十五个典型湖区底泥中耐药基因污染的初步研究
发布时间：2018-11-08 16:37
【摘要】：[目的]抗生素的广泛应用,导致细菌的耐药性出现并不断增强。水体中检测出的残留抗生素的种类和数量不断增加,使得水中微生物催生耐药基因,成为耐药基因演化媒介,促进了耐药性的扩展。由于已有的研究手段单一,缺乏对水环境中耐药基因库、抗生素残留和耐药基因种类与数量的相互关系和水体中耐药基因是如何影响水体微生物及其携带的耐药基因的整体了解。本实验采用普通PCR技术与荧光定量技术研究湖泊水环境中耐药性基因流行情况,了解水体中抗菌药物残留对耐药基因的影响,以及耐药基因在水环境微生物之间的传播转移机制,对水环境中耐药基因对环境和人类的影响进行风险评估。[方法]收集15个国内典型湖泊的底泥作为研究对象,采用间接提取法和直接提取法获得底泥微生物基因组DNA,通过普通PCR技术与荧光定量PCR技术扩增耐药基因和传播元器件整合子及相关耐药基因盒,检测耐药基因相对含量,分析我国湖泊底泥中耐药基因的流行情况及耐药基因的传播机制。[结果]1采用蛋白酶K+SDS法直接提取底泥微生物基因组DNA,并结合OGMEA纯化试剂盒去除残存在内的腐殖质、重金属离子等相关杂质,可以获得大量的,高质量的,完整性强的基因组DNA。2采用普通PCR技术对28种耐药基因进行检测发现,水环境中耐药性基因主要集中在长江中下游地区(洞庭湖,鄱阳湖,巢湖,太湖,阳澄湖)、淮河流域(洪泽湖)、黄河下游地区(微山湖)以及西南地区(滇池)。相对来说,高原高寒地区(巴松错湖,青海湖,纳木错,茶卡盐湖)与高纬度高寒地区的兴凯湖耐药基因检出率较低。3采用荧光定量PCR技术检测耐药基因的相对含量中发现：长江中下游地区(洞庭湖,鄱阳湖,巢湖,太湖,阳澄湖)、淮河流域(洪泽湖)、黄河下游地区(微山湖)以及西南地区(滇池)底泥样品种耐药基因的相对含量明显高于高原高寒地区(巴松错湖,青海湖,纳木错,茶卡盐湖)与高纬度高寒地区的兴凯湖底泥样品中耐药基因的相对含量。4采用普通PCR技术检测耐药基因传播元器件整合子及耐药基因盒,以直接法提取底泥微生物基因组DNA为模板的检测Ⅰ、Ⅱ、Ⅲ类整合子,检出率分别为80%,100%,46.67%。并检测到Ⅰ类整合子一个aadA2基因盒。[结论]对14种四环素类耐药基因、12种β-内酰胺酶类耐药基因和2种氨基糖苷类耐药基因进行研究发现：水环境中耐药性基因主要集中在长江中下游地区(洞庭湖,鄱阳湖,巢湖,太湖,阳澄湖)、淮河流域(洪泽湖)、黄河下游地区(微山湖)以及西南地区(滇池)。相对来说,高原高寒地区(巴松错湖,青海湖,纳木错,茶卡盐湖)与高纬度高寒地区的兴凯湖耐药基因的相对含量较低。
[Abstract]:[objective] the widespread use of antibiotics has led to the emergence and increasing of bacterial resistance. The kinds and the quantity of residual antibiotics detected in the water are increasing continuously, which makes the microbes in the water produce resistance genes, become the evolution medium of drug resistance genes, and promote the expansion of drug resistance. Because of the single research methods available, there is a lack of gene pool for drug resistance in the water environment. The relationship between the types and the number of antibiotic residues and drug resistance genes and how the drug resistance genes in water affect the understanding of microbes and drug resistance genes in water. In this study, PCR and fluorescence quantitative techniques were used to study the prevalence of drug resistance genes in lake water environment, and to understand the effect of antimicrobial residues on drug resistance genes. The mechanism of transmission and transfer of drug resistance genes between microorganisms in water environment is also discussed. The risk assessment of the effects of drug resistance genes on the environment and human beings in water environment is carried out. [methods] the sediment of 15 typical lakes in China was collected as the research object, and the genomic DNA, of sediment microorganism was obtained by indirect extraction and direct extraction. Drug resistance genes, integron and related drug resistance gene boxes were amplified by PCR and fluorescence quantitative PCR, and the relative content of drug resistance genes was detected. To analyze the prevalence of drug resistance genes and the transmission mechanism of drug resistance genes in lake sediments of China. [results] 1 using protease K SDS method to directly extract genomic DNA, from sediment microorganism and combine with OGMEA purification kit to remove the remaining humus, heavy metal ions and other related impurities, a large amount of high quality can be obtained. Genomic DNA.2 with strong integrity was used to detect 28 drug-resistant genes using common PCR technique. It was found that resistance genes in water environment were mainly located in the middle and lower reaches of the Yangtze River (Dongting Lake, Poyang Lake, Chaohu Lake, Taihu Lake, Yangcheng Lake). Huaihe River Basin (Hongze Lake), Lower Yellow River region (Weishan Lake) and Southwest region (Dianchi Lake). Relatively speaking, the plateau and alpine regions (Basongcao Lake, Qinghai Lake, Namuco), The detection rate of drug-resistant genes in Xingkai Lake was lower than that in high-latitude alpine areas. 3 in the middle and lower reaches of the Yangtze River (Dongting Lake, Poyang Lake, Chaohu Lake, Taihu Lake), fluorescence quantitative PCR technique was used to detect the relative content of drug-resistant genes. In Yangcheng Lake, Huaihe River Basin (Hongze Lake), Lower Yellow River (Weishan Lake) and Southwest area (Dianchi Lake), the relative contents of drug resistance genes in sediment samples were significantly higher than those in highland alpine regions (Basongcao Lake, Qinghai Lake, Namuco). The relative content of drug resistance genes in the sediment samples of Xingkai Lake in high latitude and cold area. 4 the common PCR technique was used to detect the components integron and drug resistance gene box of drug resistance gene transmission. Class 鈪，
本文编号：2319057
[Abstract]:[objective] the widespread use of antibiotics has led to the emergence and increasing of bacterial resistance. The kinds and the quantity of residual antibiotics detected in the water are increasing continuously, which makes the microbes in the water produce resistance genes, become the evolution medium of drug resistance genes, and promote the expansion of drug resistance. Because of the single research methods available, there is a lack of gene pool for drug resistance in the water environment. The relationship between the types and the number of antibiotic residues and drug resistance genes and how the drug resistance genes in water affect the understanding of microbes and drug resistance genes in water. In this study, PCR and fluorescence quantitative techniques were used to study the prevalence of drug resistance genes in lake water environment, and to understand the effect of antimicrobial residues on drug resistance genes. The mechanism of transmission and transfer of drug resistance genes between microorganisms in water environment is also discussed. The risk assessment of the effects of drug resistance genes on the environment and human beings in water environment is carried out. [methods] the sediment of 15 typical lakes in China was collected as the research object, and the genomic DNA, of sediment microorganism was obtained by indirect extraction and direct extraction. Drug resistance genes, integron and related drug resistance gene boxes were amplified by PCR and fluorescence quantitative PCR, and the relative content of drug resistance genes was detected. To analyze the prevalence of drug resistance genes and the transmission mechanism of drug resistance genes in lake sediments of China. [results] 1 using protease K SDS method to directly extract genomic DNA, from sediment microorganism and combine with OGMEA purification kit to remove the remaining humus, heavy metal ions and other related impurities, a large amount of high quality can be obtained. Genomic DNA.2 with strong integrity was used to detect 28 drug-resistant genes using common PCR technique. It was found that resistance genes in water environment were mainly located in the middle and lower reaches of the Yangtze River (Dongting Lake, Poyang Lake, Chaohu Lake, Taihu Lake, Yangcheng Lake). Huaihe River Basin (Hongze Lake), Lower Yellow River region (Weishan Lake) and Southwest region (Dianchi Lake). Relatively speaking, the plateau and alpine regions (Basongcao Lake, Qinghai Lake, Namuco), The detection rate of drug-resistant genes in Xingkai Lake was lower than that in high-latitude alpine areas. 3 in the middle and lower reaches of the Yangtze River (Dongting Lake, Poyang Lake, Chaohu Lake, Taihu Lake), fluorescence quantitative PCR technique was used to detect the relative content of drug-resistant genes. In Yangcheng Lake, Huaihe River Basin (Hongze Lake), Lower Yellow River (Weishan Lake) and Southwest area (Dianchi Lake), the relative contents of drug resistance genes in sediment samples were significantly higher than those in highland alpine regions (Basongcao Lake, Qinghai Lake, Namuco). The relative content of drug resistance genes in the sediment samples of Xingkai Lake in high latitude and cold area. 4 the common PCR technique was used to detect the components integron and drug resistance gene box of drug resistance gene transmission. Class 鈪，
本文编号：2319057
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