在复杂的临床和环境样本使用寡核苷酸加上荧光微多元检测细菌
Summary
我们描述了一个复用的微生物样本内使用寡核苷酸耦合的荧光珠的检测方法。从样本内的所有生物体的扩增杂交的探针耦合珠小组。一个Luminex公司或生物复杂的仪器是用于查询每个珠珠型和杂交信号。
Abstract
细菌性阴道病（BV）是一个经常性的多种微生物综合征的特点是在“正常” 菌群改变乳酸菌占主导地位， 其中包括阴道加德纳菌，Atopobium阴道的细菌种类占主导地位的菌群， 及其他1-3。这种情况是关联与对健康的负面结果，包括艾滋病毒收购4范围内，它可能难以管理临床 5 。此外，BV的诊断依赖于使用的各6,7数值标准取得的阴道拭子涂片革兰氏污渍。虽然这个诊断是简单，价格低廉，适合资源有限的环境，它可以受到相关的主观解释，并没有给出一个详细的个人资料的阴道菌群8的组成问题。最近的深度测序工作已经透露了一个丰富，形式多样的阴道菌群明确诊断相比，那些被认为正常9,10个人，这导致在11,12 BV的分子诊断的潜在目标识别与BV的个人采取的样本之间的差异。这些研究提供了丰富的有用信息，但深度测序尚未实际在临床上的诊断方法。最近，我们描述的方法，迅速 分析多重格式的阴道菌群，使用寡核苷酸耦合的荧光珠检测在 Luminex公司平台13。像目前的革兰氏染色的方法，这种方法是快速和简单，但增加了额外的优势，利用分子测序探针设计研究产生的知识。因此，这种方法提供了一种方法来配置文件的阴道拭子，可用于诊断BV具有较高的特异性和灵敏度比赛中的主要微生物ared革兰氏染色同时提供更多的信息物种的存在和丰富的半定量和快速的方式。这种复用的方法是，远远超出了目前定量PCR分析，特别是生物，目前仅限于5或6个不同的检测单个样品14范围扩展。更重要的是，该方法不仅限于细菌在阴道拭子检测，可以很容易地适应快速分析几乎所有的微生物群落的兴趣。例如，我们最近开始申请污水处理厂使用这种方法的诊断工具的发展。
Protocol
这种方法被用于Dumonceaux 等的研究。J。临床。微生物 47，4067-4077，DOI：10.1128/jcm.00112-09（2009年）。 图1描绘了整个过程的示意图。 1。珠耦合这说明了耦合聚苯乙烯Luminex公司珠的寡核苷酸探针（见表2）使用方法。卷稍微适应新的捕获探针的评价试行;括号中表示这些卷。 取出-20℃dessicator 1 – 乙基-3 – （3 – dimethylamio…
Discussion
特异性的信号的产生是至关重要的，你必须有信心，信号观察真实地反映了从该生物体产生的扩增检测。如PrimerPlex（总理Biosoft）的软件可以帮助设计探针，将有效地杂交，但他们可能会或可能不会跨非目标物种杂交。如在本议定书中所述，当使用通用引物进行PCR，重要的是要牢记，代表所有的生物样品中扩增产生。它是重要的，因此，分析潜在的探针交叉杂交的可能性比较探针设计软件建议c…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
我们感谢这个手稿与实验发展和批评性意见帮助阿尔贝托Severini和Vanessa Goleski。这项工作是由加拿大公共卫生署和工业研究援助计划（加拿大国家研究理事会）。从萨斯喀彻温大学出版基金获得额外的支持。
Materials
| Oligonucleotide name | Company | Sequence1 |
| H279BP | Invitrogen, IDT, or other | Biotin-OEFOGAIIIIGCIGGIGAYGGIACIACIAC |
| H280 | YKIYKITCICCRAAICCIGGIGCYTT | |
| H1612BP | Biotin-OEFOGAIIIIGCIGGYGACGGYACSACSAC | |
| H1613 | CGRCGRTCRCCGAAGCCSGGIGCCTT | |
| 1O, phosphorothioate-C; E, phosphorothioate-G; F, phosphorothioate-A; I, inosine; Y, C or T; R, A or G; K, T or G; S, C or G. | ||
Table 1. Sequences of the modified oligonucleotides for universal cpn60 PCR.
| Name of the reagent | Company | Catalogue number | Comments |
| 1-Ethyl-3-(3-dimethylamiopropyl) carbodiimide HCl (EDC) | Pierce | 22980 | |
| Fluorescent polystyrene beads: MicroPlex Microspheres (Luminex), or Bio-Plex COOH Bead (Bio-Rad) | Luminex or Bio-Rad | Bio-Rad: 171-506xxx where xxx corresponds to the bead identifier | Magnetic beads are becoming available for oligonucleotide coupling and may offer certain advantages. Have not been tried by these authors. |
| Capture oligonucleotides (5′ amino C12 modified) | Invitrogen, IDT, or other | various | Desalted purity level is acceptable. Sequences of the capture probes used to characterize vaginal swabs are provided in the manuscript on which this protocol is based13. |
| T7 exonuclease | New England Biolabs | M0263S | |
| Streptavidin-R-phycoerythrin (SA-PE) | Invitrogen | S-866 | Be careful to obtain high purity SA-PE; this catalog number is recommended |
| Thermowell 96 well PCR plates | Fisher | CS006509 | fit into both 96-well thermocycler and BioPlex machine |
| Thermowell sealing mat | Fisher | CS006555 | Can be re-used; wash with soapy water, rinse well, and dry |
| 5M TMAC | Sigma | T3411 | |
| Bio-Plex or Luminex instrument | Bio-Rad or Luminex | Bio-Rad : 171-000201 | |
| PrimerPlex software for probe design | Premier Biosoft | www.premierbiosoft.com | Suggested for Luminex probe design, although other software platforms may be used |
Table 2. Specific reagents and equipment.
| component | μl/assay | μl/100 assays | final concentration |
| 10x PCR buffer (Invitrogen) | 5 | 500 | 1x |
| 50 mM MgCl2 (Invitrogen) | 2.5 | 250 | 2.5 mM |
| 10 mM dNTP | 1 | 100 | 0.2 mM each |
| H279BP, 25 μM | 0.25 | 25 | 375 nM |
| H1612BP, 25 μM | 0.75 | 75 | 125 nM |
| H280, 25 μM | 0.25 | 25 | 375 nM |
| H1613, 25 μM | 0.75 | 75 | 125 nM |
| Water | 34 | 3400 | — |
| Totals | 44.5 | 4450 |
Table 3. Suggested mixtures for PCR with modified cpn60 UT primers (Table 1). The assay is set up for 5 μl of template DNA and 0.5 μl (2.5U) of Taq DNA polymerase (Invitrogen). Normally large volumes are prepared (e.g. sufficient for 100 assays) and stored at -20°C.
Referencias
- Hale, L. P., Swidsinski, A., Mendling, W. Bacteria associated with bacterial vaginosis. N. Engl. J. Med. 354, 202-203 (2006).
- Hay, P. Life in the littoral zone: lactobacilli losing the plot. Sex. Transm. Infect. 81, 100-102 (2005).
- Morris, M., Nicoll, A., Simms, I., Wilson, J., Catchpole, M. Bacterial vaginosis: a public health review. Brit. J. Obstet. Gynecol. 108, 439-450 (2001).
- Myer, L., Kuhn, L., Stein, Z. A., Wright, T. C., Denny, L. Intravaginal practices, bacterial vaginosis, and women’s susceptibility to HIV infection: epidemiological evidence and biological mechanisms. Lancet. Infect. Dis. 5, 786-794 (2005).
- Wilson, J. Managing recurrent bacterial vaginosis. Sex. Transm. Infect. 80, 8-11 (2004).
- Nugent, R. P., Krohn, M. A., Hillier, S. L. Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation. J. Clin. Microbiol. 29, 297-301 (1991).
- Ison, C. A., Hay, P. E. Validation of a simplified grading of Gram stained vaginal smears for use in genitourinary medicine clinics. Sex. Transm. Infect. 78, 413-415 (2002).
- Money, D. The laboratory diagnosis of bacterial vaginosis. Can. J. Infect. Dis. Med. Microbiol. 16, 77-79 (2005).
- Schellenberg, J. Pyrosequencing of the chaperonin-60 universal target as a tool for determining microbial community composition. Appl. Environ. Microbiol. 75, 2889-2898 (2009).
- Spear, G. T. Comparison of the diversity of the vaginal microbiota in HIV-infected and HIV-uninfected women with or without bacterial vaginosis. J. Infect. Dis. 198, 1131-1140 (2008).
- Brotman, R. M., Ravel, J. Ready or not: the molecular diagnosis of bacterial vaginosis. Clin. Infect. Dis. 47, 44-46 (2008).
- Fredricks, D. N., Fiedler, T. L., Marrazzo, J. M. Molecular identification of bacteria associated with bacterial vaginosis. N. Engl. J. Med. 353, 1899-1911 (2005).
- Dumonceaux, T. J. Multiplex detection of bacteria associated with normal microbiota and with bacterial vaginosis in vaginal swabs by use of oligonucleotide-coupled fluorescent microspheres. J. Clin. Microbiol. 47, 4067-4077 (2009).
- Molenkamp, R., van der Ham, A., Schinkel, J., Beld, M. Simultaneous detection of five different DNA targets by real-time Taqman PCR using the Roche LightCycler480: Application in viral molecular diagnostics. J. Virol. Meth. 141, 205-211 (2007).
- Nikiforov, T. T., Rendle, R. B., Kotewicz, M. L., Rogers, Y. H. The use of phosphorothioate primers and exonuclease hydrolysis for the preparation of single-stranded PCR products and their detection by solid-phase hybridization. PCR. Methods. Appl. 3, 285-291 (1994).
- Hill, J. E., Penny, S. L., Crowell, K. G., Goh, S. H., Hemmingsen, S. M. cpnDB: A Chaperonin Sequence Database. Genome. Res. 14, 1669-1675 (2004).
- Bradshaw, C. S. The association of Atopobium vaginae and Gardnerella vaginalis with bacterial vaginosis and recurrence after oral metronidazole therapy. J. Infect. Dis. 194, 828-836 (2006).
- Dumonceaux, T. J. Enumeration of specific bacterial populations in complex intestinal communities using quantitative PCR based on the chaperonin-60 target. J. Microbiol. Meth. 64, 46-62 (2006).
Tags
Multiplex DetectionBacteriaClinical SamplesEnvironmental SamplesOligonucleotide-coupled Fluorescent MicrospheresBacterial VaginosisPolymicrobial SyndromeLactobacillusGardnerella VaginalisAtopobium VaginaeNegative Health OutcomesHIV AcquisitionClinical ManagementDiagnosisGram StainsVaginal Swab SmearsSubjective InterpretationsVaginal Microbiota CompositionDeep SequencingMolecular Diagnosis
Citar este artículo
Dumonceaux, T. J., Town, J. R., Hill, J. E., Chaban, B. L., Hemmingsen, S. M. Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres. J. Vis. Exp. (56), e3344, doi:10.3791/3344 (2011).