计算溶液所需的质量、体积或浓度。
为什么选择此级别
，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
-20°C储存,避免反复冻融。超低温运输 。请查阅批次 COA 获取详细规格。
质量文档
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
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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。
本产品将人型结核分枝杆菌（MTBC）鉴定、非结核分枝杆菌（MOTT）及模板质量鉴定、结核分枝杆菌北京家族菌株 鉴定，和后续的9位点VNTR分型鉴定整合在一个试剂盒中，使用户仅需一个试剂盒、12个PCR反应即可获得待测菌株基因 型鉴定所需的完整信息。检测的分辨力指数（Hunter-Gaston index，HGI）可达0.9891）。并且，基因型鉴定结果可数字化记录，使得不同批次样本，不同地区，不同时间，不同研究者之间的实验数据可以很方便地进行比对和汇集分析，极大地提高了数据的使用效率。
对鉴定为VNTR-9基因型成簇（所有9个位点具有相同重复数）的样本，若要判定菌株间是否存在近期传播关系，需使用配套产品TB基因分型试剂盒HV-3，做进一步的分型鉴定。VNTR-9与HV-3两个产品的结合使用可将检测的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. 为避免污染，建议制备样本和配制 PCR Mix 在不同的地点内进行，并使用不同的移液器。
2. 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。
3. 常用试剂和耗材在实验前需高压灭菌。
4. 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。
5. 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。
6. 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。
7. 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 μl 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 EP 管盖子可能会爆开，要尽量避免，扣紧 EP 管，不要让水进入管中），立即置于冰上 2 分钟，12,000 rpm（~13,400×g）离心 10 分钟后，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 VNTR-9，待液体平衡到室温后，轻微摇晃 3-4 次混匀，12,000 rpm 离心 5 秒，使盖上的液体收集到管内。
2.2 结核分枝杆菌基因型分析：
2.2.1 鉴定样本是否为人型结核分枝杆菌（重要！此步骤不可省略！）：
A．PCR 扩增：反应体系为 20 μl。
在每个 PCR 管中分别加入 19 μl Mtb 鉴定 PCR Mix ，1 μl DNA 模板，混匀。
B．反应程序：
-1.png?access_token=7e9b26d2-1a34-48c6-9c78-0636b649bc22)
C. 制胶，电泳
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，12×6 cm 胶盘制胶，每块胶为 40 ml。
1）称取 0.4 g 琼脂糖，加入 40 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，以保持胶凝胶的浓度不受影响。2）待融化的凝胶冷却至 55℃左右时加入 2 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 温热的凝胶浇灌入胶盘。
3）待凝胶完全凝结（室温下放置 30 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。
4）PCR 产物上样：每个孔中加入 3 μl PCR 产物，每块胶上留一个孔加入 5 μl MarkerⅠ，每块胶上加一个 H37Rv 作 为质量控制。电压 150V，电泳时间为 45 分钟。
结果显示：
-2.png?access_token=d2cf244f-8321-4b47-8d31-6e57b82e87bf)
D．结果分析：
1）如果出现了 850 和 361 bp 两条带，则说明模板 DNA 是人型结核分枝杆菌，可进入第 F 步（见后面）进行结核分枝杆 菌北京基因型的分析鉴定，并进一步使用本试剂盒对该菌株进行 9 位点 VNTR 基因型分析鉴定。
2）如果只扩增出 850 bp 的条带，则说明该菌株属于结核分枝杆菌复合群，但不是人型结核分枝杆菌，不适用本产品进行 基因型分析鉴定。
4）如果没有特异性条带扩增，则说明该菌株是非结核分枝杆菌或模板 DNA 质量不好，不适用本产品进行基因型分析鉴定， 可放弃该样本，或进入第 E 步（见后面），检测 DNA 模板的质量。
E．DNA 模板质量鉴定：
若在以上步骤 A-D 中没有特异性条带扩增，则说明模板 DNA 质量不佳或该菌株并非结核分枝杆菌，出现此种情况请 进行如下操作。
1）PCR 扩增：反应体系为 20 μl。
在每个 PCR 管中分别加入 19 μl 16S rRNA PCR Mix ，1 μl DNA 模板，混匀。
2）反应程序：-3.png?access_token=4d142c03-a3d0-4310-b667-62165d17f7e5)
3）电泳
1%琼脂糖凝胶电泳，150V 电泳 45 分钟；
4）结果显示：
-4.png?access_token=23006f84-3642-4bd0-aec7-98127baa56a9)
5）结果分析：
所有细菌中都存在 16S rRNA 序列，如果样本无扩增产物，说明模板DNA 的数量或质量不足以进行 VNTR 基因分型检测。如果有扩增产物条带出现，则说明此菌株为非结核分枝杆菌，不应使用本试剂盒进行基因分型鉴定。
F．北京基因型菌株鉴定方法：
在确定菌株为结核分枝杆菌后，进一步区分是否属于北京基因型菌株。
1）PCR 扩增：反应体系为 20 μl。
在每个PCR 管中分别加入 19 μl RD105 PCR Mix ，1 μl DNA 模板，混匀。
2）反应程序：
-5.png?access_token=15f9e290-95e6-4451-9969-5428fc563e38)
3）电泳
1%琼脂糖凝胶电泳，150V 电泳 45 分钟。
4）结果显示：
-6.png?access_token=dba0c449-ac59-414c-b9bd-b24881235d6e)
5）结果分析：
如果扩增产物为 1495 bp，该菌株为非北京基因型菌株；
如果扩增产物为 786 bp，该菌株为北京基因型菌株。
2.2.2 结核分枝杆菌 9 位点 VNTR 基因型分型法： 对结核分枝杆菌临床菌株进行基因型分析，初步鉴定成簇菌株。
A．PCR 扩增：反应体系为 20 μl。
取 9 个 PCR 反应管，在每个 PCR 管中分别加入 19 μl QUB-11b，QUB-18，QUB-26，MIRU26，MIRU31，MIRU40， Mtub21，Mtub04，VNTR2372 的 PCR Mix，加入 1 μl DNA 模板，混匀。
B . 反应程序：
-7.png?access_token=736f6a77-8ca3-44e4-8f35-a08e8eb8b4a5)
C. 制胶、电泳：
C-1：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，为了使不同实验室结果能准确的相互比较， 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：
1） 制胶所用梳子为 18 孔。
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。
3） PCR 扩增产物进行首次电泳并使用 Marker I 时，凝胶浓度为 1%，应使用两电极间距离不小于 30 厘米的电泳槽，电 压为 150V，时间为 100-120 分钟。
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%，应使用两电极间距离不 小于 30 厘米的电泳槽，电压 150V，时间为 150 分钟。
C-2：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。
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。
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。
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。
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，加一个 H37Rv 作为质量控制（点样孔分布见下图）。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。
5）部分位点（QUB-18 及 QUB-26）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 MarkerⅡ 作为条带大小对照，电压 150V，电泳时间 150 分钟。
-8.png?access_token=84a33da4-2539-4007-89d8-22c2f46c98d5)
D. 结果分析：
根据各临床菌株扩增条带与 Marker 相对位置，利用凝胶分析软件读出每个条带的大小。利用各 VNTR 位点重复单元 读数表及 VNTR 位点读数规则，计算出各菌株在该位点的重复单元数，每个 VNTR 位点重复单元读数表及 VNTR 位点读数规则附后。
结果报告： Excel 文件（以下表格为结果报告输出格式举例）
-9.png?access_token=42efc693-f98f-42eb-9d59-1894302ca57d)
比较不同菌株 9 个 VNTR 位点的重复数，进行成簇分析：如果两个或两个以上菌株具有相同 9 位点基因型，则初步鉴 定为成簇菌株，如有必要，可使用配套产品 TB 基因分型试剂盒 HV-3，做更精细的进一步分型鉴定。如果 分离的菌株具有特异的 9 位点基因型，则鉴定为单一菌株。
附录 1：VNTR 位点读数规则
-10.png?access_token=d833fafb-b2c2-45d6-80fc-71ea4048b062)
附录 2 ：VNTR 位点重复单元读数表
-11.png?access_token=ceff3f85-e6f4-48f0-a4b9-8983cd4d1dc1)
-12.png?access_token=2d2db7be-12a2-4e59-9ae3-233701b88a3b)
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Product Introduction
This kit is a genotyping product for human Mycobacterium tuberculosis based on the latest advances 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 for 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 between strains prevalent in China1,2,3), and is therefore particularly suited to 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 product integrates the identification of human-type Mycobacterium tuberculosis (MTBC), the identification of non-Mycobacterium tuberculosis (MOTT) and template mass, the identification of Mycobacterium tuberculosis Beijing family bacteria, and the subsequent identification of 9-locus VNTR typing in a single kit, so that the user only needs a single kit and 12 PCR reactions to obtain the complete information necessary for the genotypic identification of the strain to be tested. The resolution index (Hunter-Gaston index (HGI) of the assay can reach 0.9891). Moreover, the genotype identification results can be digitally recorded, which makes it easy to compare and pool the experimental data between different batches of samples, different regions, different times, and different researchers, and greatly improves the efficiency of data use.
For samples identified as clustered (all 9 loci with the same number of repeats) of the VNTR-9 genotype, further typing is required to determine whether there is a recent transmission relationship between strains using the companion TB Genotyping Kit HV-3. (The combination of the two products, VNTR-9 and HV-3, can be used to increase the HGI of the assay to 0.9931). For more information about the TB Genotyping Kit HV-3, please refer to its datasheet.
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
3）Mycobacterium marinum. Vet Microbiol. 2011 Aug 26;152(1-2)
4）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. To avoid contamination, it is recommended that sample preparation and PCR Mix preparation be performed within separate locations and with separate pipettes.
2. Care should be taken to label the samples at all stages of DNA collection, extraction and amplification, and to prevent cross-contamination between different samples.
3. Commonly used reagents and consumables need to be autoclaved before experimentation.
4. Each tube of PCR Mix contains different primers and cannot be mixed. It can be dispensed into different quantities at once according to the experimental needs to avoid repeated freezing and thawing.
5. 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.
6. Be careful not to cross-contaminate the PCR Mix when aspirating, and it is recommended to wipe the pipette head with 75% alcohol 2 times before taking Mix each time.
7. Pre-experiment preparation: 1×TE buffer (PH=8.0), 0.5×TBE buffer, agarose, ethidium bromide (EB), common PCR instrument, DNA electrophoresis equipment and gel imager, 0.2 ml of PCR reaction tubes, octreotide rows or 96-well PCR tubes, and 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 the solid medium, resuspend in 100 μl of TE, and inactivate at 80°C for 30 minutes.
1.2 After inactivation, take the strain out of the P3 laboratory and do the following: boil at 100°C for 10 minutes (the cap of the EP tube may burst open during boiling, try to avoid this, fasten the EP tube tightly and do not allow water to enter the tube), place it on ice immediately for 2 minutes, centrifuge it at 12,000 rpm (~13,400 x g) for 10 minutes, then take the supernatant and put it into another sterile EP tube, mark it and - 20°C.
2. Testing procedures:
2.1 Remove the TB Genotyping Kit VNTR-9, allow the liquid to equilibrate to room temperature, mix by shaking slightly 3-4 times, and centrifuge at 12,000 rpm for 5 seconds to allow the liquid on the cap to collect in the tube.
2.2 Mycobacterium tuberculosis genotype analysis:
2.2.1 Identify the sample as human Mycobacterium tuberculosis (Important! This step should not be omitted!) :
A. PCR amplification: the reaction system was 20 μl.
Add 19 μl Mtb Identification PCR Mix, 1 μl DNA template to each PCR tube separately and mix well.
B. Reaction procedure:-1e.png?access_token=ee05e659-ec62-4576-9cbb-a045b10c8127)
C. Gluing, Electrophoresis
PCR amplification products were electrophoresed using a 1% agarose gel.
To prepare 1% agarose gel, 12×6 cm gel tray was used to make the gel, each gel was 40 ml.
(1) Weigh 0.4g agarose, add 40ml 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 replenish it with an appropriate amount of double-distilled water to keep the concentration of the gelatinous gel unaffected.
2) When the melted gel was cooled to about 55°C, 2 μl of ethidium bromide (10ug/ml) was added and gently rotated to mix well. Use an 18-tooth comb to make the gel and pour the warm gel into the gel tray.
3) When the gel is completely solidified (left at room temperature for 30 minutes), carefully pull out the comb, remove the tray and put it into the electrophoresis tank. Add 0.5×TBE buffer to the electrophoresis tank, not exceeding the gel surface by 1-2mm.
4) PCR product up-sampling: 3μl PCR product was added to each well, one well was left to add 5μl MarkerⅠ to each piece of gel, and one H37Rv was added to each piece of gel as quality control. The voltage was 150V and the electrophoresis time was 45 minutes.
The result shows:
D. Result analysis:
(1) If two bands of 850 and 361bp appear, it means that the template DNA is human-type Mycobacterium tuberculosis, and you can proceed to step F (see later) for the analysis and identification of Mycobacterium tuberculosis Beijing genotype, and further use this kit for the identification of 9-locus VNTR genotype analysis of this strain.
(2) If only a band of 850 bp is amplified, it means that the strain belongs to Mycobacterium tuberculosis complex group, but is not human-type Mycobacterium tuberculosis, and this product is not suitable for genotypic analysis and identification.
(3) If there is no specific band amplification, it means that the strain is a non-Mycobacterium tuberculosis or the quality of template DNA is not good enough for this product to be used for genotyping and identification, so you can either abandon the sample or go to step E (see later) and test the quality of the DNA template.
E. DNA template quality characterization:
If no specific bands are amplified in steps A-D above, the template DNA is of poor quality or the strain is not Mycobacterium tuberculosis, in which case proceed as follows.
1) PCR amplification: the reaction system was 20 μl.
Add 19 μl 16S rRNA PCR Mix, 1 μl DNA template to each PCR tube separately and mix well.
2) Reaction procedure:-3e.png?access_token=06bbe3c8-2889-45b6-939e-1b88ed546264)
3) Electrophoresis
1% agarose gel electrophoresis at 150 V for 45 min;
4) The result shows:
5) Result analysis:
16S rRNA sequences are present in all bacteria, and if no amplification product is present in the sample, the amount or quality of template DNA is not sufficient for VNTR genotyping. If an amplification product band is present, the strain is a non-Mycobacterium tuberculosis and should not be genotyped using this kit.
F. Methods for identification of genotyped strains in Beijing:
After the strain was identified as Mycobacterium tuberculosis, it was further distinguished whether it was a Beijing genotype strain.
1) PCR amplification: the reaction system was 20 μl.
Add 19 μl RD105 PCR Mix, 1 μl DNA template to each PCR tube separately and mix well.
2) Reaction procedure:-5e.png?access_token=c28b5709-f808-40fa-82a3-cd888d5ccda4)
3) Electrophoresis
1% agarose gel electrophoresis at 150 V for 45 min.
4) The result shows:
5) Analysis of results:
If the amplification product is 1495 bp, the strain is a non-Beijing genotype strain;
If the amplification product is 786 bp, the strain is a Beijing genotype strain.
2.2.2 Mycobacterium tuberculosis 9-locus VNTR genotyping method: genotyping of clinical strains of Mycobacterium tuberculosis and preliminary identification of cluster-forming strains.A. PCR amplification: the reaction system was 20 μl.
Take 9 PCR reaction tubes, add 19 μl of PCR Mix of QUB-11b, QUB-18, QUB-26, MIRU26, MIRU31, MIRU40, Mtub21, Mtub04, VNTR2372 to each PCR tube, add 1 μl of DNA template and mix well.
B Reaction procedure:-7e.png?access_token=87b9b8a4-df51-4991-87c5-118064536608)
C. Gluing and electrophoresis:
C-1: 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 of different laboratories accurately comparable with each other, it is necessary to follow a unified standard operation in this step of electrophoresis, and the following points should be noted:
1) The comb used for glue making is 18 holes.
(2) The two holes on the left and right sides of the gel are easily deformed during electrophoresis, which affects the interpretation of the results, so they are discarded or a negative control is placed in one of the holes, and the remaining 16 holes are 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.
(3) When PCR amplification products are subjected to the first electrophoresis and Marker I is used, the gel concentration is 1%, and an electrophoresis tank with a distance between two electrodes of not less than 30 centimeters should be used, with a voltage of 150 V and a time of 100-120 minutes.
4) If the fragment of the amplification product is too large (>1000bp), and it is necessary to electrophoresis again and use Marker II, the concentration of the gel is 0.8%, and an electrophoresis tank with a distance of not less than 30 cm between the two electrodes should be used with a voltage of 150V and a time of 150 minutes.
C-2: Gel making as well as 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.
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 replenish it with an appropriate amount of double-distilled water to keep the concentration of the gum unaffected.
2) Add 4 μl of ethidium bromide (10ug/ml) when the melted gel is cooled to about 55°C, and gently rotate 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.
3) When the gel is completely solidified (40 minutes at room temperature), carefully pull out the comb, remove the tray and put it into the electrophoresis tank. Add 0.5×TBE buffer to the electrophoresis tank, not exceeding the gel surface by 1-2mm.
4) Sampling electrophoresis: add 12 samples to each piece of gel (the topmost wells are not sampled), add 3-5 μl PCR products to each well, meanwhile add three 5 μl DNA MarkerⅠ to each piece of gel, and add one H37Rv as a quality control (the distribution of spotting wells is shown in the following figure). The voltage is 150V and the electrophoresis time is 100-120 minutes. This step is the key to the accuracy of the final readout of each spot and needs to be followed uniformly.
5) Some of the loci (QUB-18 and QUB-26) had amplification products greater than 1000bp in clinical strains, and these amplification products were then electrophoresed using 0.8% agarose gel, while DNA Marker Ⅱ was added as a control for the size of the bands, with a voltage of 150V and an electrophoresis time of 150 minutes.
According to the relative position of the amplified bands and Marker of each clinical strain, the size of each band was read out using gel analysis software. The number of repeat units of each strain at the VNTR site was calculated using the table of repeat unit readings for each VNTR site and the rules for reading VNTR sites, and the table of repeat unit readings for each VNTR site and the rules for reading VNTR sites are attached.
Results report: Excel file (the following table is an example of the results report output format)-9e.png?access_token=3dc1bb57-de5d-4d12-b55d-aedb20050e80)
Compare the number of repeats of the 9 VNTR loci of different strains for cluster analysis: if two or more strains have the same 9-locus genotype, they are initially identified as clustered strains, and if necessary, the companion product, TB Genotyping Kit HV-3, can be used to do further typing identification in a more refined way. If the isolated strains have specific 9-locus genotypes, they will be identified as a single strain.
Appendix 1: VNTR site readout rules
-10e.png?access_token=b923c302-35c2-4b10-a12a-32cf022459ef)
Appendix 2: VNTR Site Repeat Unit Readout Table
-11e.png?access_token=a85955b9-5c8e-4af6-adb9-02e9f209e1c1)
-12e.png?access_token=59d4c040-5508-4f59-8f7c-badabcb893e5)
规格
英文名称
TB Typing Kit VNTR-9
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