatant before trypsinization. Centrifuge at 1,500 rpm for 5 minutes. Collect the supernatant for detection.
2.2 Suspension Cells
During subculturing, collect 0.2-1 mL of the cell culture medium. Centrifuge at 1,500 rpm for 5 minutes. Collect the supernatant for detection.
2.3 Thawed Cells
After thawing frozen cells, add them to fresh complete medium. Culture for 1-2 hours, then collect 0.2-1 mL of the cell culture medium. Centrifuge at 1,500 rpm for 5 minutes. Collect the supernatant for detection.
Notes:
① The mycoplasma detection signal may decrease after cell passaging or trypsinization. If sampling after passaging or trypsinization, sample at least 24 hours after the process is complete.
② Supernatant samples are best detected immediately after collection. They can also be stored at 4°C and detected on the same day, or stored at -80°C for detection within six months. For detection, equilibrate frozen samples to room temperature before use.
3. Assay Steps
3.1 Add 50 μL of each test sample, Positive Control, and negative control (e.g., sterile water, PBS, fresh culture medium) into separate wells of a 96-well solid black or solid white microplate.
3.2 Add 50 μL of Mycoplasma Reagent A to each well. Mix well and incubate at room temperature for 5 minutes. Measure the chemiluminescence value (RLUA) using a multifunctional microplate reader.
3.3 Add 50 μL of Mycoplasma Reagent B to each well. Mix well and incubate at room temperature for 10 minutes. Measure the chemiluminescence value (RLUB) using a multifunctional microplate reader.
3.4 Calculate the Ratio = RLUB / RLUA. Refer to Table 1. A Ratio greater than 1.2 indicates mycoplasma contamination. A Ratio less than 0.9 indicates no mycoplasma contamination. If the Ratio is between 0.9 and 1.2, continue culturing the original sample for 24-48 hours and retest.
Table 1. Result Analysis for the Luminescence Mycoplasma Detection Kit
| Ratio | Result | Recommended Action |
| ＜0.9 | Negative | No action required. |
| 0.9-1.2 | Suspect | Continue isolated culture for 24-48 hours and retest. |
| ＞1.2 | Positive | Discard cells after sterilization, or isolate and treat with specific mycoplasma prevention/removal reagents. |
Notes:
① Set the reading time per well of the multifunctional microplate reader to 1,000 ms. Detection must be performed strictly 10 minutes after adding Mycoplasma Reagent B. Do not measure earlier or later, as this may affect the interpretation of results for samples with ratios near the cutoff value.
② The optimal temperature for this assay is 20-25°C. Ensure all reagents naturally equilibrate to room temperature before use. Do not heat reagents using a water bath or other methods. For long-term storage, aliquot reagents according to the amount needed per experiment and avoid repeated freeze-thaw cycles.
③ Skin surfaces contain significant ATP. Wear gloves when handling samples and performing the experiment to prevent contamination that could lead to false positives or negatives.
④ Some samples, such as those containing special additives or using certain culture media, might contain components that inhibit or enhance the reaction, potentially causing false negatives or positives. In such cases, dilute the sample 10-fold before detection and determine mycoplasma contamination based on the Ratio obtained from the diluted sample.
⑤ The Ratio for the same sample may vary when using different kit batches or different instruments, but this does not affect the qualitative determination.
4. Result Demonstration
Table 2. Example Detection Data for the Luminescence Mycoplasma Detection Kit
Frequently Asked Questions
| Question | Answer |
| Are there sensitivity requirements for the luminometer used with this product? | A high-sensitivity instrument is recommended. If the ratio is consistently approximately 1, it might be related to the instrument's sensitivity. Try increasing the integration time from 1 second to an interval between 1-10 seconds. |
| How long can the prepared Mycoplasma Reagent A solution be stored? | It can be stored at -20°C protected from light for up to 3 months, or at -80°C protected from light for up to 12 months. Aliquoting is recommended to avoid repeated freeze-thaw cycles. |
| How does the luminescence method differ from PCR detection? | The chemiluminescence method is faster, allowing for convenient and high-throughput detection of mycoplasma contamination in cell culture supernatants. |
| Can the reaction time be extended to increase the signal for easier calculation? | No. The procedure must be followed strictly. Do not initiate reading earlier or later than specified, as this may affect the interpretation of results near the cutoff values. |
| Can the sample volume be changed to increase detection accuracy? | For a 96-well plate, 50 μL of sample is recommended. The sample volume can be increased (up to 100 μL maximum), with Mycoplasma Reagent A and B volumes increased proportionally. Reducing sample, Reagent A, or Reagent B volumes is not recommended, as detection values near the background may affect the ratio and result interpretation. If necessary, the sample can be diluted and tested. |
Precautions
This product is for research use only. Not for use in diagnostic procedures.
规格
别名
支原体检测试剂盒（发光法）
英文别名
Mycoplasma Detection Kit (Luminescent)
规格或纯度
BioReagent, 用于细胞培养, 用于化学发光, 无菌
英文名称
Luminescent Mycoplasma Detection Kit
应用
Cell Culture, 生物发光
敏感性
Light sensitive
储存与运输
储存条件
避光,-20°C储存
运输条件
超低温运输
稳定性与储存
Store at -20℃ long term (12 months). Store in the dark.
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0332732 |
分析证书 | L1506760 |
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