食品筛查为1类整合和基因盒
Summary
This protocol describes the detection of class 1 integrons and their associated gene cassettes in foodstuffs.
Abstract
Antibiotic resistance is one of the greatest threats to health in the 21st century. Acquisition of resistance genes via lateral gene transfer is a major factor in the spread of diverse resistance mechanisms. Amongst the DNA elements facilitating lateral transfer, the class 1 integrons have largely been responsible for spreading antibiotic resistance determinants amongst Gram negative pathogens. In total, these integrons have acquired and disseminated over 130 different antibiotic resistance genes. With continued antibiotic use, class 1 integrons have become ubiquitous in commensals and pathogens of humans and their domesticated animals. As a consequence, they can now be found in all human waste streams, where they continue to acquire new genes, and have the potential to cycle back into humans via the food chain. This protocol details a streamlined approach for detecting class 1 integrons and their associated resistance gene cassettes in foodstuffs, using culturing and PCR. Using this protocol, researchers should be able to: collect and prepare samples to make enriched cultures and screen for class 1 integrons; isolate single bacterial colonies to identify integron-positive isolates; identify bacterial species that contain class 1 integrons; and characterize these integrons and their associated gene cassettes.
Introduction
抗生素的发现是20 世纪最伟大的科学成就之一。然而，抗生素的使用和滥用，导致对抗生素有抗药性的细菌的快速发展，而这些现在在构成21 世纪严重威胁到公众健康。细菌耐药株大多数治疗方案的兴起，引起了我们正在进入一个时代，抗菌药物不再有效1,2的可能性。
赋予抗生素抗性的遗传机制是一个古老的制度，由百万年3早于人类和抗生素的选择压力。移动遗传元件，如质粒，转座子，基因组岛，一体化接合元件和整合子可以传播抗生素抗性基因（ARG）内和细菌物种4之间。其中，整合都起到ARG传播的核心作用，尽管事实上，他们依靠质粒和转座子动员和插入到细菌基因组中5。整合捕获使用整合子整合酶基因盒，然后表达使用整合编码启动子6,7-（ 图1）盒。 整合基因盒是由单个开放阅读框（ORF），其产品能够赋予对抗生素或消毒剂8的小型移动元件。 1类整合是从临床分离5，在那里他们已经集体过130个不同的抗生素耐药基因盒9收购最常用的回收整合。
1类整合传播到人类的相关共生菌和致病菌产生含有大量的这些遗传元件10的人类废物流。据估计，10 19的细菌包含1类整合是通过污水污泥每年我发布n中的英国11。因此，毫不奇怪，1类整合赋予抗生素抗性现在正在野生鸟类，鱼类和其他野生动物12-14菌群检测。整合释放回环境构成了显著的公共健康威胁，因为收购新基因盒和复杂的重组与其他移动元素仍然存在，特别是在污水处理厂和其他水体15-18。自然环境就变成一个招兵的新的阻力因素和条件致病菌19,20。新型含整合细菌和新的ARG可以绕回到人类社会通过被污染的水和食物21,22。 ARG游戏环境监测是理解和未来23管理抗生素耐药性的关键战略。特别是，要注意食品要被生吃或轻熟，因为这些本作的新要素流动和病原体传播的最大威胁。
在这个协议中，用于检测，鉴定和表征1类整合并在食品及其相关基因盒一个简化的方法概述（ 图2）。使用的培养和聚合酶链反应（PCR）的组合，整合可迅速在复杂细菌群落和个体分离物检测。方法来识别细菌和整合相关基因盒的构象和身份的物种中给出。该方法适用于范围广泛的植物和动物的食物和典型的工作流程的例子给出了每个这些食物类型。
Protocol
Representative Results
Discussion
整合及其相关基因盒的识别可能是在预测的新机会病原体的出现，跟踪途径病原体进入人类食物链，并确定新的抗性的关键步骤和毒力决定8,21,26。本文的目的是描述筛选样品1类整合，其特征阵列盒，并确定其所在的细菌种类精简的办法。在协议中的关键步骤涉及微生物好做法，并防止污染的PCR将产生误报。
这里所描述的协议可以很容易地修改，以检测其他临床相关?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
感谢麦克拉厅，拉里萨Bispo的古斯塔沃和塔瓦雷斯的技术援助。
Materials
| GoTaq Colourless Mastermix | Promega | M7132 | Used in all PCRs |
| RNAse (Ribonuclease A from bovine pancreas) | Sigma | R6513-10MG | Used in all PCRs |
| HinFI restriction enzyme | Promega | R6201 | Used to digest 16S rDNA PCR poducts. Enzyme comes with optimal buffer and BSA |
| 100 bp ladder | GE Healthcare | 27400701 | Used as a size standard on all agarose gels |
| GelRed DNA stain | Biotium | 41003 | CAUTION: Personal protection must be worn when handling this material |
| Guanidinium Thiocyanate | Life Technologies | AM9422 | CAUTION: Personal protection must be worn when handling this material |
| CLS-TC Solution | MP Biomedicals | 6540409 | Resuspension solution used at the begining of the genomic DNA extraction |
| Lysing Matrix E FastPrep tubes | MP Biomedicals | 116914500 | Tube required for mechanical disruption of bacterial cell walls. This code is used for packs of 500 tubes, smaller quantities are available. |
| binding matrix | MP Biomedicals | 116540408 | Diluted 1:5 with 6M guanidinium thiocyanate and used in the genomic DNA extraction method. |
| Fast Prep machine | MP Biomedicals | Number of options available | MP Biomedicals has a number of FastPrep machines available to purchase. Visit http://www.mpbio.com for more information |
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