【摘要】：[目的]抗生素的廣泛應(yīng)用,導(dǎo)致細(xì)菌的耐藥性出現(xiàn)并不斷增強(qiáng)。水體中檢測(cè)出的殘留抗生素的種類和數(shù)量不斷增加,使得水中微生物催生耐藥基因,成為耐藥基因演化媒介,促進(jìn)了耐藥性的擴(kuò)展。由于已有的研究手段單一,缺乏對(duì)水環(huán)境中耐藥基因庫(kù)、抗生素殘留和耐藥基因種類與數(shù)量的相互關(guān)系和水體中耐藥基因是如何影響水體微生物及其攜帶的耐藥基因的整體了解。本實(shí)驗(yàn)采用普通PCR技術(shù)與熒光定量技術(shù)研究湖泊水環(huán)境中耐藥性基因流行情況,了解水體中抗菌藥物殘留對(duì)耐藥基因的影響,以及耐藥基因在水環(huán)境微生物之間的傳播轉(zhuǎn)移機(jī)制,對(duì)水環(huán)境中耐藥基因?qū)Νh(huán)境和人類的影響進(jìn)行風(fēng)險(xiǎn)評(píng)估。[方法]收集15個(gè)國(guó)內(nèi)典型湖泊的底泥作為研究對(duì)象,采用間接提取法和直接提取法獲得底泥微生物基因組DNA,通過(guò)普通PCR技術(shù)與熒光定量PCR技術(shù)擴(kuò)增耐藥基因和傳播元器件整合子及相關(guān)耐藥基因盒,檢測(cè)耐藥基因相對(duì)含量,分析我國(guó)湖泊底泥中耐藥基因的流行情況及耐藥基因的傳播機(jī)制。[結(jié)果]1采用蛋白酶K+SDS法直接提取底泥微生物基因組DNA,并結(jié)合OGMEA純化試劑盒去除殘存在內(nèi)的腐殖質(zhì)、重金屬離子等相關(guān)雜質(zhì),可以獲得大量的,高質(zhì)量的,完整性強(qiáng)的基因組DNA。2采用普通PCR技術(shù)對(duì)28種耐藥基因進(jìn)行檢測(cè)發(fā)現(xiàn),水環(huán)境中耐藥性基因主要集中在長(zhǎng)江中下游地區(qū)(洞庭湖,鄱陽(yáng)湖,巢湖,太湖,陽(yáng)澄湖)、淮河流域(洪澤湖)、黃河下游地區(qū)(微山湖)以及西南地區(qū)(滇池)。相對(duì)來(lái)說(shuō),高原高寒地區(qū)(巴松錯(cuò)湖,青海湖,納木錯(cuò),茶卡鹽湖)與高緯度高寒地區(qū)的興凱湖耐藥基因檢出率較低。3采用熒光定量PCR技術(shù)檢測(cè)耐藥基因的相對(duì)含量中發(fā)現(xiàn)：長(zhǎng)江中下游地區(qū)(洞庭湖,鄱陽(yáng)湖,巢湖,太湖,陽(yáng)澄湖)、淮河流域(洪澤湖)、黃河下游地區(qū)(微山湖)以及西南地區(qū)(滇池)底泥樣品種耐藥基因的相對(duì)含量明顯高于高原高寒地區(qū)(巴松錯(cuò)湖,青海湖,納木錯(cuò),茶卡鹽湖)與高緯度高寒地區(qū)的興凱湖底泥樣品中耐藥基因的相對(duì)含量。4采用普通PCR技術(shù)檢測(cè)耐藥基因傳播元器件整合子及耐藥基因盒,以直接法提取底泥微生物基因組DNA為模板的檢測(cè)Ⅰ、Ⅱ、Ⅲ類整合子,檢出率分別為80%,100%,46.67%。并檢測(cè)到Ⅰ類整合子一個(gè)aadA2基因盒。[結(jié)論]對(duì)14種四環(huán)素類耐藥基因、12種β-內(nèi)酰胺酶類耐藥基因和2種氨基糖苷類耐藥基因進(jìn)行研究發(fā)現(xiàn)：水環(huán)境中耐藥性基因主要集中在長(zhǎng)江中下游地區(qū)(洞庭湖,鄱陽(yáng)湖,巢湖,太湖,陽(yáng)澄湖)、淮河流域(洪澤湖)、黃河下游地區(qū)(微山湖)以及西南地區(qū)(滇池)。相對(duì)來(lái)說(shuō),高原高寒地區(qū)(巴松錯(cuò)湖,青海湖,納木錯(cuò),茶卡鹽湖)與高緯度高寒地區(qū)的興凱湖耐藥基因的相對(duì)含量較低。
[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，

本文編號(hào)：2319057