沙门氏菌筛选的高通量平台.
Summary
沙门氏菌是腹泻的常见病原体。在这里, 我们描述了一个高通量平台, 用于筛选沙门氏菌spp./志贺氏菌pp. 结合引导培养。
Abstract
急性胃肠炎的粪便-口腔传播不时发生, 特别是当处理食物和水的人感染沙门氏菌时. 沙门氏菌的金标准检测方法是直接培养, 但劳动强度大 , 耗时。在这里, 我们描述了一个高通量平台的沙门氏菌 spp/志贺氏菌筛选, 利用实时聚合酶链反应 (pcr) 结合引导培养。主要有两个阶段: 实时 pcr 和引导培养。对于第一阶段 (实时 pcr), 我们解释了该方法的每个步骤: 样本采集、预富集、dna 提取和实时 pcr。如果实时 pcr 结果为阳性, 则进行第二阶段 (引导培养): 选择性培养、生化鉴定和血清学表征。我们还说明了由此产生的代表性成果。这里描述的协议将是一个有价值的平台, 用于沙门氏菌的快速、特异、敏感和高通量筛选。
Introduction
腹泻仍然是一个常见的健康问题, 在全球范围内发病率很高, 为1,2。虽然死亡率相对较低, 但一些患者出现了数周的各种症状 (如大便松动、水汪汪, 紧急去洗手间), 这使得社会经济影响非常高3,4.更严重的是, 一些患者甚至可能发展肠易激综合征,如果不治疗5。有各种各样的细菌、病毒和寄生虫会引起腹泻.沙门氏菌是传播急性肠胃炎 7、8、9、10、11的最常见细菌之一。因此, 许多县颁布了法律或条例, 对处理食物和水的人进行定期沙门氏菌检查。例如, 中国政府每年颁布一次强制性沙门氏菌筛查法律.
沙门氏菌的金标准检测方法是细菌培养.通过细菌培养和连续的生化鉴定和血清学鉴定, 可以识别细菌的种类, 这可以促进疾病爆发管理和抗菌分析, 帮助患者治疗 “12. 它还可以帮助追踪沙门氏菌疫情期间的感染源. 然而, 这种方法是劳动密集型的 (需要手动操作) 和耗时 (需要几天), 特别是用于测试大量的样品 7.此外,在一些粪便样本中可能存在可行但不可培养的沙门氏菌.鉴于这些缺点, 许多实验室试图开发检测沙门氏菌的新技术. ,19,20,21,22,23,24,25. 所有这些方法都采用了核酸扩增试验, 其中最常见的是聚合酶链反应。这些基于 naat 的方法的一个主要局限性是, 死细菌, 甚至含有不完整基因组 dna 的细菌碎片, 可以显示出阳性的结果26, 这可能会在很大程度上影响疾病的准确诊断。blanco等人表示, 分子检测是高度敏感的, 不仅对培养中可行的沙门氏菌, 而且对部分基因组和死细菌或过去污染的不可行细菌也是如此.因此, 应开发新技术。
在这里, 我们描述了一种结合基于 naat 的方法和培养的新方法。如图 1所示, 这种新方法首先应用实时 pcr 筛选, 然后发送阳性样本进行细菌培养和鉴定。
Protocol
该协议遵循珠海国际旅游医疗中心人类研究伦理委员会制定的指导方针。请在实验过程中使用标准的无菌操作。 1. 文化媒体的构成与准备 准备营养肉汤: 溶解1% 的肽, 0.3% 牛肉提取物, 0.5% 氯化钠, 0.1% 葡萄糖在 h2o, 调整 ph 值7.5, 并在121°c 高压灭菌15分钟。 制备硒酸盐培养基: 溶解0.5% 肽, 0.4% 乳糖, 1%Na 2hco 3, 0.4% 钠硒酸钠,0.001% l-胱氨酸在 h2…
Representative Results
该程序用于筛选处理食物和水的人的肛门粪便样本中的沙门氏菌。 在实时 pcr 步骤中, 如图 5a 所示, 在 hex 通道中进行了成功的扩增, 这意味着混合样品对沙门氏菌的治疗呈阳性。然后对单个样本进行了进一步的实时 pcr, 组成了阳性样本。如图 5…
Discussion
由于沙门氏菌经常与食物中毒和急性胃肠炎的粪便-口腔传播有关, 常规方法要么劳动密集型, 要么耗时7、利用实时 pcr 与引导培养相结合, 描述了沙门氏菌筛查的高通量平台。
有几个步骤需要考虑, 以最大限度地提高该平台的能力。第一个是营养肉汤 (步骤 2.2) 中样品的浓缩前步骤。虽然?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了珠海科技项目 (赠款编号 20171009e030064)、广东省科技项目 (赠款编号 2015a0202211004) 和综合管理科学技术计划的支持。中华人民共和国质量监督检验检疫局 (补助金编号 2016ik302, 2017ik224)。
Materials
| Tris | Sigma | 10708976001 | |
| EDTA | Sigma | 798681 | |
| NP40 | Sigma | 11332473001 | |
| ddH2O | Takara | 9012 | |
| PrimeSTAR HS (Premix) | Takara | R040Q | |
| Nutrient Broth | LandBridge | CM106 | |
| Nutrient agar | LandBridge | CM107 | |
| Selenite Cystine medium | LandBridge | CM225 | |
| XLD | LandBridge | CM219 | |
| MAC | LandBridge | CM908 | |
| Salmonella chromogenic agar | CHROMagar | SA130 | |
| Salmonella diagnostic serum | Tianrun | SAL60 | |
| Shigella diagnostic serum | Tianrun | SHI54 | |
| anal swab (collecting tube plus) | Huachenyang | ||
| slide | Mingsheng | 7102 | |
| micro-loop | Weierkang | W511 | |
| incubator | Jinghong | DNP-9082 | |
| autoclave | AUL | SS-325 | |
| dry bath | Jinghong | KB-20 | |
| automated microbial identification system | bioMérieux | VITEK2 | other equivalent system could be used |
| fluorescent real-time PCR machine | ThermoFisher | ABI7500 | other equivalent machine could be used |
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Cite This Article
Yang, Z., Chen, X., Tu, C., Su, Y., Wang, H. A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.. J. Vis. Exp. (141), e58200, doi:10.3791/58200 (2018).