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概述
组分说明
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产品简介
本试剂盒是以分子流行病学最新研究进展1）为基础，经工艺优化后形成的人型结核分枝杆菌基因分型产品。本产品利用 结核分枝杆菌基因组中可变数目串联重复序列（variable-number tandem repeats, VNTR）多态性进行基因型分型区分临 床菌株，是研究结核分枝杆菌分子流行病学和监测结核病传播状况的有力工具。与现有其它基于VNTR原理的结核分枝杆菌 VNTR分型系统相比，这一分型系统对中国流行的菌株具有更强的分辨能力1，2，3），因此特别适合于中国用户的需求。
通过对各PCR反应引物序列和预混反应液成份进行精心优化，使得本产品具有很强的抗干扰力。与用户自配试剂相比， 本产品显著提升了特异条带信号强度，降低了使用粗制模板（煮沸菌液）时非特异条带的出现率，使实验操作更加简便、快 捷的同时，提高了检测成功率。本产品的预混反应液化学稳定性良好，能有效抵抗反复冻融（10次）和较长时间（一周） 的室温环境，更好地适应了用户检测工作中的灵活性需求。
本试剂盒是 TB 基因分型试剂盒 VNTR-9的配套产品。对 VNTR-9 试剂盒鉴定为成簇或相同菌株的 样本，如有必要，可使用本产品做更精细的进一步分型鉴定。本产品中的 3 个高分辨率检测位点 VNTR3820，VNTR4120 和 VNTR3232 与 VNTR-9 中的 9 个检测位点结合使用可将检测的分辨率指数（Hunter-Gaston index，HGI)提升至 0.9931）。
参考文献
1） Luo T et al. Development of a hierarchical variable-number tandem repeat typing scheme for Mycobacterium tuberculosis in China. PLoS One. 2014 Feb 25;9(2)
2） Sun G et al. Discriminatory potential of a novel set of Variable Number of Tandem Repeats for genotyping Mycobacterium marinum. Vet Microbiol. 2011 Aug 26;152(1-2)
3） Zhang L et al. Highly polymorphic variable-number tandem repeats loci for differentiating Beijing genotype strains of Mycobacterium tuberculosis in Shanghai, China. FEMS Microbiol Lett. 2008 May;282(1):22-31.
注意事项
1. 本产品是 TB 基因分型试剂盒 VNTR-9的配套产品。待测菌株应先经过 VNTR-9 分型检测，再使用 本产品检测。且本产品的检测结果应与 VNTR-9 的检测结果进行整合分析。
2. 为避免污染，建议制备生物时与配制 PCR Mix 在不同的地点内进行，并使用不同的移液器。
3. 在样本 DNA 的收集，抽提和扩增的所有环节都应注意作好标记，同时防止不同样本间发生交叉污染。
4. 常用试剂和耗材在实验前需高压灭菌。
5. 每管 PCR Mix 中均含有不同的引物，不可混用。可根据实验需求一次性分装为不同的量，避免反复冻融。
6. 为避免打开反应管时，反应液飞溅，开盖前请短暂离心，收集液体于管底。若不小心溅到手套或桌面上，应立刻更换手 套并用 75%酒精或稀酸擦拭桌面。
7. 吸取时注意不要交叉污染 PCR Mix，建议每次取 Mix 前用 75%酒精擦拭移液器头 2 次。
8. 实验前准备：1×TE 缓冲液（PH=8.0）、0.5×TBE 缓冲液、琼脂糖、溴化乙锭（EB）、普通 PCR 仪、DNA 电泳设备和 凝胶成像仪、0.2 ml PCR 反应管、八联排或 96 孔 PCR 管、不同规格的移液器：0.5-10 μl 和 20-200 μl。
操作步骤
1. DNA 模板制备：
1.1 从固体培养基上刮取少量(1-2 接种环)样本，重悬于 100 ul TE 中，80°C 灭活 30 分钟。
1.2 灭活后的菌株拿出 P3 实验室进行如下操作： 100°C 煮沸 10 分钟(煮沸时注意避免 EP 管盖子爆开，避免让水进入管中)，立即置于冰上 2 分钟，12,000 rpm (~13,400×g)离心 10 分钟，取上清置于另一无菌 EP 管中，做上标记，-20°C 保存。
2 . 检测程序：
2.1 取出 TB 基因分型试剂盒 HV-3，待液体平衡到室温后，轻微摇晃 3-4 次混匀，然后 12,000 rpm (~13,400×g)离心 5 秒，使盖上的液体回落到管内。
2.2 三位点 VNTR 分型：对于 12 位点结果相同的菌株需进一步进行 VNTR 分型，即增加以下 4 个位点进行比较。
1）PCR 扩增：反应体系为 20 μl。 在每个 PCR 管里分别加入 19 μl VNTR3820、VNTR4120、VNTR3232 的 PCR Mix，加入 1 μl DNA 模板，混匀。
2）扩增条件：
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3）制胶、电泳：
a：注意事项：
重要！每次实验需要设置阳性（H37Rv 菌株 DNA）和阴性对照（去离子水）。
关键！本实验是以琼脂糖凝胶电泳为基础来判读 VNTR 位点基因型，因此，为了使结果准确，在电泳这一步必须要按 照统一的标准操作，应注意以下几点：
a-1：制胶所用梳子为 18 孔。
a-2：凝胶左右边上的两个孔由于在电泳过程中容易使条带变形，影响结果判读，舍弃不用，或者在其中一个孔点上阴 性对照。剩余 16 孔分为 12 个样本，3 个 DNA Marker 和 1 个阳性对照。点样顺序分别为“1，2，M，3，4，5， 6， M，7，8，9，10，M，11，12，H37Rv”，数字代表样本，M 代表 DNA Marker。
a-3：PCR 扩增产物进行首次电泳并使用 Marker I 时，凝胶浓度为 1%，电压为 150V，时间为 100-120 分钟。
a-4：如果扩增产物片段过大（>1000bp），需要再次电泳并使用 Marker II 时，凝胶浓度为 0.8%，电压 150V，时间为 150 分钟。
b：制胶以及电泳过程：
使用 1%的琼脂糖凝胶对 PCR 扩增产物进行电泳。
配制 1%的琼脂糖凝胶，12×12 cm 胶盘制胶，每块胶为 80 ml。
b-1：称取 0.8 g 琼脂糖，加入 80 ml 0.5×TBE，在天平上称重后放入微波炉，高火加热 2-3 分钟，使琼脂糖完全溶化， 摇匀，观察为均一透明溶液，无颗粒，再在天平称量，补入适量的双蒸水，以保持胶的浓度不受影响。
b-2：待融化的凝胶冷却至 55℃左右时加入 4 μl 溴化乙锭（10 ug/ml），轻轻旋转以充分混匀。用 18 齿的梳子制胶， 将温热的凝胶浇灌入 12×12 cm 胶盘。
b-3：待凝胶完全凝结（室温下放置 40 分钟），小心拔出梳子，取出托盘，放入电泳槽中。电泳槽中加入 0.5×TBE 缓冲 液，没过胶面 1-2 mm。
b-4：上样电泳：每块胶中加入 12 个样本（最边上的孔不加样），每个孔中加入 3-5 μl PCR 产物，同时每块胶上加三 个 5 μl DNA MarkerⅠ。电压 150V，电泳时间为 100-120 分钟。本步骤是各位点最终读数是否准确的关键，需 要统一按照此标准操作。
b-5：部分位点在临床菌株中存在扩增产物大于 1000 bp 的情况，对这些扩增产物再利用 0.8%琼脂糖凝胶进行电泳， 同时加入 DNA Marker II 作为条带大小对照，电压 150V，电泳时间 150 分钟。
4）结果显示：
-2.png?access_token=9651bf4f-f2d6-425a-8e7a-ab666b716d21)
5）结果分析：
a. 如果不同菌株的 3 个高变位点基因型也相同，可确定为成簇菌株；
b. 如果高变位点读数高度相似，即只有 1-2 个高变位点有差别，需结合流行病学数据鉴定是否为成簇菌株；
c. 如果 3 个高变位点基因型都不一致，鉴定为单一菌株。
附录 1：VNTR 位点读数规则
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附录 2 ：VNTR 位点重复单元读数表
-4.png?access_token=22da9050-5b33-47e0-8c89-fe8d30ea99e1)
Component Description
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Product Introduction
This kit is a genotyping product for human Mycobacterium tuberculosis based on the latest research progress in molecular epidemiology1) and optimized by process. It utilizes variable-number tandem repeats (VNTR) polymorphisms in the Mycobacterium tuberculosis genome for genotyping to differentiate clinical strains, and is a powerful tool for studying the molecular epidemiology of Mycobacterium tuberculosis and monitoring the status of tuberculosis transmission. Compared with other existing Mycobacterium tuberculosis VNTR typing systems based on the VNTR principle, this typing system has a stronger ability to discriminate strains prevalent in China1,2,3), and is therefore particularly suitable for the needs of Chinese users.
By carefully optimizing the primer sequences of each PCR reaction and the composition of the premixed reaction solution, this product has a strong anti-interference power. Compared with the user's own reagents, this product significantly improves the signal intensity of specific bands and reduces the appearance of non-specific bands when using crude templates (boiling bacterial solution), which makes the experimental operation easier and quicker, and at the same time, improves the success rate of the test. The premixed reaction solution is chemically stable and can effectively withstand repeated freezing and thawing (10 times) and a longer period of time (one week) at room temperature, which is better adapted to the user's need for flexibility in the detection work.
This kit is a companion product to the TB Genotyping Kit VNTR-9. For samples identified as clustered or identical strains by the VNTR-9 kit, this product can be used for finer further typing identification if necessary. The three high-resolution detection sites VNTR3820, VNTR4120 and VNTR3232 in this product can be used in combination with the nine detection sites in the VNTR-9 to increase the resolution index (Hunter-Gaston index (HGI) to 0.9931).
References
1) Luo T et al. Development of a hierarchical variable-number tandem repeat typing scheme for Mycobacterium tuberculosis in China. PLoS One. 2014 Feb 25. 9(2)
2)Sun G et al. Discriminatory potential of a novel set of Variable Number of Tandem Repeats for genotyping Mycobacterium marinum. Vet Microbiol. 2011 Aug Vet Microbiol. 2011 Aug 26;152(1-2)
3) Zhang L et al. Highly polymorphic variable-number tandem repeats loci for differentiating Beijing genotype strains of Mycobacterium tuberculosis in Shanghai, China. FEMS Microbiol Lett. 2008 May;282(1):22-31.
matters needing attention
1.This product is a companion to the TB genotyping kit VNTR-9. The strains to be tested should be tested by VNTR-9 typing test first, and then use this product for testing. And the results of this product should be integrated and analyzed with the results of VNTR-9.
2.To avoid contamination, it is recommended that the preparation of the organisms be done within a different location than the preparation of the PCR Mix and that different pipettes be used.
3.Care should be taken at all stages of sample DNA collection, extraction and amplification to ensure proper labeling and to prevent cross-contamination between different samples.
4.Commonly used reagents and consumables need to be autoclaved before experimentation.
5.Each tube of PCR Mix contains different primers and cannot be mixed. It can be dispensed into different amounts at once according to the experimental needs to avoid repeated freezing and thawing.
6.To avoid splashing the reaction solution when opening the reaction tube, centrifuge briefly before opening the cap and collect the liquid at the bottom of the tube. In case of accidental splashing on gloves or table, change gloves immediately and wipe the table with 75% alcohol or dilute acid.
7.Be careful not to cross-contaminate the PCR Mix when aspirating, and it is recommended that the pipette tip be wiped with 75% alcohol 2 times before taking Mix each time.
8.Pre-experiment preparation: 1×TE buffer (PH=8.0), 0.5×TBE buffer, agarose, ethidium bromide (EB), normal PCR instrument, DNA electrophoresis equipment and gel imager, 0.2 ml PCR reaction tubes, octuplex or 96-well PCR tubes, pipettes of different sizes: 0.5-10 μl and 20-200 μl.
Operation steps
1. DNA template preparation:
1.1. scrape a small amount (1-2 inoculation loops) of sample from solid medium, resuspend in 100ul TE and inactivate at 80°C for 30 minutes.
1.2. The inactivated strain was taken out of the P3 laboratory as follows:
Boil at 100°C for 10 minutes (be careful to avoid bursting the cap of the EP tube during boiling to avoid letting water into the tube), place immediately on ice for 2 minutes, centrifuge at 12,000 rpm (~13,400 × g) for 10 minutes, take the supernatant and place in another sterile EP tube, label it, and store at -20°C.
2. Testing procedures:
2.1. Remove the TB Genotyping Kit HV-3, allow the liquid to equilibrate to room temperature, mix by shaking slightly 3-4 times, and then centrifuge at 12,000 rpm (~13,400 x g) for 5 seconds to allow the capped liquid to fall back into the tube.
2.2.Three-locus VNTR typing: strains with identical results at 12 loci need to be further VNTR typed, i.e., the following four loci are added for comparison.
1）PCR amplification: the reaction system was 20 μl. 19 μl of PCR Mix of VNTR3820, VNTR4120, and VNTR3232 were added to each PCR tube, 1 μl of DNA template was added, and mixed well.
2）Amplification conditions:
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-e2.png?access_token=69521148-ef1c-4eda-89ff-c30ed35d21ef)
3) Gel preparation and electrophoresis:
a: Notes:
Important! Positive (H37Rv strain DNA) and negative controls (deionized water) need to be set up for each experiment.
Key! This experiment is based on agarose gel electrophoresis to interpret the genotype of VNTR locus, therefore, in order to make the results accurate, it is necessary to follow the unified standard operation in this step of electrophoresis, and the following points should be noted:
a-1: The comb used for glue making is 18 holes.
a-2: The two wells on the left and right sides of the gel were discarded due to the tendency to distort the bands during electrophoresis, affecting the interpretation of the results, or a negative control was spotted in one of the wells. The remaining 16 wells were divided into 12 samples, 3 DNA Markers and 1 positive control. The order of spotting was "1, 2, M, 3, 4, 5, 6, M, 7, 8, 9, 10, M, 11, 12, H37Rv", the numbers represent samples, and M represents DNA Marker.
a-3: When PCR amplification products are subjected to the first electrophoresis and Marker I is used, the gel concentration is 1%, the voltage is 150 V, and the time is 100-120 min.
a-4: If the amplification product fragment is too large (>1000bp) and needs to be electrophoresed again and Marker II is used, the gel concentration is 0.8%, the voltage is 150V and the time is 150 minutes.
b: Gluing as well as the electrophoresis process:
PCR amplification products were electrophoresed using a 1% agarose gel.
To prepare 1% agarose gel, 12×12 cm gel tray was used to make the gel, each gel was 80 ml.
b-1: Weigh 0.8g of agarose, add 80ml of 0.5×TBE, weigh it on the balance and put it into the microwave oven, heat it on high for 2-3 minutes to make the agarose dissolve completely, shake it well, and observe it as a homogeneous and transparent solution without particles, then weigh it again on the balance and make up the appropriate amount of double-distilled water to keep the concentration of the gum unaffected.
b-2: When the melted gel was cooled to about 55°C add 4 μl of ethidium bromide (10ug/ml) and gently swirl to mix well. The gel was made with an 18-tooth comb and the warm gel was poured into a 12 × 12 cm gel tray.
b-3: Allow the gel to completely set (40 minutes at room temperature), carefully pull out the comb, remove the tray, and place it in the electrophoresis tank. Add 0.5× TBE buffer to the electrophoresis tank, not exceeding the gel surface by 1-2mm.
b-4: Sample electrophoresis: add 12 samples to each gel (the topmost wells are not sampled), add 3-5μl PCR products to each well, and at the same time add three 5μl DNA MarkerⅠ to each gel. The voltage is 150V and the electrophoresis time is 100-120 minutes. This step is the key to the accuracy of the final readings of each point, and needs to be operated uniformly according to this standard.
b-5: Some loci have amplification products greater than 1000bp in clinical strains, and these amplification products were then electrophoresed using 0.8% agarose gel, with DNA Marker II added as a control for the band size, voltage 150V, electrophoresis time 150 minutes.
4) Results display:
a. If the genotypes of the three highly variable loci are also the same in different strains, they can be identified as clustered strains;
b. If the high variant readings are highly similar, i.e., only 1-2 high variant sites are different, they need to be combined with epidemiologic data to identify if they are clustered strains;
c. If all 3 high variant loci are genotypically discordant, identify as a single strain.
Appendix 1: Rules for reading VNTR loci-3e.png?access_token=e2e7ebf5-bc86-4d5d-adcc-6769ca3ca422)
Appendix 2: VNTR locus repeat unit readout table
-4e.png?access_token=39434807-3afa-485c-925d-eb58ad1543eb)
规格
英文名称
TB Typing Kit HV-3
储存与运输
储存条件
-20°C储存,避免反复冻融
运输条件
超低温运输
技术文档
高级数据
质检证书（CoA,COO,BSE/TSE 和分析图谱)
C of A & Other Certificates(BSE/TSE, COO):
输入批号以搜索分析图谱:
溶液计算器
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