本试剂盒适用于从宫颈拭子保护液、全血、血浆、痰液、肺泡灌洗液、脑脊液等样本中提取病原微生物核酸，该方法采用磁珠纯化技术，提取过程中无需使用有毒的酚氯仿抽提，也无需进行耗时的醇类沉淀。该方法纯化的核酸包括病毒、支原体、细菌、真菌等微生物总核酸，得到的产物可直接用于PCR、QPCR、二代测序等实验。其处理后的产物用于科研检测使用。
| 产品号 | 组件名称 | 物理外观 | 48T | 储存温度 | | M1522636A | 蛋白酶K | 液体 | 1.1 mL | 2-8℃. | | M1522636B | 核酸保护液MP | 液体 | 12 mL | RT. | | M1522636C | 研磨管 (ZB_M1) | — | 1 EA×48 | RT. | | M1522636D | 消泡剂 | 液体 | 200 μL | RT. | | M1522636E | 裂解结合液RLC | 液体 | 33 mL | RT. | | M1522636F | 磁珠MXVB | 液体 | 1.1 mL | 2-8℃. | | M1522636G | 洗涤液PW1 | 液体 | 40 mL | RT. | | M1522636H | 洗涤液DW2 | 液体 | 80 mL | RT. | | M1522636I | 洗脱液DE | 液体 | 6 mL | RT. |
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储存条件
M1522636A, M1522636F：2-8℃保存，12个月。
M1522636B, M1522636C, M1522636D, M1522636E, M1522636G, M1522636H, M1522636I：室温保存，12个月。
适配仪器
手工提取
注意事项
1. 使用前请仔细阅读本说明书。
2. 本试剂盒中的多种缓冲液含有刺激性的胍盐，务必戴上手套，并按照安全标准预防措施来处理。不要让缓冲液接触到皮肤、眼睛以及黏膜，如果确实发生，请立即用大量清水清洗并就医。
3. 如因气温较低，溶液出现沉淀，需要30℃水浴至沉淀完全溶解后方可使用。
4.如因气温较低，用力振荡后磁珠无法重悬，请勿使用。
5. 本试剂盒中的多种缓冲液含胍盐，请勿用氧化性消毒剂如次氯酸钠进行处理，否则会释放有毒气体，须按医疗废物进行处理。
6. 本试剂盒需配套使用，不同批号试剂盒中各组分不可以互换或混用。
使用方法
1. 样本要求
- 适用标本类型：宫颈拭子、全血、血浆、痰液、肺泡灌洗液、脑脊液等样本。
- 标本保存和运输：标本可立即用于测试，也可以保存于-70℃或更低温度待测，保存期为6个月，应避免反复冻融。标本运输采用冷链运输。
- 冻融要求：速冻速融，避免反复冻融。
2. 自备试验器材
3. 试剂准备
4. 操作步骤
4.1 根据不同样本类型选择对应步骤进行处理
- 痰液样本：取适量痰液至2 mL离心管中，加入20~100 μL 1M DTT（自备，视样本粘稠程度适量添加），70℃温浴至充分液化，转移400 μL加入研磨管（ZB_M1）；或减少取样量，加入生理盐水进行稀释，转移400 μL至研磨管（ZB_M1）中。加入200 μL核酸保护液MP、20 μL蛋白酶K，在涡旋仪最大转速涡旋或转移至震荡破碎仪高速研磨样品10分钟（不同品牌震荡破碎仪，请选择仪器推荐程序）。
- 拭子类样本或者其他含保护液（胍盐大于1M）的生物液体样本：取400 μL液体样本至研磨管（ZB_M1）中，再加入20 μL蛋白酶K，在涡旋仪最大转速涡旋或转移至震荡破碎仪高速研磨样品10分钟（不同品牌震荡破碎仪，请选择仪器推荐程序）。注意：若样本体积＜400 μL，使用核酸保护液MP补齐至400 μL。
- 全血样本：取400 μL液体样本至研磨管（ZB_M1）中，加入200 μL核酸保护液MP、20 μL蛋白酶K，在涡旋仪最大转速涡旋或转移至震荡破碎仪高速研磨样品10分钟（不同品牌震荡破碎仪，请选择仪器推荐程序）。注意：1) 全血样本：如脐带血，骨髓血这些细胞含量高的全血，提取过程中可能会出现磁珠残留或颜色残留，建议将样本用生理盐水或PBS进行稀释后再进行核酸提取。2) 痰液样本：提取过程中容易出现磁珠残留，建议将样本用生理盐水或PBS进行稀释后再进行核酸提取。
4.2 瞬时离心，吸取300 μL上清溶液至新的离心管，加入600 μL裂解结合液RLC中，小心吸取，避免吸到沉淀或玻璃珠。
4.3 加入20 μL磁珠MXVB，涡旋混匀30秒，振荡或涡旋5~7分钟使磁珠吸附核酸。
4.4 转移至磁力架上进行磁分离至溶液澄清，吸弃溶液。
4.5 加入700 μL洗涤液PW1，涡旋60~180秒后转移至磁力架上吸附至溶液澄清，吸弃溶液。
4.6 加入700 μL洗涤液DW2，涡旋30秒打散磁珠后转至磁力架上进行磁分离至溶液澄清，吸弃溶液。
4.7 再次加入700 μL洗涤液DW2，涡旋30秒打散磁珠后转至磁力架上进行磁分离至溶液澄清，吸弃溶液。
4.8 掌上离心机瞬时离心后，转至磁力架上吸附至溶液澄清，充分吸弃所有溶液，打开管盖，空气干燥3~5分钟。
注意：乙醇残留会抑制后续的酶反应，所以晾干时要确保乙醇挥发干净。切勿干燥过久，以免影响后续洗脱效果。
4.9 加入30~100 µL洗脱液DE，高速涡旋2~3分钟打散磁珠。
4.10 60℃振荡温浴5分钟，如恒温仪无振荡功能，可在温浴期间取出，高速涡旋打散磁珠2~3次，每次10~30秒。
4.11 瞬时离心收集管盖液滴至管中，转移至磁力架上进行磁分离。
4.12 把核酸溶液转移至新的离心管中待用，如果不立即使用，请存储于-15~-25℃，长期保存请放置于-70℃或更低温度。
注意：在进行下游PCR或qPCR检测前，对于双链RNA病毒如轮状病毒、传染性胰坏死病毒、传染性法氏囊病病毒、果蝇的X病毒、双瓣软体动物的Tellina病毒（TV）和牡蛎病毒（Oyster virus，OV）等，建议先将双链RNA经90℃变性5分钟后，迅速降温至4℃，再进行逆转录及后续PCR。
方法的局限性
1. 对于某些取样不佳的样本，提取产物中可能存在少量杂质抑制PCR扩增。
2. 样本核酸的提取效果与样本收集、处理、运输和保存质量密切相关，其中任何失误都将会导致结果不准确。
This kit is suitable for the extraction of pathogenic microbial nucleic acids from samples such as cervical swab preservation solution, whole blood, plasma, sputum, bronchoalveolar lavage fluid, and cerebrospinal fluid. The method employs magnetic bead purification technology, eliminating the need for toxic phenol‑chloroform extraction or time‑consuming alcohol precipitation. Nucleic acids purified with this method include total microbial nucleic acids from viruses, mycoplasma, bacteria, fungi, and other microorganisms. The resulting products can be used directly in PCR, qPCR, next‑generation sequencing, and other experiments. The purified nucleic acids are intended for research use only.
| Product Code | Component | Appearance | 48T | Storage | | M1522636A | Proteinase K | Liquid | 1.1 mL | 2–8 °C | | M1522636B | Nucleic Acid Preservation Solution MP | Liquid | 12 mL | RT | | M1522636C | Grinding Tube (ZB_M1) | — | 1 EA × 48 | RT | | M1522636D | Antifoam Agent | Liquid | 200 μL | RT | | M1522636E | Lysis/Binding Buffer RLC | Liquid | 33 mL | RT | | M1522636F | Magnetic Beads MXVB | Liquid | 1.1 mL | 2–8 °C | | M1522636G | Wash Buffer PW1 | Liquid | 40 mL | RT | | M1522636H | Wash Buffer DW2 | Liquid | 80 mL | RT | | M1522636I | Elution Buffer DE | Liquid | 6 mL | RT |
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Storage Conditions
M1522636A, M1522636F: Store at 2–8 °C for 12 months.
M1522636B, M1522636C, M1522636D, M1522636E, M1522636G, M1522636H, M1522636I: Store at room temperature for 12 months.
Compatible Instrumentation
Manual extraction.
Precautions
Read the entire manual carefully before use.
Several buffers contain irritating guanidine salts. Always wear gloves and follow standard safety precautions. Avoid contact with skin, eyes, and mucous membranes. If contact occurs, rinse immediately with plenty of water and seek medical attention.
If precipitates appear due to low temperature, warm the solution at 30 °C until precipitates are completely dissolved before use.
If magnetic beads cannot be resuspended by vigorous shaking due to low temperature, do not use.
Several buffers contain guanidine salts. Do not treat with oxidizing disinfectants (e.g., sodium hypochlorite) as this may release toxic gas. Dispose as medical waste.
This kit is intended for use as an integrated system. Do not interchange or mix components from different lot numbers.
Protocol
1. Sample Requirements
Applicable sample types: Cervical swabs, whole blood, plasma, sputum, bronchoalveolar lavage fluid, cerebrospinal fluid, and other samples.
Sample preservation and transport: Samples can be processed immediately or stored at –70 °C or lower for up to 6 months. Avoid repeated freeze‑thaw cycles. Transport samples under cold chain conditions.
Freeze‑thaw requirements: Quick freeze and quick thaw; avoid repeated freeze‑thaw cycles.
2. Materials to Be Prepared by User
Water bath (for dissolving precipitates), dry thermostat bath
Magnetic stand
1.5/2.0 mL sterile, nuclease‑free centrifuge tubes
Various pipettes and tips (20 µL, 200 µL, 1000 µL)
3. Reagent Preparation
Check solutions for precipitates and ensure magnetic beads can be resuspended.
Before using this kit for the first time, add 20 μL of Antifoam Agent to the Nucleic Acid Preservation Solution MP and mix by inversion.
4. Procedure
4.1 Sample processing according to sample type
Sputum samples: Transfer an appropriate amount of sputum to a 2 mL centrifuge tube. Add 20–100 μL of 1 M DTT (prepare by user; adjust amount according to sample viscosity). Incubate at 70 °C until fully liquefied. Transfer 400 μL to a Grinding Tube (ZB_M1). Alternatively, reduce the sample volume and dilute with physiological saline, then transfer 400 μL to a Grinding Tube (ZB_M1). Add 200 μL of Nucleic Acid Preservation Solution MP and 20 μL of Proteinase K. Vortex at maximum speed or transfer to a bead beater and grind at high speed for 10 minutes (follow the instrument‑recommended program for different brands).
Swab samples or other biological fluid samples containing preservation solution (guanidine salt >1 M): Transfer 400 μL of liquid sample to a Grinding Tube (ZB_M1), then add 20 μL of Proteinase K. Vortex at maximum speed or transfer to a bead beater and grind at high speed for 10 minutes (follow the instrument‑recommended program). Note: If the sample volume is less than 400 μL, make up to 400 μL with Nucleic Acid Preservation Solution MP.
Whole blood samples: Transfer 400 μL of liquid sample to a Grinding Tube (ZB_M1). Add 200 μL of Nucleic Acid Preservation Solution MP and 20 μL of Proteinase K. Vortex at maximum speed or transfer to a bead beater and grind at high speed for 10 minutes (follow the instrument‑recommended program). Notes:
For whole blood samples with high cell content (e.g., cord blood, bone marrow blood), magnetic bead or color residue may occur during extraction. It is recommended to dilute the sample with physiological saline or PBS before nucleic acid extraction.
For sputum samples, magnetic bead residue may occur. It is recommended to dilute the sample with physiological saline or PBS before nucleic acid extraction.
4.2 Briefly centrifuge. Transfer 300 μL of the supernatant to a new centrifuge tube containing 600 μL of Lysis/Binding Buffer RLC. Carefully pipette to avoid transferring precipitate or glass beads.
4.3 Add 20 μL of Magnetic Beads MXVB. Vortex for 30 seconds to mix, then shake or vortex for 5–7 minutes to allow the beads to bind nucleic acids.
4.4 Place the tube on a magnetic stand for magnetic separation until the solution is clear. Aspirate and discard the supernatant.
4.5 Add 700 μL of Wash Buffer PW1. Vortex for 60–180 seconds, then return to the magnetic stand and allow the solution to become clear. Aspirate and discard the supernatant.
4.6 Add 700 μL of Wash Buffer DW2. Vortex for 30 seconds to resuspend the beads. Return to the magnetic stand, separate until clear, and aspirate the supernatant.
4.7 Add another 700 μL of Wash Buffer DW2. Vortex for 30 seconds to resuspend the beads. Separate magnetically until clear, then aspirate the supernatant.
4.8 Briefly centrifuge in a microcentrifuge. Place the tube on the magnetic stand and allow the solution to become clear. Aspirate and discard all remaining liquid thoroughly. Open the cap and air‑dry for 3–5 minutes.
Note: Residual ethanol will inhibit downstream enzymatic reactions. Ensure that ethanol is completely evaporated during drying. Do not over‑dry, as this may affect elution efficiency.
4.9 Add 30–100 μL of Elution Buffer DE. Vortex vigorously for 2–3 minutes to fully resuspend the beads.
4.10 Incubate at 60 °C with shaking for 5 minutes. If the incubator does not have a shaking function, remove the tube during incubation and vortex vigorously to resuspend the beads 2–3 times, each for 10–30 seconds.
4.11 Briefly centrifuge to collect droplets from the cap. Return the tube to the magnetic stand for magnetic separation.
4.12 Transfer the nucleic acid solution to a new centrifuge tube for immediate use. If not used immediately, store at –15 to –25 °C. For long‑term storage, keep at –70 °C or lower.
Note: Before performing downstream PCR or qPCR, for double‑stranded RNA viruses (e.g., rotavirus, infectious pancreatic necrosis virus, infectious bursal disease virus, Drosophila X virus, Tellina virus (TV) of bivalve mollusks, and oyster virus (OV)), it is recommended to denature the double‑stranded RNA at 90 °C for 5 minutes, then rapidly cool to 4 °C before reverse transcription and subsequent PCR.
Limitations of the Method
For certain poorly collected samples, the extracted product may contain trace impurities that inhibit PCR amplification.
The efficiency of nucleic acid extraction is closely related to sample collection, processing, transport, and storage. Any error in these steps may lead to inaccurate results.
J2614479
危险品化学品经营许可证（带存储）