展现着多药耐药<em>结核分枝杆菌</em>扩增基因芯片
Summary
扩增微阵列结合的不对称PCR扩增和微阵列杂交到一个单一的腔室，其显著简化了微阵列的工作流程最终用户。简化工作流程芯片是一个用于创建可在低资源环境中经常使用的芯片为基础的诊断是必要的第一步。
Abstract
简化工作流程芯片是一个用于创建可在低资源环境中经常使用的MDR-TB芯片为基础的诊断是必要的第一步。扩增基因芯片结合了不对称PCR扩增，目标尺寸选择，目标标签和芯片杂交在一个单一的解决方案，并成一个单一的微流体室。批次处理方法表现出了9复杂不对称的预混和低密度凝胶元素的微阵列基因分型耐多药结核分枝杆菌 （MDR-TB）。这里描述的协议可在6小时内完成，并提供正确的基因分型与基因组DNA的至少1000细胞当量。结合片上的洗涤步骤是可行的，这将导致在一个完全封闭的扩增子的方法和系统。复用的扩增微阵列的程度最终是由引物对的，能够被组合成一个单一的主米的数量受限九，并且仍然获得期望的灵敏度和特异性的性能度量，而不是被固定在阵列上的探针的数目。同样地，总分析时间可以缩短或加长取决于特定的预期用途，研究问题，并且检测期望的限制。然而，一般的方法显著简化了微阵列的工作流程，通过减少手动密集和费时的加工步骤的数量，最终用户，并且提供了一个简化的生物化学和微流体通道用于翻译的微阵列为基础的诊断成常规的临床实践。
Introduction
早期发现和快速处理被认为是最有效的控制策略，以降低结核分枝杆菌 （MTB）传输1，和现在有在结核病的社区，一个点的护理（POC）或接近POC测试，同时诊断结核病的广泛共识和耐药性（DR）是必要的。技术，如Cepheid公司的的GeneXpert和其他核酸扩增试验的时间减少到诊断为许多结核病患者，并提供快速的读出指示的耐利福平或所选突变赋予对其他第一或第二线药物2。虽然实时性和等温核酸扩增试验的目的是确定药物的耐药突变，导致耐多药结核病，他们发现突变的频谱往往不足，设计对应于患者的耐药性分布的个体化药物治疗方案，及相关技术的限制光学串扰或扩增和报告的化学复杂性5月3日至7日限制位点或检测到的突变的数量。因此，检测技术具有较高的复用能力需要解决的差距称为耐多药结核病的POC诊断。
微阵列和世界卫生组织认可的海恩线性探针检测可以解决“多基因，多基因突变”诊断耐多药结核病8-29的挑战。不幸的是，这些杂交为基础的，复用检测平台使用多步骤，复杂的，需要显著培训和熟练掌握分子生物学技术的开放式扩增子协议。扩增芯片30的目的是要解决一些芯片的工作流程和运营的担忧。该简化的流体原则是扩增，杂交和单微流体腔室中检测靶核酸。用户引入了核酸扩增和马斯之三混合成流体室用吸管，并开始热循环程序。对于此处所示的批量处理方法，微阵列，其后洗涤散装溶液，干燥，并成像。这项研究表明，使用MDR-TB芯片测试的rpoB（30突变），katG的 （2突变） 的inhA（4突变） 的rpsL（2突变），embB（1突变），IS1245，IS6110扩增芯片的功能和一个内部的扩增和杂交的控制。至少有一个匹配的一对基因芯片的探针（野生型（WT）和单核苷酸突变（MU））被列入为每个感兴趣的突变。从耐多药M.纯化核酸结核病是由结核台湾存托凭证应变银行31。凝胶元素微阵列在玻璃基板上通过共聚如其他地方32描述制造基本上，除了我们用4％的单体和0.05毫每个探头在polymeriz通报BULLETIN混合物。阵列被包围在50ml的垫圈在使用之前。后的热循环，杂交和洗涤步骤，扩增微阵列上成像的Akonni便携式分析。背景校正，集成信号强度从原料取得，使用一个固定的圆的算法TIF图像。噪音对每个凝胶元素的计算公式为当地现货背景的三倍标准偏差。靶基因通常被认为是检测到的信号噪声比（SNR）值≥3。为了确定在每个基因的密码子或特定突变的存在或不存在下，生成的判别比是从SNR值作为（WT-MU）/（WT + MU）计算。判别比值<0顷指示耐药突变的位点的，而比值> 0是指示性的野生型序列中。
Protocol
对于遵循通用的PCR预防措施实验室，在运作上更有效率，包括几个放大芯片和基板每垫圈和在散装容器同时清洗所有放大芯片，如下所述。耗材格式可用于执行在一个完全密封的，封闭的扩增试验扩增后芯片的洗涤步骤，因为在别处30,33报。 1。设置提取并在适当的生物安全条件，选择的方法纯化核酸从样品。的要求是，核酸具有足够的纯度为不对称的，多重P…
Representative Results
定性图像分析可以提供洞察实验噪音或变化的挑战是识别通过自动图像分析软件生成的数据表的来源。因此，它可以是有用的在视觉上确定：1）所有的凝胶元素都完好无损，2）全球背景是不受任何可能影响个人的信噪比（SNR）值荧光文物，3）没有证据气泡的形成或不均匀的放大/杂交在阵列中，和4），该软件可准确识别的微阵列图像上的所有点。例如MDR-TB的扩增微阵列图像示于图1，</strong…
Discussion
复用的扩增微阵列的程度最终是由多重不对称PCR的效率，而不是在微阵列所决定的。在我们的经验，10-12独特的引物对能够容易地复用在一个扩增微阵列格式。因此，常规的引物和探针的设计准则适用于新的测定法，不同之处在于1也需要考虑溶液相的核酸和固定化微阵列的探针，该热循环仪的热效率，并且在PCR缓冲液探针杂交行为之间可能的相互作用也含有PCR引物和低分子量的扩增假象。高?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由美国国立卫生研究院（NIH）根据授予RC3 AI089106支持。
耐多药结核病核酸是由联合国儿童基金/联合国开发计划署/世界银行/世界卫生组织特别规划热带病（TDR），日内瓦，瑞士提供的研究和培训。
我们感谢美国疾病控制和预防的汤姆Shinnick博士对特定基因的突变和指导在原型试验，以包括。
Materials
| MDR-TB amplification microarrays, with applied gasket and pre-cut cover slips | Akonni Biosystems | Inquire | |
| Multiplex PCR kit, containing 2X PCR buffer with HotStar Taq plus | Qiagen | #206143 | |
| Taq polymerase | Qiagen | #201207 | |
| RNAse-free water | Qiagen | #206143 | |
| Formamide | Thermo Fisher Scientific, Inc. | #BP227-500 | |
| 20 mg mL-1 non-acetlyated bovine serum albumin (BSA) | Sigma-Aldrich | #3B6917 | |
| 5X concentrated MDR-TB primer mix | Akonni Biosystems | Inquire | |
| 500 fg uL-1 amplification and inhibition control | Akonni Biosystems | Inquire | |
| 20X SSPE | Thermo Fisher Scientific, Inc. | #BP1328-4 | |
| Triton X-100 | Thermo Fisher Scientific, Inc. | #BP151-500 | |
| Disinfecting Spray | Current Technologies, Inc. | #BRSPRAY128 | |
| 70% Isopropyl Alcohol | Decon Labs, Inc. | #8416 | |
| Equipment | Company | Catalog Number | |
| Microarray imager, with automated image and data analysis software | Akonni Biosystems | 100-20011 | |
| Thermal cycler with flat block insert | Akonni Biosystems | 100-10021 | |
| High-throughput wash station and slide holder | ArrayIt | HTW | |
| Dissecting forceps | Thermo Fisher Scientific, Inc. | #10-300 | |
| Mini Vortexer | VWR | #3365040 | |
| Mini-centrifuge | VWR | #93000-196 | |
| Airbrush Compressor Kit | Central Pneumatic | #95630 |
References
- Lemaire, J. -. F., Casenghi, M. New diagnostics for tuberculosis: fulfilling patient needs first. J. Int. AIDS Soc. 13, 40 (2010).
- World Health Organization. . Tuberculosis diagnostic technology landscape. 42, (2012).
- Notomi, T., Okayama, H., et al. Loop-mediated isothermal amplification of DNA. Nucl. Acids Res. 28, e63 (2000).
- Vincent, M., Xu, Y., Kong, H. Helicase-dependent isothermal DNA amplification. EMBO Rep. 5, 795-800 (2004).
- Pritchard, C. G., Stefano, J. E. Amplified detection of viral nucleic acid at subattomole levels using Q beta replicase. Ann. Biol. Clin. 48, 492-497 (1990).
- Compton, J. Nucleic acid sequence-based amplification. Nature. 350, 91-92 (1991).
- Guatelli, J. C., Whitfield, K. M., et al. Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication. Proc. Natl. Acad. Sci. U.S.A. 87, 1874-1878 (1990).
- Shi, X. C., Liu, X. Q., Xie, X. L., Xu, Y. C., Zhao, Z. X. Gene chip array for differentiation of mycobacterial species and detection of drug resistance. Chin. Med. J. , 3292-3297 (2012).
- Troesch, A., Nguyen, H., et al. Mycobacterium species identification and rifampin resistance testing with high-density DNA probe arrays. J. Clin. Microbiol. 37, 49-55 (1999).
- Caoili, J. C., Mayorova, A., et al. Evaluation of the TB-biochip oligonucleotide microarray system for rapid detection of rifampin resistance in Mycobacterium tuberculosis. J. Clin. Microbiol. 44, 2378-2381 (2006).
- Waddell, S., Laing, K., Senner, C., Butcher, P. Microarray analysis of defined Mycobacterium tuberculosis populations using RNA amplification strategies. BMC Genomics. 9, 94 (2008).
- Fukushima, M., Kakinuma, K., et al. Detection and identification of mycobacterium species isolates by DNA microarray. J. Clin. Microbiol. 41, 2605-2615 (2003).
- Park, H., Jang, H., et al. Detection and genotyping of Mycobacterium species from clinical isolates and specimens by oligonucleotide array. J. Clin. Microbiol. 43, 1782-1788 (2005).
- Yao, C., Zhu, T., et al. Detection of rpoB, katG and inhA gene mutations in Mycobacterium tuberculosis clinical isolates from Chongqing as determined by microarray. Clin. Microbiol. Infect. 16, 1639-1643 (2010).
- Sun, A. H., Fan, X. L., et al. Rapid detection of rpoB gene mutations in rif-resistant M. tuberculosis isolates by oligonucleotide microarray. Biomed. Environ. Sci. 22, 253-258 (2009).
- Volokhov, D. V., Chizhikov, V. E., Denkin, S., Zhang, Y. Molecular detection of drug-resistant Mycobacterium tuberculosis with a scanning-frame oligonucleotide microarray. Methods Mol. Biol. 465, 395-417 (2009).
- Fu, L., Shinnick, T. M. Understanding the action of INH on a highly INH-resistant Mycobacterium tuberculosis strain using Genechips. Tuberculosis. 87, 63-70 (2007).
- Fu, L. M., Shinnick, T. M. Genome-wide exploration of the drug action of capreomycin on Mycobacterium tuberculosis using Affymetrix oligonucleotide GeneChips. J. Infect. 54, 277-284 (2007).
- Denkin, S., Volokhov, D., Chizhikov, V., Zhang, Y. Microarray-based pncA genotyping of pyrazinamide-resistant strains of Mycobacterium tuberculosis. J. Med. Microbiol. 54, 1127-1131 (2005).
- Gryadunov, D. A., Mikhailovich, V., et al. Evaluation of hybridisation on oligonucleotide microarrays for analysis of drug-resistant Mycobacterium tuberculosis. Clin. Microbiol. Infect. 11, 531-539 (2005).
- Tang, X., Morrix, S. L., Langone, J. J., Bockstahler, L. E. Microarray and allele specific PCR detection of point mutations in Mycobacterium tuberculosis genes associated with drug resistance. J. Microbiol. Methods. 63, 318-330 (2005).
- Wade, M. M., Volokhov, D., Peredelchuk, M., Chizhikov, V., Zhang, Y. Accurate mapping of mutations of pyrazinamide-resistant Mycobacterium tuberculosis strains with a scanning-frame oligonucleotide microarray. Diagn. Microbiol. Infect. Dis. 49, 89-97 (2004).
- Yue, J., Shi, W., et al. Detection of rifampin-resistant Mycobacterium tuberculosis strains by using a specialized oligonucleotide microarray. Diagn. Microbiol. Infect. Dis. 48, 47-54 (2004).
- Kim, S. Y., Park, Y. K., et al. Evaluation of the CombiChip Mycobacteria Drug-Resistance detection DNA chip for identifying mutations associated with resistance to isoniazid and rifampin in Mycobacterium tuberculosis. Diagn. Microbiol. Infect. Dis. 54, 203-210 (2006).
- Brown, T. J., Herrera-Leon, L., Anthony, R. M., Drobniewski, F. A. The use of macroarrays for the identification of MDR Mycobacterium tuberculosis. J. Microbiol. Methods. 65, 294-300 (2006).
- Brossier, F., Veziris, N., Truffot-Pernot, C., Jarlier, V., Sougakoff, W. Performance of the genotype MTBDR line probe assay for detection of resistance to rifampin and isoniazid in strains of Mycobacterium tuberculosis with low- and high-level resistance. J. Clin. Microbiol. 44, 3659-3664 (2006).
- Padilla, E., Gonzalez, V., et al. Comparative evaluation of the new version of the INNO-LiPA Mycobacteria and GenoType Mycobacterium assays for identification of Mycobacterium species from MB/BacT liquid cultures artificially inoculated with mycobacterial strains. J. Clin. Microbiol. 42, 3083-3088 (2004).
- Barnard, M., Gey van Pittius, N. C., et al. The diagnostic performance of the GenoType MTBDRplus Version 2 line probe assay is equivalent to that of the Xpert MTB/RIF assay. J. Clin. Microbiol. 50, 3712-3716 (2012).
- Sekiguchi, J. -. I., Nakamura, T., et al. Development and evaluation of a line probe assay for rapid identification of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis strains. J. Clin. Microbiol. 45, 2802-2807 (2007).
- Chandler, D. P., Bryant, L., et al. Integrated amplification microarrays for infectious disease diagnostics. Microarrays. 1, 107-124 (2012).
- Vincent, V., Rigouts, L., et al. The TDR Tuberculosis Strain Bank: a resource for basic science, tool development and diagnostic services. Int. J. Tuberc. Lung Dis. 16, 24-31 (2012).
- Golova, J. B., Chernov, B. K., et al. Non-volatile copolymer compositions for fabricating gel element microarrays. Anal. Biochem. 421, 526-533 (2012).
- Cooney, C. G., Sipes, D., Thakore, N., Holmberg, R., Belgrader, P. A plastic, disposable microfluidic flow cell for coupled on-chip PCR and microarray detection of infectious agents. Biomed. Microdevices. 14, 45-53 (2012).
- Lane, S., Evermann, J., Loge, F., Call, D. R. Amplicon secondary structure prevents target hybridization to oligonucleotide microarrays. Biosens. Bioelectron. 20, 728-735 (2004).
- El-Hajj, H. H., Marras, S. A. E., et al. Use of sloppy molecular beacon probes for identification of Mycobacterial species. J. Clin. Microbiol. 47, 1190-1198 (2009).
- Boehme, C. C., Nabeta, P., et al. Rapid molecular detection of tuberculosis and rifampin resistance. N. Engl. J. Med. 363, 1005-1015 (2010).
Tags
Cite This Article
Linger, Y., Kukhtin, A., Golova, J., Perov, A., Qu, P., Knickerbocker, C., Cooney, C. G., Chandler, D. P. Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray. J. Vis. Exp. (86), e51256, doi:10.3791/51256 (2014).