- ￥327 - 557
- 翌圣生物（Yeasen）
- 40601ES
- 上海
- 2025年11月07日
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- 详细信息
- 文献和实验
- 技术资料
- 库存：
现货
- 供应商：
翌圣生物科技（上海）股份有限公司
- 英文名：
GMyc-PCR Mycoplasma Test Kit
- 规格：
10 assays(40601ES10)/20 assays(40601ES20)
| 规格： | 10 assays(40601ES10) | 产品价格： | ￥327.00 |
|---|---|---|---|
| 规格： | 20 assays(40601ES20) | 产品价格： | ￥557.00 |
研究表明，至少二十多种支原体能污染细胞，其中最常见的有：口腔支原体（M.orale）、精氨酸支原体（M.arginini）、猪鼻支原体（M.hyorhinis）、发酵支原体（M. fermentans）、人型支原体（M. hominis）、唾液支原体（M.salivarium）、肺支原体（M.pulmonis）和梨支原体（M.pirum）等。培养细胞的支原体污染率在4%-92%不等，其污染来源包括工作环境、操作者本身（一些支原体是人体的正常菌群）、培养基、血清、细胞交叉污染、实验器材和用来制备细胞的原始组织或器官的污染。
确认细胞培养过程中出现问题的潜在原因是一项困难且耗时的任务，在细胞培养中，任何突然的变化都应该被怀疑，同时良好的试验规范和定期检测支原体污染也十分必要。支原体检测的方法有很多种，如直接培养、DNA荧光染色、ELISA和PCR法等。
GMyc-PCR支原体检测试剂盒主要采用PCR方法对各种生物材料（如细胞培养物、实验动物分泌物、动物血清等）支原体感染的检测。它综合了几个优势：灵敏、特异、快速并且可以用直接用细胞培养上清液检测。本制品通过PCR方法检测培养细胞等生物材料中的支原体，所用引物为根据支原体16S-23S rRNA序列保守区域设计，只特异性扩增支原体DNA，检出灵敏度与特异性均较高。PCR扩增及电泳分析只需数个小时，操作方便简单。
产品组分
| 组分编号 | 组分名称 | 产品编号/规格 | |
| 40601ES10 | 40601ES20 | ||
| 40601-A | GMyc-1st PCR Mix | 250 µL | 2×250 µL |
| 40601-B | GMyc-2nd PCR Mix | 250 µL | 2×250 µL |
| 40601-C | Positive Control Template | 20 µL | 20 µL |
- 40601-C长期不用时，可-85~-65℃冷冻保存。
- PCR反应极其灵敏，为防止假阳性，加样时，最后加阳性对照。
实验流程此PCR反应为巢式PCR，第一轮PCR结束后，取其反应产物为第二轮PCR模板。根据两轮PCR产物电泳条带之有无及片段大小来分析结果（第一轮阳性对照PCR条带大小为448bp、第二轮阳性对照PCR条带大小为304bp）。细胞培养3-6天后，直接取其上清液进行PCR反应。为排除细胞培养基抑制PCR反应，阳性对照中需加入等量的细胞上清液。
第一轮PCR：
1. 充分融解GMyc-1st PCR Mix（40601-A），按下列表格配制反应液：
| 试剂 | 实验组 | 阳性对照 | 阴性对照 |
| GMyc-1st PCR Mix（40601-A） | 25 µL | 25 µL | 25 µL |
| 样品 | 4 µL | 4 µL | / |
| ddH2O | 21 µL | 20 µL | 25 µL |
| Positive Control Template（40601-C） | / | 1 µL | / |
2. 按下列条件进行PCR反应：
94℃ 5 min
94 ℃ 30 s
58 ℃ 30 s 30~35 cycles
72 ℃ 30 s
72℃ 7 min
第二轮PCR：
1. 充分融解PCR Mix，按下列表格配制反应液：
| 试剂 | 实验组 | 阳性对照 | 阴性对照 |
| GMyc-2nd PCR Mix | 25 µL | 25 µL | 25 µL |
| ddH2O | 24 µL | 24 µL | 24 µL |
| 1st PCR反应液（1000倍稀释液） | 1 µL | 1 µL | 1 µL |
2. 按下列条件进行PCR反应：
94 ℃ 5 min
94 ℃ 30 s
58 ℃ 30 s 30~35 cycles
72 ℃ 30 s
72 ℃ 7 min
反应结束后，取7 μL 的PCR反应液（不需要加loading buffer）直接进行1.5%琼脂糖凝胶电泳，确认PCR扩增产物。
【注】: 跑胶时不需要额外添加loading buffer，但需要额外加入染料，避免出现没有条带的情况。
推荐使用频率
| 新细胞进入实验室 | 必检 |
| 液氮保存前 | 必检 |
| 定期常规 | 每月检测一次 |
| 发现污染后 | 每周检测一次 |
| 发现细胞异常 | 随时检测 |
注意事项1）使用本试剂前请仔细阅读本说明书全文。
2）操作时应尽量少说话，因口腔中也含有支原体，可能引起样品污染，而造成假阳性；整个检测过程中，反应体系的配制、样本处理及加样、PCR扩增应分区域进行，以避免交叉污染。
3）实验时，试剂盒组分中的试剂使用前应充分融化并混匀（混匀时禁止激烈振荡，只需要进行上下倒置多次进行混匀）。
4）反应管中加好所有的试剂后，应尽快上PCR仪进行扩增，以免形成过多的二聚体。
5）细胞培养物中含有青霉素和链霉素等抗生素不会影响本品的检测结果。如果用户需要进一步提高检测灵敏度，建议细胞在不含青霉素和链霉素等抗生素中进行培养2-3天后送样检测。
6）为了您的健康，实验操作时请穿实验服和带一次性手套。
7）本产品仅作科研用途！HB230301
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