Bacitracin

一、欧洲药典近期更新了2.6.7支原体检测（MYCOPLASMAS）的相关内容，新增NAT（核酸扩增技术）方法，将于2026.04.01生效。具体变化、措施及中英文对照翻译见下文。 二、变化及措施章节内容 备注 措施 引言及检测策略1. 检测对象明确为“柔膜体纲”，涵盖更广菌属。2. 培养法与指示细胞法应联合使用，除非风险评估或主管当局允许。3. 引入风险评估选择方法，需考虑抑制物、生产工艺、潜在污染菌等。4. 建议取样时“尽可能同时取细胞和上清液”。新规不再强制“必须联合使用”，改为基于风险的“应联合使用”。若现行SOP强制两种方法，虽更严格，但未体现灵活性，且可能不适用新方法（如NAT）引入。1. 更新SOP，增加基于风险评估选择检测方法的章节。2. 在检测策略中明确取样要求（尽可能同时取细胞和上清液）。2.1 培养基选择菌种示例列表调整，部分菌株编号删除。菌种选择原则未变，仅表述和部分编号调整。若公司已建立合适阳性对照菌库，无实质影响。无需立即行动。下次更新SOP时同步更新附录中的菌株编号。2.4 抑制物测试1. 明确稀释法可作为消除抑制物手段。2. 强调中和后必须验证有效性。新规对已有要求细化，未引入颠覆性变化。核对现行SOP，确认已包含抑制物测试及中和后验证流程。如有缺失则补充。3 指示细胞法新增“抑制物测试”章节：1. 指示细胞培养法需进行抑制物测试。2. 使用中和措施（如抗血清）时，必须验证其不抑制支原体生长。 全新要求。旧版仅培养法有抑制物测试，新版明确指示细胞法也需要。若现行SOP未包含，存在重大合规差距。立即行动，更新SOP，新增指示细胞培养法的抑制物测试流程；更新检验记录表单。4.3 NAT验证验证标准重大更新：1. 新增对参考标准品制备和表征要求：指数生长期收获；同时测定CFU和GC；GC/CFU比值须小于10（除非论证）。2. 验证用菌种列表新增 M. arginini, M.   salivarium，明确列出 S.   citri。3. NAT替代培养法灵敏度：10 CFU/mL 或 <100 GC/mL。4. NAT替代指示细胞法灵敏度：100 CFU/mL 或 <1000 GC/mL。5. NAT同时替代两种方法灵敏度：10 CFU/mL 或 <100 GC/mL。核心技术要求变更。若已使用或计划使用NAT，必须重新验证。未用NAT：此项不适用。 已用NAT：GC/CFU比值<10可能是新要求，需检查现有参考品表征；灵敏度以GC/mL量化是新增。1. 若已使用NAT，立即评估现有验证数据与新规差距，特别是GC/CFU比值。2. 制定验证计划，重新确认检测限。3. 若未使用NAT，暂不行动，但需关注。4.4 抑制物测试NAT抑制物测试细化：1. 要求使用至少3批产品进行测试。2. 明确加标水平（≤10 CFU/mL 或 100   GC/mL）和回收率要求（如95%）。若公司已进行NAT抑制物测试，但未使用3批产品，或未明确规定加标量和回收率标准，则存在差距。更新NAT方法相关SOP，明确抑制物测试的批次数、加标量和验收标准。附录（验证指南）NAT验证指南细化：1. 对检测限测定方法进行更详细规定（如要求24个测试结果进行统计分析）。2. 明确可使用WHO IS作为参考品。此为验证执行层面的技术指南，非强制条款，对内部验证方案有参考价值。可作为内部技术文件更新的参考，非紧急。 三、翻译全文 2.6.7. MYCOPLASMAS 2.6.7.支原体 1. INTRODUCTION This general chapter is intended to guide the testing for bacteria of the class Mollicutes (trivial name mycoplasmas), e.g. the genera Mycoplasma, Mycoplasmodes, Mesomycoplasma, Metamycoplasma, Mycoplasmopsis, Ureaplasma, Acholeplasma and Spiroplasma. 本通则旨在指导柔膜体纲（俗名支原体）细菌的检测，例如支原体属、Mycoplasmodes属、Mesomycoplasma属、Metamycoplasma属、Mycoplasmopsis属、脲原体属、无胆甾原体属和螺原体属。 The methods described are adequate for detecting Mollicutes with a growth optimum of 35‑38°C such as Mollicutes that could contaminate mammalian or avian cells. Testing for the presence of product‑relevant mycoplasma species that are not indicated in the chapter, or testing of other cell production systems such as insect, fish or plant cell lines, may require different conditions. 所述方法足以检测最适生长温度为 35‑38°C 的柔膜体纲，例如可能污染哺乳动物或禽类细胞的柔膜体纲。对于本通则未列出的与产品相关的支原体种类的检测，或对于其他细胞生产系统（如昆虫、鱼类或植物细胞系）的检测，可能需要不同的条件。 Three methods for mycoplasma testing are described in this chapter: the culture method, the indicator cell culture method, and nucleic acid amplification techniques (NAT). The culture method and the indicator cell culture method should be used conjointly, unless otherwise prescribed in a monograph or if justified by a risk assessment and authorised by the competent authority, to ensure detection of both “cultivable” and “non‑cultivable”mycoplasmas. NAT may be used as an alternative method to one or both of the other methods after suitable validation (see Guidelines below). 本章描述了三种支原体检测方法：培养法、指示细胞培养法和核酸扩增技术（NAT）。培养法和指示细胞培养法应联合使用，以确保检测到“可培养”和“不可培养”的支原体，除非各论中另有规定，或经风险评估证明并得到主管当局授权。NAT 在适当验证后（参见下文指南）可作为替代方法替代其中一种或两种方法。 The selection of the most suitable method(s) is based on a risk assessment that considers a number of factors, including the culture media, manufacturing process, overall testing strategy (including production stage) and/or potentially contaminating mycoplasma species. The presence of inhibitory substances is also taken into account as the sample may interfere with the test or the interpretation of the result (e.g. it might not be possible to efficiently neutralise products consisting of virus material). Suitable alternative approaches, such as adapting the prescribed methods or using an NAT‑based method, may be applied in such cases, as well as in cases where the culture method and the indicator cell culture method are not suitable for the detection of potentially relevant contaminants (e.g. where plant‑derived raw materials or starting materials that may be contaminated with Spiroplasma citri are used in the manufacturing process). 最合适方法的选择基于风险评估，该风险评估考虑了多种因素，包括培养基、生产工艺、整体检测策略（包括生产阶段）和/或潜在污染支原体种类。还需要考虑抑制性物质的存在，因为样品可能干扰检测或结果解读（例如，可能无法有效中和由病毒材料组成的产品）。在这种情况下，以及在培养法和指示细胞培养法不适合检测潜在相关污染物的情况下（例如，在生产过程中使用了可能被柑橘螺原体污染的植物源性原料或起始物料），可以采用合适的替代方法，例如调整规定方法或使用基于 NAT 的方法。 Given that these contaminants may adhere to cells or be present in the cells, both cells and supernatant should be sampled for mycoplasma testing, whenever possible. 鉴于这些污染物可能粘附于细胞或存在于细胞内，只要可能，支原体检测时应同时取细胞和上清液进行检测。 2. CULTURE METHOD 2.培养法 2.1. CHOICE OF CULTURE MEDIA 2.1.培养基的选择 The test is carried out using a sufficient number of both solid and liquid media to ensure growth under the chosen incubation conditions of small numbers of mycoplasmas that may be present in the product to be examined. Liquid media may contain phenol red. The range of media chosen is shown to have satisfactory nutritive properties for at least the micro‑organisms shown below. The nutritive properties of each new batch of medium are verified for the appropriate micro‑organisms in the list. 试验使用足量的固体和液体培养基进行，以确保在所选培养条件下，待检产品中可能存在的少量支原体能够生长。液体培养基可含酚红。所选的培养基范围应证明对至少下列微生物具有满意的营养特性。每一新批次培养基的营养特性应使用列表中的适当微生物进行验证。 Example species that could be used are presented below: - Acholeplasma laidlawii; - Mycoplasma fermentans; - Mycoplasma gallisepticum (e.g. where avian material has been used during production or where a vaccine is intended for use in poultry); - Mycoplasma hyorhinis; - Mycoplasma orale; - Mycoplasma pneumoniae; - Mycoplasma synoviae (e.g. where avian material has been used during production or where a vaccine is intended for use in poultry). 可以使用以下示例菌种： -莱氏无胆甾原体； -发酵支原体； -鸡毒支原体（例如，生产过程中使用了禽源材料，或疫苗拟用于家禽时）； -猪鼻支原体； -口腔支原体； -肺炎支原体； -滑液支原体（例如，生产过程中使用了禽源材料，或疫苗拟用于家禽时）。 Test strains should not have been cultured for more than 15 passages, and they should be stored frozen or freeze‑dried. Examples of type cultures and collections providing appropriate strains are listed below: A. laidlawii NCTC 10116  CIP 75.27   ATCC 23206 M. fermentans NCTC 10117  ATCC 19989 M. gallisepticum NCTC 10115 ATCC 19610 M. hyorhinis NCTC 10130  ATCC 17981 M. orale NCTC 10112   ATCC 23714 M. pneumoniae NCTC 10119  ATCC 15531 M. synoviae NCTC 10124  ATCC 25204 试验菌株的传代次数不应超过 15 代，并应冷冻或冻干保存。提供适当菌株的模式培养物和菌种保藏中心的示例如下： 莱氏无胆甾原体 NCTC 10116   CIP 75.27  ATCC 23206 发酵支原体 NCTC 10117   ATCC 19989 鸡毒支原体 NCTC 10115   ATCC 19610 猪鼻支原体 NCTC 10130   ATCC 17981 口腔支原体 NCTC 10112   ATCC 23714 肺炎支原体 NCTC 10119   ATCC 15531 滑液支原体 NCTC 10124   ATCC 25204 2.2. INCUBATION CONDITIONS 2.2.培养条件 Incubate liquid media in tightly stoppered containers at 35‑38°C. Incubate solid media under microaerophilic conditions (nitrogen containing 5-10 per cent of carbon dioxide and sufficient humidity to prevent desiccation of the agar surface) at 35‑38°C. 液体培养基在 35‑38°C 下置于密封容器中培养。固体培养基在微需氧条件（含 5‑10% 二氧化碳的氮气，并保持足够湿度以防止琼脂表面干燥）下于 35‑38°C 培养。 2.3. NUTRITIVE PROPERTIES 2.3.营养特性 Carry out the test for nutritive properties for each new batch of medium. Inoculate the chosen media with the appropriate test micro‑organisms; use not more than 100 CFU per plate containing solid medium and per 100 mL container of liquid medium; use a separate plate and container for each species of micro‑organism. Incubate the media and make subcultures from 0.2 mL of liquid medium on to solid medium at the specified intervals (see section 2.5). The solid medium complies with the test if adequate growth is found for each test micro‑organism (growth obtained does not differ by a factor greater than 5 from the value calculated with respect to the inoculum). The liquid medium complies with the test if growth on agar plates is observed for at least one subculture for each test micro‑organism. 每一新批次培养基均应进行营养特性试验。将适当的试验微生物接种至所选培养基中；每个固体培养基平板（含 9 mL 培养基）和每个 100 mL 液体培养基容器中接种不超过 100 CFU；每种微生物使用单独的平板和容器。按规定的间隔（见 2.5 节）培养培养基，并从 0.2 mL 液体培养基转种至固体培养基。若每种试验微生物均生长良好（所获生长量相对于接种量计算值的差异不超过 5 倍），则固体培养基符合要求。若每种试验微生物至少有一次转种后在琼脂平板上观察到生长，则液体培养基符合要求。 2.4. INHIBITORY SUBSTANCES 2.4.抑制性物质 The test for inhibitory substances is carried out for a given product and is repeated whenever there is a change in the production process that may affect the detection of mycoplasmas. 抑制性物质试验针对特定产品进行，当生产工艺发生可能影响支原体检测的变化时，应重复进行。 To demonstrate absence of inhibitory substances, carry out the test for nutritive properties in the presence and absence of the product to be examined. If growth of a test micro‑organism occurs more than 1 subculture sooner in the absence of the product to be examined than in its presence, or if plates directly inoculated with the product to be examined have fewer than 1/5 of the number of colonies of those inoculated without the product to be examined, inhibitory substances are present. 为证明无抑制性物质，应在存在和不存在待检产品的情况下进行营养特性试验。若某种试验微生物在不存在待检产品时比存在待检产品时提前一个以上传代周期出现生长，或直接接种待检产品的平板上菌落数少于未接种待检产品平板上菌落数的 1/5，则存在抑制性物质。 Dilution of the sample or other measures (e.g. passage in substrates not containing inhibitors) can be used to neutralise the effect of inhibitory substances. When dilution is used, larger medium volumes may be used or the inoculum volume may be divided among several 100 mL containers. The effectiveness of the neutralisation is verified by repeating the test for inhibitory substances after neutralisation. 可使用样品稀释或其他措施（例如在不含抑制剂的基质中传代）来中和抑制性物质的影响。当使用稀释法时，可使用较大体积的培养基或将接种体积分至数个 100 mL 容器中。中和后通过重复抑制性物质试验来验证中和的有效性。 2.5. TEST FOR MYCOPLASMAS IN THE PRODUCT TO BE EXAMINED 2.5.待检产品中支原体的检查 The following procedure is recommended. In justified cases (e.g. limited batch size), a smaller sample volume may be used if authorised by the competent authority. 推荐使用以下程序。在合理的情况下（例如批次规模有限），若经主管当局批准，可使用较小的样品体积。 Inoculate 10 mL of the product to be examined per 100 mL of each liquid medium. If a substantial change in pH occurs upon the addition of the product to be examined, the liquid medium is restored to its original pH value by the addition of a solution of either sodium hydroxide or hydrochloric acid. Inoculate 0.2 mL of the product to be examined on each plate of each solid medium. Incubate liquid media for 20‑21 days. Incubate solid media for not less than 14 days, except those corresponding to the 20‑21 day subculture, which are incubated for 7 days. At the same time, incubate an un‑inoculated 100 mL portion of each liquid medium and solid medium plates, as a negative control. On days 2‑4 after inoculation, subculture each liquid medium by inoculating 0.2 mL on at least 1 plate of each solid medium. Repeat the procedure between the 6th and 8th days using the same incubation conditions, then again between the 13th and 15th days and again between the 19th and 21st day of the incubation of the liquid media (enrichment culture). Observe the liquid media regularly for colour change. If a colour change occurs, subculture. If a liquid medium shows bacterial or fungal contamination, the test is invalid. The test is valid if at least 1 plate per medium and per inoculation day can be read. Include in the test positive controls prepared by inoculation of not more than 100 CFU of at least 1 test micro‑organism on solid medium and into liquid medium. The test micro‑organisms used are those listed in section 2.1. Depending on the type of product and manufacturing process, additional strains might be included, based on a risk assessment. Where the test for mycoplasmas is carried out regularly, it is recommended to rotate the test micro‑organisms used as positive controls. 每 100 mL 每种液体培养基中接种 10 mL 待检产品。若加入待检产品后 pH 发生显著变化，应通过加入氢氧化钠溶液或盐酸溶液将液体培养基调回原 pH 值。在每个固体培养基平板上接种 0.2 mL 待检产品。液体培养基培养 20‑21 天。固体培养基培养不少于 14 天，但对应于第 20‑21 天转种的固体培养基培养 7 天。同时，将每种液体培养基和固体培养基平板的未接种部分作为阴性对照进行培养。接种后第 2‑4 天，从每种液体培养基中取 0.2 mL 转种至每种固体培养基的至少 1 个平板上。在第 6‑8 天以相同培养条件重复该操作，然后在第 13‑15 天再次重复，并在液体培养基培养的第 19‑21 天再次重复（增菌培养）。定期观察液体培养基的颜色变化。若发生颜色变化，则进行转种。若液体培养基出现细菌或真菌污染，则试验无效。若每种培养基、每个转种日至少有 1 个平板可供读取，则试验有效。试验中应包含阳性对照，通过将不超过 100 CFU 的至少一种试验微生物接种至固体培养基和液体培养基中制备。所用试验微生物为 2.1 节中列出的菌种。根据产品类型和生产工艺，基于风险评估可能还需加入其他菌株。若定期进行支原体检查，建议轮换使用作为阳性对照的试验微生物。 2.6. INTERPRETATION OF RESULTS 2.6.结果解释 At the end of the prescribed incubation period, examine all inoculated solid media microscopically for the presence of mycoplasma colonies. The product to be examined complies with the test if growth of characteristic mycoplasma colonies has not occurred. It does not comply with the test if growth of characteristic mycoplasma colonies has occurred on any of the solid media. The test is invalid if one or more of the positive controls do not show growth of mycoplasmas on at least one subculture plate. The test is invalid if one or more of the negative controls show growth of mycoplasmas. If suspect colonies are observed, a suitable validated method may be used to determine whether they are due to mycoplasmas. 在规定培养期结束时，显微镜检查所有接种的固体培养基上是否存在支原体菌落。若未出现典型支原体菌落生长，则待检产品符合规定。若任何固体培养基上出现典型支原体菌落生长，则待检产品不符合规定。若一个或多个阳性对照在至少一个转种平板上未显示支原体生长，则试验无效。若一个或多个阴性对照显示支原体生长，则试验无效。若观察到可疑菌落，可使用适当的验证过的方法确定其是否由支原体引起。 2.7. RECOMMENDED MEDIA FOR THE CULTURE METHOD 2.7.培养法的推荐培养基 The following media are recommended. Other media may be used, provided that their ability to sustain the growth of mycoplasmas has been demonstrated on each batch in the presence and absence of the product to be examined. 推荐使用以下培养基。也可使用其他培养基，前提是每一批培养基在存在和不存在待检产品的情况下均能证明其支持支原体生长的能力。 HAYFLICK MEDIA (RECOMMENDED FOR THE GENERAL DETECTION OF MYCOPLASMAS)** HAYFLICK培养基（推荐用于支原体的一般检测） Liquid medium Beef heart infusion broth (1) 90.0 mL  Horse serum 20.0 mL  Yeast extract (250 g/L) 10.0 mL  Phenol red (0.6 g/L solution) 5.0 mL  Penicillin (20 000 IU/mL) 0.25 mL  Deoxyribonucleic acid (2 g/L solution) 1.2 mL  Adjust to pH 7.8. 液体培养基  牛肉心浸液肉汤 (1) 90.0 mL  马血清 20.0 mL  酵母提取物 (250 g/L) 10.0 mL  酚红 (0.6 g/L 溶液) 5.0 mL  青霉素 (20 000 IU/mL) 0.25 mL  脱氧核糖核酸 (2 g/L 溶液) 1.2 mL  调 pH 至 7.8。 Solid medium Prepare as described above replacing beef heart infusion broth by beef heart infusion agar containing 15 g/L of agar. 固体培养基 按上述方法制备，但将牛肉心浸液肉汤替换为含 15 g/L 琼脂的牛肉心浸液琼脂。 FREY MEDIA (RECOMMENDED FOR THE DETECTION OF M. SYNOVIAE) FREY培养基（推荐用于滑液支原体的检测） Liquid medium Beef heart infusion broth (1) 90.0 mL  Essential vitamins (2) 0.025 mL  Glucose monohydrate (500 g/L solution) 2.0 mL  Swine serum 12.0 mL  β‑Nicotinamide adenine dinucleotide (10 g/L solution) 1.0 mL  Cysteine hydrochloride (10 g/L solution) 1.0 mL  Phenol red (0.6 g/L solution) 5.0 mL  Penicillin (20 000 IU/mL) 0.25 mL  Mix the solutions of β‑nicotinamide adenine dinucleotide and cysteine hydrochloride and after 10 min add to the other ingredients. Adjust to pH 7.8. 液体培养基  牛肉心浸液肉汤 (1) 90.0 mL  必需维生素 (2) 0.025 mL  一水葡萄糖 (500 g/L 溶液) 2.0 mL  猪血清 12.0 mL  β‑烟酰胺腺嘌呤二核苷酸 (10 g/L 溶液) 1.0 mL   盐酸半胱氨酸 (10 g/L 溶液) 1.0 mL  酚红 (0.6 g/L 溶液) 5.0 mL  青霉素 (20 000 IU/mL) 0.25 mL  将 β‑烟酰胺腺嘌呤二核苷酸溶液和盐酸半胱氨酸溶液混合，10 分钟后加入其他成分。调 pH 至7.8。 Solid medium  Beef heart infusion broth (1) 90.0 mL  Agar, purified (3) 1.4 g  Adjust to pH 7.8, sterilise by autoclaving then add:  Essential vitamins (2) 0.025 mL  Glucose monohydrate (500 g/L solution) 2.0 mL  Swine serum 12.0 mL  β‑Nicotinamide adenine dinucleotide (10 g/L solution) 1.0 mL  Cysteine hydrochloride (10 g/L solution) 1.0 mL  Phenol red (0.6 g/L solution) 5.0 mL  Penicillin (20 000 IU/mL) 0.25 mL 固体培养基  牛肉心浸液肉汤 (1) 90.0 mL  纯化琼脂 (3) 1.4 g  调 pH 至 7.8，高压灭菌后加入：  必需维生素 (2) 0.025 mL  一水葡萄糖 (500 g/L 溶液) 2.0 mL  猪血清 12.0 mL  β‑烟酰胺腺嘌呤二核苷酸 (10 g/L 溶液) 1.0 mL   盐酸半胱氨酸 (10 g/L 溶液) 1.0 mL  酚红 (0.6 g/L 溶液) 5.0 mL  青霉素 (20 000 IU/mL) 0.25 mL FRIS MEDIA (RECOMMENDED FOR THE DETECTION OF NON‑AVIAN MYCOPLASMAS) FRIS培养基（推荐用于非禽源支原体的检测） Liquid medium Hanks’ balanced salt solution (modified) (4) 800 mL   Distilled water 67 mL  Brain heart infusion (5) 135 mL  PPLO Broth (6) 248 mL  Yeast extract (170 g/L) 60 mL  Bacitracin 250 mg  Meticillin 250 mg  Phenol red (5 g/L) 4.5 mL  Horse serum 165 mL  Swine serum 165 mL  Adjust to pH 7.40‑7.45. 液体培养基  Hanks平衡盐溶液（改良）(4) 800 mL   蒸馏水 67 mL  脑心浸液 (5) 135 mL  PPLO肉汤(6) 248 mL   酵母提取物 (170 g/L) 60 mL  杆菌肽 250 mg  甲氧西林 250 mg  酚红 (5 g/L) 4.5 mL  马血清 165 mL  猪血清 165 mL  调 pH 至 7.40‑7.45。 Solid medium  Hanks’ balanced salt solution (modified) (4) 200 mL   DEAE‑dextran 200 mg   Agar, purified (3) 15.65 g  Mix well and sterilise by autoclaving. Cool to 100°C. Add to 1740 mL of liquid medium as described above. 固体培养基 Hanks平衡盐溶液（改良）(4) 200 mL   DEAE‑葡聚糖 200 mg   纯化琼脂 (3) 15.65 g  混匀后高压灭菌。冷却至 100°C。加入至上述 1740 mL液体培养基中。 (1) Beef heart infusion broth  Beef heart (for preparation of the infusion) 500 g  Peptone 10 g  Sodium chloride 5 g  Distilled water to 1000 mL  Sterilise by autoclaving. (1)牛肉心浸液肉汤   牛心（用于制备浸液）500 g  蛋白胨 10 g  氯化钠 5 g  蒸馏水加至 1000 mL  高压灭菌。 (2) Essential vitamins  Biotin 100 mg  Calcium pantothenate 100 mg  Choline chloride 100 mg  Folic acid 100 mg  i‑Inositol 200 mg   Nicotinamide 100 mg  Pyridoxal hydrochloride 100 mg  Riboflavine 10 mg  Thiamine hydrochloride 100 mg  Distilled water to 1000 mL (2)必需维生素   生物素 100 mg  泛酸钙 100 mg  氯化胆碱 100 mg  叶酸 100 mg  肌醇 200 mg  烟酰胺 100 mg  盐酸吡哆醛 100 mg  核黄素 10 mg  盐酸硫胺 100 mg  蒸馏水加至 1000 mL (3) Agar, purified  A highly refined agar for use in microbiology and immunology, prepared by an ion‑exchange procedure that results in a product having superior purity, clarity and gel strength. It contains about:  Water 12.2 per cent  Ash 1.5 per cent  Acid‑insoluble ash 0.2 per cent   Chlorine 0 Phosphate (calculated as P₂O₅) 0.3 per cent   Total nitrogen 0.3 per cent  Copper 8 ppm  Iron 170 ppm  Calcium 0.28 per cent  Magnesium 0.32 per cent (3)纯化琼脂   一种高度精制的琼脂，用于微生物学和免疫学，通过离子交换工艺制备，产品具有优异的纯度、透明度和凝胶强度。其含量约为：  水分 12.2%  灰分 1.5%  酸不溶性灰分 0.2%  氯 0  磷酸盐（以 P₂O₅计）0.3%  总氮 0.3%  铜 8 ppm  铁 170 ppm  钙 0.28%  镁 0.32% (4) Hanks’ balanced salt solution (modified)   Sodium chloride 6.4 g  Potassium chloride 0.32 g  Magnesium sulfate heptahydrate 0.08 g  Magnesium chloride hexahydrate 0.08 g  Calcium chloride, anhydrous 0.112 g  Disodium hydrogen phosphate dihydrate 0.0596 g  Potassium dihydrogen phosphate, anhydrous 0.048 g  Distilled water to 800 mL (4) Hanks 平衡盐溶液（改良）   氯化钠 6.4 g  氯化钾 0.32 g  七水硫酸镁 0.08 g  六水氯化镁 0.08 g  无水氯化钙 0.112 g  二水磷酸氢二钠 0.0596 g  无水磷酸二氢钾 0.048 g  蒸馏水加至 800 mL (5) Brain heart infusion  Calf‑brain infusion 200 g   Beef‑heart infusion 250 g   Proteose peptone 10 g  Glucose monohydrate 2 g  Sodium chloride 5 g  Disodium hydrogen phosphate, anhydrous 2.5 g  Distilled water to 1000 mL (5)脑心浸液   小牛脑浸液 200 g  牛心浸液 250 g  示蛋白胨 10 g  一水葡萄糖 2 g  氯化钠 5 g  无水磷酸氢二钠 2.5 g  蒸馏水加至 1000 mL (6) PPLO broth  Beef‑heart infusion 50 g   Peptone 10 g  Sodium chloride 5 g  Distilled water to 1000 mL (6) PPLO 肉汤   牛心浸液 50 g  蛋白胨 10 g  氯化钠 5 g  蒸馏水加至 1000 mL 3. INDICATOR CELL CULTURE METHOD 3.指示细胞培养法 Cell cultures are stained with a fluorescent dye that binds to DNA. Mycoplasmas are detected by their characteristic particulate or filamentous pattern of fluorescence on the cell surface and, if contamination is heavy, in surrounding areas. Mitochondria in the cytoplasm may be stained but are readily distinguished from mycoplasmas. 细胞培养物用与 DNA 结合的荧光染料染色。支原体通过其在细胞表面（若污染严重，则在周围区域）的特征性颗粒状或丝状荧光模式进行检测。细胞质中的线粒体可能被染色，但易于与支原体区分。 3.1. VERIFICATION OF THE SUBSTRATE 3.1.基质的确认 Use Vero cells or another cell culture (for example, the production cell line) that is equivalent in effectiveness for detecting mycoplasmas. Test the effectiveness of the cells to be used by applying the procedure shown below and inoculating not more than 100 CFU or CFU‑like micro‑organisms of suitable reference strains of M. hyorhinis and M. orale. The following strains have been found to be suitable: M. hyorhinis ATCC 29052 M. orale NCTC 10112 ATCC 23714 The cells are suitable if both reference strains are detected. 使用 Vero 细胞或其他在检测支原体方面等效的细胞培养物（例如生产细胞系）。按以下程序测试待用细胞的有效性，接种不超过 100 CFU 或类 CFU 的猪鼻支原体和口腔支原体适当参考菌株。以下菌株已被证明适用： 猪鼻支原体 ATCC 29052 口腔支原体 NCTC 10112 ATCC 23714 若两种参考菌株均被检出，则细胞适用。 The indicator cells must be subcultured without an antibiotic before being used in the test. 指示细胞在使用前必须在无抗生素条件下传代。 3.2. INHIBITORY SUBSTANCES 3.2.抑制性物质 The test for inhibitory substances is carried out for a given product to be examined and is repeated whenever there is a change in the production process that may affect the detection of mycoplasmas. 抑制性物质试验针对特定待检产品进行，当生产工艺发生可能影响支原体检测的变化时，应重复进行。 To demonstrate absence of inhibitory substances, carry out the test for the verification of the substrate in the presence and absence of the product to be examined. Inhibitory substances are present if one or both reference strain(s) are detected in the absence but not in the presence of the product to be examined or if the indicator cells show signs of growth inhibition in the presence of the product to be examined. 为证明无抑制性物质，应在存在和不存在待检产品的情况下进行基质确认试验。若在不存在待检产品时检出一种或两种参考菌株，而在存在待检产品时未检出，或指示细胞在存在待检产品时出现生长抑制迹象，则存在抑制性物质。 In some cases, neutralisation of inhibitory substances is needed. When dilution of the sample is performed, larger medium volumes may be used or the inoculum volume may be divided among several cell culture containers. 在某些情况下，需要对抑制性物质进行中和。当进行样品稀释时，可使用较大体积的培养基或将接种体积分至数个细胞培养容器中。 If for viral suspensions the interpretation of results is affected by marked cytoplasmic effects, the virus may be neutralised using a specific antiserum that has no inhibitory effects on mycoplasmas. 若对于病毒悬液，结果解读受到明显细胞病变效应的影响，可使用对支原体无抑制作用的特异性抗血清中和病毒。 The absence of inhibitory effects due to the neutralisation procedure must be demonstrated. Alternative approaches, such as testing the control cells, must be justified and authorised by the competent authority. 必须证明中和过程未引入抑制效应。替代方法（如测试对照细胞）必须经过论证并获得主管当局授权。 3.3. TEST METHOD 3.3.检验方法 1. Seed the indicator cell culture at a suitable density (for example, 2 × 10⁴–2 × 10⁵ cells/mL, 4 × 10³– 2.5 × 10⁴ cells/cm²) that will yield confluence after 3 days of growth. Inoculate 1 mL of the product to be examined into the cell culture vessel and incubate at 35‑38 °C. 2. After at least 3 days of incubation, when the cells have grown to the desired confluence, make a subculture on cover slips in suitable containers or on some other surface (for example, chambered slides) suitable for the test procedure. Seed the cells at low density so that they reach about 50 per cent confluence after 3‑5 days of incubation. Complete confluence impairs visualisation of mycoplasmas after staining and must be avoided. 3. Remove the medium and rinse the indicator cells with phosphate buffered saline pH 7.4 R, then add a suitable fixing solution (a freshly prepared mixture of 1 volume of glacial acetic acid R and 3 volumes of methanol R is suitable when bisbenzimide R is used for staining). 4. Remove the fixing solution and wash the cells with sterile water R. Dry the slides completely if they are to be stained more than 1 h later (particular care is needed for staining of slides after drying due to the potential for artefact formation). 5. Add a suitable DNA stain and allow to stand for a suitable time (bisbenzimide working solution R and a standing time of 10 min are suitable). 6. Remove the stain and rinse the monolayer with water R. 7. Mount each coverslip, where applicable (a mixture of equal volumes of glycerol R and phosphate‑citrate buffer solution pH 5.5 R is suitable). Examine by fluorescence (for bisbenzimide stain a 330 nm/380 nm excitation filter and an LP 440 nm barrier filter are suitable) at 400×magnification or greater. 8. Compare the microscopic appearance of the test cultures with that of the negative and positive controls, examining for extranuclear fluorescence. Mycoplasmas produce pinpoints or filaments over the indicator cell cytoplasm. They may also produce pinpoints and filaments in the intercellular spaces. Multiple microscopic fields are examined according to the protocol established during validation. 1.以合适的密度（例如 2×10⁴～2×10⁵个细胞/mL，4×10³～2.5×10⁴个细胞/cm²）接种指示细胞培养物，使其培养 3 天后达到汇合。向细胞培养容器中接种 1 mL 待检产品，于 35‑38°C 培养。 2.培养至少3 天，待细胞生长至所需汇合度后，将细胞传代至盖玻片（置于合适容器中）或其他适合试验程序的表面（例如腔室玻片）。以低密度接种细胞，使其在培养 3‑5 天后达到约 50% 汇合。完全汇合会影响染色后支原体的观察，应避免。 3.吸去培养基，用磷酸盐缓冲液 pH 7.4 R 冲洗指示细胞，然后加入合适的固定液（当使用双苯甲亚胺 R 染色时，新配制的 1 体积冰醋酸 R 与 3 体积甲醇 R 的混合液适用）。 4.吸去固定液，用无菌水 R 洗涤细胞。若要在 1 小时以上后进行染色，则将玻片完全干燥（干燥后染色需特别注意可能产生的假象）。 5.加入合适的 DNA 染色剂，静置适当时间（双苯甲亚胺工作液 R 和 10 分钟静置时间适用）。 6.吸去染色剂，用水 R 冲洗单层细胞。 7.如有盖玻片，则封片（等体积甘油 R 和磷酸‑柠檬酸盐缓冲液 pH 5.5 R 的混合液适用）。在 400 倍或更高放大倍数下进行荧光检查（对于双苯甲亚胺染色，适用 330 nm/380 nm 激发滤光片和 LP 440 nm 阻光滤光片）。 8.将试验培养物的显微外观与阴性和阳性对照进行比较，检查核外荧光。支原体在指示细胞胞质上产生点状或丝状荧光，也可见于细胞间隙。根据验证时建立的方案检查多个显微镜视野。 3.4. INTERPRETATION OF RESULTS 3.4.结果解释 The product to be examined complies with the test if fluorescence typical of mycoplasmas is not present. The test is invalid if the positive controls do not show fluorescence typical of mycoplasmas. The test is invalid if the negative controls show fluorescence typical of mycoplasmas. 若未出现支原体典型的荧光，则待检产品符合规定。若阳性对照未显示支原体典型的荧光，则试验无效。若阴性对照显示支原体典型的荧光，则试验无效。 4. NUCLEIC ACID AMPLIFICATION TECHNIQUES (NAT) 4.核酸扩增技术 (NAT) 4.1. INTRODUCTION 4.1.引言 Nucleic acid amplification techniques (2.6.21) may be used for the detection of species of the Mollicutes class by amplification of nucleic acids extracted from a test sample with specific primers that reveal the presence of the target nucleic acid. NAT indicate the presence of a particular nucleic acid sequence and not necessarily the presence of viable mycoplasmas. A number of different techniques are available. This general chapter does not prescribe a particular method for the test. The procedure applied must be validated as described, taking account of the guidelines presented at the end of this section. Where a commercial kit is used, certain elements of the validation may be carried out by the manufacturer and information provided to the user. 核酸扩增技术 (2.6.21) 可用于通过特异性引物扩增从待检样品中提取的核酸来检测柔膜体纲的种，该引物能揭示靶核酸的存在。NAT 指示特定核酸序列的存在，而不一定是活支原体的存在。有多种不同的技术可供使用。本通则未规定特定的检测方法。所采用的方法必须按所述进行验证，并考虑本节末尾提供的指南。当使用商业试剂盒时，验证的某些要素可由制造商完成并提供信息给用户。 NAT are applied where prescribed in a monograph. They may also be used as a replacement for one or both of the culture method or the indicator cell culture method after validation. NAT在各论规定时应用。经验证后，它们也可用作培养法或指示细胞培养法中一种或两种的替代方法。 Direct NAT: the nucleic acids are extracted from the test sample and used directly for detection by NAT (without cell‑ or media‑based enrichment). 直接 NAT：从待检样品中提取核酸，直接用于 NAT 检测（不经细胞或培养基富集）。 Cell‑based enrichment followed by NAT**: the test sample and a suitable cell substrate (as described under the indicator cell‑culture method) are cultured together for a suitable period. The nucleic acids are then extracted from the cells and supernatant and used for detection by NAT. 细胞富集后 NAT：将待检样品与合适的细胞基质（如指示细胞培养法中所述）共同培养适当时间。然后从细胞和上清液中提取核酸，用于 NAT 检测。 Media‑based enrichment followed by NAT**: the test sample is inoculated into liquid media suitable to support the growth of the species listed in section 2 and/or section 3 and incubated for a suitable period. The nucleic acids are then extracted from the enrichment culture and used for detection of mycoplasmas by NAT. 培养基富集后 NAT：将待检样品接种至适合支持第 2 节和/或第 3 节中所列菌种生长的液体培养基中，培养适当时间。然后从富集培养物中提取核酸，用于 NAT 检测支原体。 4.2. CONTROLS 4.2.对照 **Internal control**  The internal control is necessary for routine verification of the absence of NAT inhibition. The internal control may contain the primer binding site, or another suitable sequence may be used. It acts as an overall control (optional pre‑treatment of the sample, extraction, reverse transcription (when applicable), amplification, and detection) and is therefore added to the sample before pre‑treatment (or before isolating the nucleic acid if pre‑treatment is not performed), whenever possible. 内部对照  内部对照对于常规验证是否存在 NAT 抑制是必要的。内部对照可包含引物结合位点，或使用其他合适的序列。它作为整体对照（样品的可选预处理、提取、逆转录（如适用）、扩增和检测），因此应尽可能在预处理前（或不进行预处理时在核酸分离前）加入样品中。 External positive control The external positive control contains a defined number of target‑sequence copies or CFUs from one or more suitable species of mycoplasma chosen from those used during validation of the test conditions. The positive control is set close to the positive cut‑off point to demonstrate that the validated sensitivity is achieved. 外部阳性对照  外部阳性对照含有确定数量的靶序列拷贝或 CFU，来自从试验条件验证期间使用的适当支原体种中选择的一个或多个菌种。阳性对照应设置在接近阳性截止点的水平，以证明达到了验证的灵敏度。 Negative control  The negative control contains no target sequence but does not necessarily represent the same matrix as the test sample. 阴性对照  阴性对照不含靶序列，但不一定代表与待检样品相同的基质。 4.3. VALIDATION 4.3.验证 Reference standards are required at various stages during validation and for use as controls during routine application of the test. The reference standards may be mycoplasmas or nucleic acids. 在验证的各个阶段以及常规试验中用作对照时，都需要参考标准品。参考标准品可以是支原体或核酸。 When using live mycoplasmas for the determination of the positive cut‑off point, the strains should be prepared and characterised as follows: - mycoplasmas should be harvested during the exponential phase of growth to guarantee a high viability of the reference organisms; - the titre in CFU should be determined prior to freezing the working suspension/dilutions; - CFU determination should be performed by using a suitable number of replicates; - for the determination of the genomic copies (GC), both fractions (i.e. the supernatant and the cellular fraction) must be considered; - the acceptance criterion for the GC/CFU ratio should be defined: the GC/CFU ratio of the reference preparations should be less than 10, unless otherwise justified. 当使用活支原体确定阳性截止点时，菌株应按以下要求制备和表征： -支原体应在指数生长期收获，以确保参考菌株的高活力； -在冷冻工作悬液/稀释液前应测定 CFU 滴度； - CFU 测定应使用足够数量的重复； -测定基因组拷贝数（GC）时，必须同时考虑上清液和细胞部分； -应定义GC/CFU 比率的接受标准：参考品的 GC/CFU 比率应小于 10，除非另有说明。 For validation of the detection limit, the following species can be used. This list is proposed based on the frequency of occurrence as contaminants and phylogenetic relationships. A. laidlawii NCTC 10116 CIP 75.27 ATCC 23206 M. arginini NCTC 10129 ATCC 23838 M. fermentans NCTC 10117 ATCC 19989 M. gallisepticum¹ NCTC 10115 ATCC 19610 M. hyorhinis NCTC 10130 (culture method) ATCC 17981 (culture method) or ATCC 29052 (indicator cell culture method) M. orale NCTC 10112 ATCC 23714 M. pneumoniae NCTC 10119 ATCC 15531 M. salivarium NCTC 10113 ATCC 23064 M. synoviae¹ NCTC 10124 ATCC 25204 S. citri or another species of the Spiroplasma genus² ATCC 27556 ¹ e.g. where there is use of or exposure to avian material during production ² e.g. where there is use of or exposure to insect or plant material during production For a generic assay for a broad range of applications, all mycoplasmas listed above should be included in the validation of the method. Otherwise, not all species need to be included in the validation study and relevant species may be selected from this list using a risk‑based approach. For example, the manufacturing process (use of mammalian, avian or insect material or expression cell lines), the product category (biopharmaceuticals, cell‑ or gene therapy products or vaccines) and the occurrence of contamination (literature research) should be taken into account. 为验证检测限，可使用以下菌种。该列表基于污染物出现的频率和系统发育关系提出。 莱氏无胆甾原体 NCTC 10116 CIP 75.27 ATCC 23206 精氨酸支原体 NCTC 10129 ATCC 23838 发酵支原体 NCTC 10117 ATCC 19989 鸡毒支原体¹ NCTC 10115 ATCC 19610 猪鼻支原体 NCTC 10130（培养法）ATCC 17981（培养法）或 ATCC 29052（指示细胞培养法） 口腔支原体 NCTC 10112 ATCC 23714 肺炎支原体 NCTC 10119 ATCC 15531 唾液支原体 NCTC 10113 ATCC 23064 滑液支原体¹ NCTC 10124 ATCC 25204 柑橘螺原体或螺原体属的其他种² ATCC 27556 ¹ 例如，生产过程中使用或接触禽源材料时 ² 例如，生产过程中使用或接触昆虫或植物材料时 对于广泛应用的通用检测方法，应将上述所有支原体种类纳入方法验证中。否则，不必将列表中的所有种类都纳入验证研究，可采用基于风险的方法从该列表中选择相关种类。例如，应考虑生产工艺（使用哺乳动物、禽类或昆虫材料或表达细胞系）、产品类别（生物制药、细胞或基因治疗产品或疫苗）以及污染发生情况（文献调研）。 Detection limit. When using live mycoplasmas for the determination of the positive cut‑off point, the strains should be prepared as recommended above and characterised both in GC and CFU content (see below Validation of nucleic acid amplification techniques (NAT) for the detection of mycoplasmas: guidelines). In addition, the WHO International Standard for mycoplasma DNA for nucleic acid amplification technique‑based assays designed for generic mycoplasma detection (WHO IS for mycoplasma DNA) can be used to compare different reference strain preparations or different PCR kits. 检测限。 当使用活支原体确定阳性截止点时，菌株应按上述建议制备，并同时表征 GC 和 CFU 含量（参见下文“用于支原体检测的核酸扩增技术验证：指南”）。此外，可用于比较不同参考菌株制备物或不同 PCR 试剂盒。 Specificity. Demonstration of specificity requires the use of a suitable range of bacterial species other than mycoplasmas. Bacterial genera with a close phylogenetic relationship to mycoplasmas could be used for this purpose (e.g. Clostridium, Lactobacillus and Streptococcus). 特异性。 证明特异性需要使用一系列合适的非支原体细菌种。与支原体亲缘关系较近的细菌属可用于此目的（例如梭菌属、乳杆菌属和链球菌属）。 Comparability studies for use of NAT as an alternative method NAT作为替代方法的比较研究 For each mycoplasma test species: - as an alternative to the culture method: the NAT test system must be shown to detect 10 CFU/mL or less than 100 GC/mL; - as an alternative to the indicator cell culture method: the NAT test system must be shown to detect 100 CFU/mL or less than 1000 GC/mL; - as an alternative to both the culture method and the indicator cell culture method: the NAT test system must be shown to detect 10 CFU/mL or less than 100 GC/mL. 对于每种支原体试验菌种： -作为培养法的替代方法：NAT 检测系统必须证明能检出 10 CFU/mL 或小于 100 GC/mL； -作为指示细胞培养法的替代方法：NAT 检测系统必须证明能检出 100 CFU/mL 或小于 1000 GC/mL； -作为培养法和指示细胞培养法两者的替代方法：NAT 检测系统必须证明能检出 10 CFU/mL 或小于 100 GC/mL。 4.4. INHIBITORY SUBSTANCES 4.4.抑制性物质 Spike a justified number of batches of the product to be examined (see examples in the section on Validation of nucleic acid amplification techniques (NAT) for the detection of mycoplasmas: guidelines, testing for inhibitory substances) with a maximum of 10 CFU/mL or 100 GC/mL, ensuring that sufficient recovery (e.g. 95 per cent) is obtained. The proposed acceptance criteria should be justified and based on the results of the method validation. 向合理数量的待检产品批次中加入不超过 10 CFU/mL 或 100 GC/mL 的支原体（参见下文“用于支原体检测的核酸扩增技术验证：指南”中的抑制性物质测试示例），确保获得足够的回收率（例如 95%）。所提出的接受标准应合理，并基于方法验证的结果。 4.5. EVALUATION AND INTERPRETATION OF RESULTS 4.5.结果评估与解释 The primers used may also amplify non‑mycoplasma bacterial nucleic acid, leading to false positive results. Procedures are established at the time of validation for dealing with confirmation of positive results, where necessary (for example, a second test may be performed using an alternative method without this specificity gap or using an official method). For more information, see 2.6.21, section 6. 所用引物也可能扩增非支原体细菌核酸，导致假阳性结果。在验证时，应建立处理阳性结果确认的程序（例如，可采用无此特异性缺口的替代方法或使用官方方法进行第二次检测）。更多信息参见 2.6.21 第 6 节。 The following section is published for information. 以下部分为信息性内容。 ### Validation of nucleic acid amplification techniques (NAT) for the detection of mycoplasmas: guidelines 用于支原体检测的核酸扩增技术验证：指南 1. SCOPE 1. 范围 Nucleic acid amplification techniques (NAT) are either qualitative or quantitative tests for the presence of nucleic acids. For the detection of mycoplasma contamination of various samples such as vaccines and cell substrates, qualitative tests are adequate and may be considered to be limit tests. 核酸扩增技术是用于检测核酸存在的定性或定量试验。对于检测疫苗和细胞基质等各种样品中的支原体污染，定性试验已足够，可视为限度试验。 These guidelines describe methods to validate qualitative NAT for the detection of mycoplasma contamination. They may also be applicable for real‑time NAT used as limit tests for the control of contaminants. 这些指南描述了验证用于检测支原体污染的定性 NAT 的方法。它们也可适用于用作污染物控制限度试验的实时 NAT。 The two characteristics regarded as the most important for validation of the analytical procedure are the specificity and the detection limit. In addition, the robustness of the analytical procedure should be evaluated. 对于分析方法的验证，最重要的两个特征是特异性和检测限。此外，还应评估分析方法的耐用性。 For the purpose of this document, an analytical procedure is defined as the complete procedure from sample pre‑treatment (or extraction of nucleic acid if pre‑treatment is not performed) to detection of the amplified products. 在本文件中，分析方法定义为从样品预处理（或不进行预处理时从核酸提取）到扩增产物检测的完整程序。 Where commercial kits are used for part or all of the analytical procedure, documented validation points already covered by the kit manufacturer can replace validation by the user. Nevertheless, the performance of the kit with respect to its intended use must be demonstrated by the user. In addition to the detection limit and robustness, cross‑detection of other classes of bacteria must be considered if relevant for the specific application. 当使用商业试剂盒进行部分或全部分析程序时，试剂盒制造商已涵盖的验证要点可替代用户验证。然而，用户必须证明试剂盒在其预期用途中的性能。除检测限和耐用性外，如果与特定应用相关，还必须考虑对其他类别细菌的交叉检测。 NAT may be used as: - a complementary test (for example, for cytotoxic viral suspensions) or for in‑process control purposes; - an alternative method to replace one or both of the indicator cell culture method or culture method. These guidelines will thus separate these two objectives by presenting first a guideline for the validation of the NAT themselves, and second, a guideline for a comparability study between NAT and official methods. NAT可用于： -作为补充试验（例如用于细胞毒性病毒悬液）或用于过程控制目的； -作为替代方法替代指示细胞培养法或培养法中的一种或两种。因此，本指南将这两个目标分开，首先介绍 NAT 本身的验证指南，其次介绍 NAT 与官方方法之间的比较研究指南。 2. MYCOPLASMA NAT VALIDATION 2.支原体NAT 验证 Three parameters should be evaluated: specificity, detection limit and robustness. 应评估三个参数：特异性、检测限和耐用性。 2‑1. Specificity 2‑1. 特异性 Specificity is the ability to unequivocally assess target nucleic acids in the presence of components that may be expected to be present. 特异性是在可能存在其他成分的情况下明确评估靶核酸的能力。 The specificity of an NAT and its ability to detect a large panel of mycoplasma species depend on the choice of primers, the choice of probes and the stringency of the test conditions (for both the amplification and detection steps). NAT的特异性及其检测多种支原体种类的能力取决于引物的选择、探针的选择以及试验条件的严格性（包括扩增和检测步骤）。 This ability should be demonstrated using characterised reference panels (e.g. reference strains provided by the EDQM or the WHO IS for mycoplasma DNA). Since NAT systems are usually based on a mix of primers and probes, the theoretical analysis of primers and probes by comparison with databases alone is not recommended, because interpretation of the results may be quite complex and may not reflect the experimental results. 这种能力应使用经过表征的参考菌株组（例如 EDQM 提供的参考菌株或 WHO 支原体 DNA 国际标准品）进行证明。由于 NAT 系统通常基于引物和探针的混合物，仅通过数据库比对进行引物和探针的理论分析不可取，因为结果的解读可能相当复杂且可能无法反映实验结果。 Moreover, as it is likely that the primers will detect other bacterial species, the potential for cross‑detection should be assessed in the validation study. Bacterial genera, such as gram‑positive bacteria, with a close phylogenetic relationship to mycoplasmas are most appropriate for this validation; these include Clostridium, Lactobacillus and Streptococcus. However, this is not an exhaustive list and species to be tested will depend on the theoretical ability (based on primer/probe sequences) of the NAT system to detect such other species. 此外，由于引物很可能检测到其他细菌种类，因此应在验证研究中评估交叉检测的可能性。与支原体亲缘关系较近的细菌属，例如革兰氏阳性菌，最适合用于此验证；这些包括梭菌属、乳杆菌属和链球菌属。然而，这并不是一个详尽的列表，待测试的菌种将取决于 NAT 系统检测这些其他菌种的理论能力（基于引物/探针序列）。 Based on the results from this validation of the specificity, if a gap in the specificity of the method is identified (such as detection of non‑mycoplasma bacterial nucleic acid), an appropriate strategy must be proposed in the validation study to allow interpretation of positive results on a routine basis. For example, a second test may be performed using an alternative method without this specificity gap or using an official method. 基于特异性验证的结果，如果发现方法的特异性存在缺口（例如检测到非支原体细菌核酸），则必须在验证研究中提出适当的策略，以便在常规基础上解释阳性结果。例如，可以使用没有此特异性缺口的替代方法或使用官方方法进行第二次检测。 2‑2. Detection limit 2‑2. 检测限 The detection limit is the lowest amount of target nucleic acid in a sample that can be detected but not necessarily quantified as an exact value. 检测限是样品中能够被检出的靶核酸的最低量，但不一定是确切的定量值。 For the establishment of the detection limit, a positive cut‑off point should be determined for the NAT. The positive cut‑off point (as defined in general chapter 2.6.21) is the minimum number of target sequences per volume of sample that can be detected in 95 per cent of the test runs. This positive cut‑off point is influenced by the distribution of mycoplasma genomes in the individual samples being tested and by factors such as enzyme efficiency, and can result in different 95 per cent cut‑off values for individual analytical test runs. 为确定检测限，应确定 NAT 的阳性截止点。阳性截止点（如通则 2.6.21 所定义）是在 95% 的测试运行中能够检出的每体积样品中靶序列的最小数量。该阳性截止点受待测样品中支原体基因组的分布以及酶效率等因素的影响，可能导致不同分析测试运行中 95% 截止值不同。 To determine the positive cut‑off point, a dilution series is performed using a panel of characterised and calibrated strains that may include in‑house prepared working strains or EDQM biological reference preparations (BRPs). A suitable combination of reference strains and mycoplasma DNA standards – for example, the WHO IS for mycoplasma DNA – may be used. This should be repeated in independent test sessions to examine variation between test runs. Cases where only DNA standards are used should be justified. 为确定阳性截止点，使用一组经过表征和校准的菌株（可能包括内部制备的工作菌株或 EDQM 生物参考品）进行稀释系列。可以使用参考菌株和支原体 DNA 标准品的适当组合——例如，WHO 支原体 DNA 国际标准品。这应在独立的测试会话中重复进行，以检查运行间的差异。仅使用 DNA 标准品的情况应进行论证。 For validation of the detection limit, the following species can be used. This list is proposed based on the frequency of occurrence as contaminants and phylogenetic relationships. A. laidlawii; M. fermentans; M. hyorhinis; M. orale; M. pneumoniae; M. arginini; M. salivarium; M. gallisepticum (e.g. where there is use of or exposure to avian material during production); M. synoviae (e.g. where there is use of or exposure to avian material during production); S. citri or another species of the Spiroplasma genus (e.g. where there is use of or exposure to insect or plant material during production). Not all species need to be included in the validation study. The selection of suitable species from this list should be risk‑based. For example, the manufacturing process (use of mammalian, avian or insect material or expression cell lines), the product category (biopharmaceuticals, cell‑ or gene therapy products, or vaccines) and the occurrence of contamination (e.g. based on literature research) should be taken into account. 为验证检测限，可使用以下菌种。该列表基于污染物出现的频率和系统发育关系提出。 莱氏无胆甾原体； 发酵支原体； 猪鼻支原体； 口腔支原体； 肺炎支原体； 精氨酸支原体； 唾液支原体； 鸡毒支原体（例如生产过程中使用或接触禽源材料时）； 滑液支原体（例如生产过程中使用或接触禽源材料时）； 柑橘螺原体或螺原体属的其他种（例如生产过程中使用或接触昆虫或植物材料时）。 并非所有菌种都必须包含在验证研究中。应根据风险从该列表中选择合适的菌种。例如，应考虑到生产工艺（使用哺乳动物、禽类或昆虫材料或表达细胞系）、产品类别（生物制药、细胞或基因治疗产品或疫苗）以及污染的发生情况（例如基于文献研究）。 When using mycoplasmas for the determination of the positive cut‑off point, the strains should be prepared and characterised as follows: - mycoplasmas should be harvested during the exponential phase of growth to guarantee a high viability of the reference organisms; - the titre in CFU should be determined prior to the freezing of the working suspension or dilutions; - CFU determination should be performed with a suitable number of replicates; - for the determination of the genomic copies (GC), both fractions (i.e. the supernatant and the cellular fraction) must be considered; - the acceptance criterion for the GC/CFU ratio should be defined: the GC/CFU ratio of the reference preparations should be less than 10, unless otherwise justified. 当使用支原体确定阳性截止点时，菌株应按以下要求制备和表征： -支原体应在指数生长期收获，以确保参考菌株的高活力； -在冷冻工作悬液或稀释液前应测定 CFU 滴度； - CFU 测定应使用足够数量的重复； -测定基因组拷贝数（GC）时，必须同时考虑上清液和细胞部分； -应定义GC/CFU 比率的接受标准：参考品的 GC/CFU 比率应小于 10，除非另有说明。 In addition, the WHO IS for mycoplasma DNA could be used to compare different reference strain preparations or different PCR‑kits. 此外，WHO 支原体 DNA 国际标准品可用于比较不同的参考菌株制备物或不同的 PCR 试剂盒。 For each strain, at least 3 independent dilution series should be tested, with a sufficient number of replicates at each dilution to give a total number of 24 test results for each dilution, to enable a statistical analysis of the results. For example, a laboratory may test 3 dilution series in independent test sessions with 8 replicates for each dilution, 4 dilution series in independent test sessions with 6 replicates for each dilution, or 6 dilution series in independent test sessions with 4 replicates for each dilution. In order to keep the number of dilutions at a manageable level, a preliminary test should be performed to obtain a preliminary value for the positive cut‑off point (i.e. the highest dilution giving a positive signal). The range of dilutions can then be chosen around the predetermined preliminary cut‑off point. The concentration of mycoplasmas (CFU or GC) that can be detected in 95 per cent of the test runs can then be calculated using an appropriate statistical evaluation. 对于每个菌株，应测试至少 3 个独立的稀释系列，每个稀释度应有足够数量的重复，使每个稀释度的总测试结果数达到24 个，以便对结果进行统计分析。例如，实验室可以在独立的测试会话中测试 3 个稀释系列，每个稀释度 8 个重复；或 4 个稀释系列，每个稀释度 6 个重复；或 6 个稀释系列，每个稀释度 4 个重复。为保持稀释度数量在可管理范围内，应进行预测试以获得阳性截止点的初步值（即产生阳性信号的最高稀释度）。然后可以在预定的初步截止点附近选择稀释度范围。随后可使用适当的统计评估计算在 95% 的测试运行中可检出的支原体浓度（CFU 或 GC）。 These results may also serve to evaluate the variability of the analytical procedure. 这些结果也可用于评估分析方法的变异性。 2‑3. Robustness 2‑3. 耐用性 The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters, and provides an indication of its reliability during normal usage. 分析方法的耐用性是衡量其在方法参数发生微小但有意的变化时保持不受影响的能力，并指示其在正常使用中的可靠性。 The evaluation of robustness should be considered during the development phase. It should show the reliability of the analytical procedure with respect to deliberate variations in method parameters. For NAT, small variations in the method parameters can be crucial. However, the robustness of the method can be demonstrated during its development when small variations in the concentrations of reagents (e.g. MgCl₂, primers or deoxyribonucleotides) are tested. Deliberate variations of extraction procedures as well as thermal cycler types may also be evaluated. 耐用性评估应在开发阶段予以考虑。它应显示分析方法在方法参数有意变化时的可靠性。对于 NAT，方法参数的微小变化可能至关重要。然而，方法的耐用性可以在其开发过程中通过测试试剂浓度（例如 MgCl₂、引物或脱氧核糖核苷酸）的微小变化来证明。提取程序以及热循环仪类型的故意变化也可以进行评估。 Finally, robustness of the method can be evaluated through collaborative studies. 最后，可以通过协作研究评估方法的耐用性。 3. COMPARABILITY STUDY 3.比较研究 NAT may be used instead of official methods (indicator cell culture method and/or culture method). In this case, a comparability study should be carried out. This comparability study should include mainly a comparison of the respective detection limits of the alternative method and official methods. However, specificity (mycoplasma panel detected, putative false positive results) should also be considered. NAT可用于替代官方方法（指示细胞培养法和/或培养法）。在这种情况下，应进行比较研究。该比较研究主要应包括替代方法和官方方法各自检测限的比较。然而，也应考虑特异性（检测到的支原体范围、假阳性结果的可能性）。 Acceptance criteria are defined as follows: - if the NAT method is proposed to replace the culture method, the NAT system must be shown to detect 10 CFU/mL or less than 100 GC/mL for the mycoplasma test species selected from the list in section 2‑2; - if the NAT method is proposed to replace the indicator cell culture method, the NAT system must be shown to detect 100 CFU/mL or less than 1000 GC/mL for the mycoplasma test species selected from the list in section 2‑2; - if the NAT method is proposed to replace both the culture method and the indicator cell culture method: the NAT test system must be shown to detect 10 CFU/mL or less than 100 GC/mL for the mycoplasma test species selected from the list in section 2‑2. 接受标准定义如下： -如果提议用 NAT 方法替代培养法，则对于从 2‑2 节列表中选择的支原体试验菌种，NAT 系统必须证明能检出 10 CFU/mL 或小于 100 GC/mL； -如果提议用 NAT 方法替代指示细胞培养法，则对于从 2‑2 节列表中选择的支原体试验菌种，NAT 系统必须证明能检出 100 CFU/mL 或小于 1000 GC/mL； -如果提议用 NAT 方法同时替代培养法和指示细胞培养法，则对于从 2‑2 节列表中选择的支原体试验菌种，NAT 系统必须证明能检出 10 CFU/mL 或小于 100 GC/mL。 4. TEST FOR INHIBITORY SUBSTANCES 4.抑制性物质测试 After validation of the method, a product‑specific test for inhibitory substances should be performed. Three different batches (if available) of the product to be tested should be included in this study to assess batch‑to‑batch variability. For the test for inhibitory substances, a reduced number of mycoplasma reference standards (mycoplasmas or nucleic acids as described under 4.3 Validation) should be used. Examples of approaches that could be used for the confirmation of the sensitivity in the presence of the product to be tested are provided below: 方法验证后，应进行针对特定产品的抑制性物质测试。该研究应包括待测产品的三个不同批次（如有），以评估批次间变异性。对于抑制性物质测试，应使用数量减少的支原体参考标准品（如 4.3 验证中所述的支原体或核酸）。可用于确认在待测产品存在下灵敏度的方法示例如下： - the selected mycoplasmas could be spiked at a known level, e.g. 10 CFU/mL (5 replicates each and per batch). All replicates must be positive. -可以将选定的支原体以已知水平加标，例如 10 CFU/mL（每批 5 个重复）。所有重复必须为阳性。 or - the selected mycoplasmas could be spiked at a concentration of 3× the previously determined detection limit for the specific micro‑organism but not more than 10 CFU/mL (8 replicates each, per batch). The results of 24 tests would be needed to confirm the positive cut‑off point. In the presence of the product, the positive cut‑off point should not be higher than 10 CFU/mL. -可以将选定的支原体以特定微生物先前确定的检测限的 3 倍浓度（但不超过 10 CFU/mL）加标（每批 8 个重复）。需要 24 次测试的结果来确认阳性截止点。在存在产品的情况下，阳性截止点不应高于 10 CFU/mL。 Other approaches could be acceptable. The rationale for the approach and the acceptance criteria must be provided. 其他方法也可接受。必须提供所选方法的原理和接受标准。

（接前文）为啥“三抗生素软膏”70多年来一直能保持抗菌活性如此稳定？（上篇） 三. 进化屏障：耐药性为何难以产生 细菌的顽固性以具备抗药性演化能力而著称。但是在本案面前，虽然已经历经70多年，杆菌肽-新霉素-多黏菌素B这三重组合形式依然能保持高效抗菌性能。这种独特的抗性效果可通过进化概率理论与适应度概念来加以解释。 1.三联疗法的数学原理 联合疗法的基础理论建立在概率独立性之上。抗生素耐药性通常通过染色体自发突变或获得可移动遗传元件产生。 设自发突变导致对药物A产生耐药性的概率为P(A)，对药物B为P(B)，对药物C为P(C)。高水平耐药性的典型突变率约为每次细胞分裂的10-7至10-9。    若耐药机制相互独立（正交），单个细菌同时获得三种药物耐药性的概率为各概率的乘积： P(Total) = P(A) x P(B)  x P(C) P(Total)≈ 10-8 x 10-8 x 10-8 = 10-24 考虑到重度感染的伤口可能含有10⁹至10¹⁰个细菌，针对3种抗生素的三联突变体从头产生的统计概率实际上为“零”。这与支撑HAART（高效抗逆转录病毒疗法）治疗HIV及结核病联合化疗成功的数学原理是一样的。 2.正交性与交叉耐药缺失 概率论论证的有效性取决于耐药机制的独立性。若单一突变即可对三种药物同时产生耐药性（交叉耐药），那耐药概率将降至10-8。然而这三类药物的耐药机制存在根本性差别： 👉 杆菌肽耐药性：通常涉及上调ABC转运蛋白（如bcrABC）将药物泵出细胞膜，或过表达十一烯醇激酶以补充C55-P池。 👉 新霉素耐药性：主要通过氨基糖苷类修饰酶（AMEs）介导，如乙酰转移酶（AAC）、磷酸转移酶（APH）和核苷酸转移酶（ANT）对药物进行化学修饰，或通过16S rRNA甲基化实现。 👉 多黏菌素耐药性：通过脂质A靶点的修饰实现。mcr-1等基因或pmrAB系统突变会导致磷酸乙醇胺或阿拉伯糖添加至脂质A，降低其负电荷，从而减弱其与阳离子多黏菌素的亲和力。 由于核糖体修饰酶无法泵出杆菌肽，且脂质A修饰对新霉素无效，故不存在交叉耐药性。该细菌必须获得三种独立的遗传性状才能存活。 3. 抗性的适应代价 生物进化是权衡取舍的过程。获得抗性常会给细菌带来代谢负担或“适应性代价”。 👉 核糖体突变：阻断新霉素结合的突变常降低蛋白质合成效率，导致细菌生长速度慢于野生型菌株。 👉 膜结构改造：为抵抗多黏菌素而改变脂多糖结构，可能削弱外膜屏障功能，使菌体更易受环境压力、宿主免疫肽（防御素）及胆汁盐的侵害。 👉 “三重负担”：若细菌同时获得三种耐药性状，其生长缺陷将极为严重，在无抗生素压力环境下反而会被非耐药菌株淘汰。 4. 新兴威胁：USA300是个别例外现象 尽管耐药性存在高门槛，但并非绝对。MRSA USA300克隆株的出现便是一个警示案例。基因组分析揭示，部分USA300谱系携带多药耐药质粒（如pUSA300-HOU-MR），该质粒串联了不同类别的耐药基因。     具体而言，这些质粒可同时携带： 👉 杆菌肽耐药性：通过bcr操纵子实现 👉 新霉素耐药性：通过氨基糖苷类修饰酶（如aphA、aadE）实现。 研究表明金黄色葡萄球菌的杆菌肽耐药性几乎普遍与同时赋予新霉素耐药性的质粒相关联。这种“共携带”特性突破了独立概率模型——单次 水平基因转移即可使菌株获得三种耐药成分中的两种。但需注意的是，此类质粒通常不携带多黏菌素耐药决定因子。 此外，局部治疗（详见下文）所形成的药物高浓度环境，往往能压制包括上述耐药机制在内的多种抗药性。 四. 局部用药的药代与药效动力学：“通杀”浓度的抑杀特点 局部抗生素的用法与全身治疗存在者根本的差异。全身给药受药物毒性阈值（如肾功能衰竭）限制剂量，而局部应用仅受限于组织耐受性与溶解度，可实现微生物学意义上的天文数字级浓度。 1. 浓度差距：全身性与局部性 市售三联抗生素软膏通常含： 📍杆菌肽：400–500单位/克（约8–10毫克/克）。 📍新霉素：3.5毫克/克。 📍多黏菌素B：5,000–10,000单位/克（约1毫克/克）。 以金黄色葡萄球菌为例说明： 📍新霉素对金黄色葡萄球菌的MIC90值（抑制90%菌株所需浓度）约为17.6µg/mL。 📍软膏中的浓度为3,500 µg/mL。 📍这意味着其治疗比值约为200:1。 对于杆菌肽，耐药菌株的MIC值可能超过512 µg/mL。该软膏提供约10,000 µg/mL的浓度（假设1单位≈20 µg），同样形成巨大的药量储备。 2. 突变预防浓度（MPC） 突变预防浓度（MPC）的概念在此至关重要。最小抑菌浓度仅表明抑制多数菌群生长的浓度阈值。然而在任何大型细菌群体中，可能存在耐药性更强的罕见突变体。MPC是即使单步耐药突变体也无法复制繁殖的浓度阈值。 👉 突变选择窗口（MSW）：危险区域即MIC与MPC之间的浓度区间。在此窗口内，药物虽能杀灭敏感菌株，却允许耐药突变体存活并扩增。 👉 关闭选择窗口：全身性抗生素常因毒性阈值限制而难以维持在MPC以上浓度。局部用药疗法通过输送远超MIC的浓度，可使TAO中的三种药物均突破 的MPC阈值。这就有效地“关闭”了细菌突变体的选择窗口，阻止了耐药亚群体的生存和富集。 3. 吸收与全身安全性 TAO中的化合物的安全性特征关键还在于其缺乏全身吸收特性。 💊杆菌肽：作为大型多肽，其通过完整皮肤甚至有效肉芽组织的吸收不良。 💊新霉素：通过完整皮肤吸收量极微。虽然在表皮缺损区域（如大面积烧伤）吸收可能增加，但常规应用于轻微割伤时血清浓度可忽略不计。 💊多黏菌素B：其对细胞膜脂质的高亲和力使其倾向于局限于创面部位。 这种低吸收特性对于TAO本身具有独特而重要的进化意义：它限制了这三种抗生素在体内的“生态足迹”。口服抗生素会扩散至全身，特别是肠道，这对微生物群中数万亿细菌都施加了选择压力，从而形成巨大的耐药性储备库。而局部使用的TAO仅在伤口部位发挥作用，这种局部化特性极大减少了接触药物的细菌总量，从而降低耐药性产生与扩散的统计概率。 五. 疗效比较：优于单一药物 现有所有临床及体外数据均一致表明，三联组合疗法优于单一药物治疗及消毒剂。 1. 体外疗效 棋盘式梯度试验显示，TAO这一三联组合配方对所有常见创面感染参考菌株均呈现协同抗菌效应。 表1 TAO三种抗生素的协同FIC指数 表中FIC指数解读： 🦠金黄色葡萄球菌：新霉素/杆菌肽=协同 🦠铜绿假单胞菌：杆菌肽/多黏菌素 = 协同 🦠粪肠球菌：新霉素/多黏菌素 = 协同作用 🦠TAO制剂：对所有菌株均呈协同作用（FIC < 1） 这种广谱协同作用确保该软膏能覆盖多种伤口的混合感染特性，其中革兰氏阳性菌和革兰氏阴性菌可能共存并形成生物膜。 2. 临床试验与伤口愈合 多项随机对照试验验证了TAO三抗软膏的临床实用性。 👉水疱创面研究：在污染性水疱创面研究中，使用三联软膏治疗的创面愈合速度显著快于使用消毒剂或未处理的创面（平均9天）。更重要的是，仅三联组合能在16-24小时内有效清除细菌污染（特别是金黄色葡萄球菌）。 👉皮肤磨削术研究：在皮肤磨削术敷料对比中，TAO优于普通纱布，能显著减少瘢痕形成。这不仅归因于其感染预防作用，更因其载体（凡士林）能维持湿润创面环境，加速上皮化过程。 👉小儿脓皮病：一项双盲研究发现，TAO在预防轻微创伤儿童链球菌性脓皮病方面优于安慰剂。TAO组感染率为15%，安慰剂组达47%。 3. 与莫匹罗星的比较 莫匹罗星常被视为局部MRSA治疗的金标准，但其仅为单一疗法（通过抑制异亮氨酰-tRNA合成酶发挥作用）。 👉 耐药性：对莫匹罗星的耐药性呈上升趋势。通过质粒上的mupA基因可获得高水平耐药性。监测研究显示莫匹罗星耐药率持续攀升，尤其在参与去定植方案的MRSA菌株中更为显著。 👉 稳定性：相比之下，TAO成分的耐药率数十年间保持相对稳定。一项1997-2002年美国研究发现，尽管TAO作为非处方药未受管制，其耐药性并未显著增加。 表2：局部制剂比较特征 特征 三抗生素软膏 莫匹罗星 夫西地酸 消毒剂（碘/银） 活性成分 3种（青霉素、新霉素、多黏菌素） 1种 1种 1种（广谱化学制剂） 作用机制 多靶点（细胞壁、核糖体、膜） 蛋白质合成（tRNA） 蛋白质合成（延伸因子G） 非特异性蛋白质变性 作用范围 广谱（革兰氏阳性菌和革兰氏阴性菌） 主要为革兰氏阳性（金黄色葡萄球菌） 主要为革兰氏阳性菌 广谱 耐药风险 低（需多重靶点作用） 中等/高（MRSA中呈上升趋势） 中等（单点突变） 低 协同效应 是（通透化作用） 否 否 否 六. 当前监测与未来展望 由杆菌肽-新霉素-多黏菌素B组成复方制剂的抗菌效果显著，耐药性70多年来几乎没有大的变化，当然这种结果也不可能是绝对的。当前监测数据既凸显了这一制剂的持续有效性，但也揭示了值得关注的领域。 1. 美国300型耐甲氧西林金黄色葡萄球菌的挑战 USA300 MRSA克隆株在北美地区的优势地位已展示了一种新的独特挑战。如前所述，该谱系常携带赋予杆菌肽和新霉素双重耐药性的质粒。 👉 体外与体内差异：尽管实验室检测显示这些菌株对药物敏感性降低（高MIC值），但轻微创伤的临床治疗失败率并未出现相应比例的上升。 这种差异可能源于前文所述的具有通杀效果的“高剂量”局部效应——新霉素的局部浓度（3.5 mg/g）仍足以抑制具有256 µg/mL“耐药”MIC值的菌株。 👉 多黏菌素的阻击：这些菌株通常对多黏菌素B不具耐药性。尽管多黏菌素对革兰氏阳性菌活性较弱，但高浓度仍可发挥一定作用，且该组合的冗余性提供了安全保障。 2. 全球差异与监管 细菌对抗生素的耐药模式受当地监管环境显著影响，本文就不再展开讨论，这里仅展示两个国家的数据： 🇦🇺 澳大利亚：TAO仅限处方使用。当地监测数据显示耐药率极低（如MRSA对TAO耐药率仅2%）。 🇺🇸 美国：尽管TAO可非处方购买，但耐药性并未爆发式增长。这表明“仅限局部使用”的场景有效隔离了耐药菌群，避免了系统性抗生素普遍存在的耐药基因扩散问题。 3. 结论：传统科学的胜利 杆菌肽-新霉素-多黏菌素B三联复方制剂印证了联合疗法与局部给药的有效性。其长久生命力并非偶然，而是设计预期的必然结果： 👉 正交性：迫使细菌同时解决三项独立生理难题。 👉 协同效应：通过膜渗透作用增强各组分的杀菌效力。 👉 浓度优势：借助局部给药的物理特性压倒细菌防御机制，彻底封堵突变选择窗口。 当新型全身性抗生素研发管道枯竭之际，这款问世七十年的制剂仍作为全球抗感染武器库中的关键工具，证明在对抗耐药性的战争中，现有抗生素武器的战略性组合可与新药研发同样有效。 参考文献(略) （全文完）