应用荧光定量PCR技术监测玄武湖蓝藻水华

应用荧光定量PCR技术和显微计数法对玄武湖蓝藻水华进行了长期监测,结果表明,荧光定量PCR法可同步监测蓝藻、微囊藻和有毒微囊藻的数量,及时准确反映玄武湖蓝藻水华优势种群微囊藻和有毒微囊藻的动态变化。与显微计数法相比,具有需要的样品量少、时效性强、检出下限较低、自动化程度高等优势,可有效地应用于蓝藻水华的监测。     

The effect of thiacloprid formulation on DNA/chromosome damage and changes in GST activity in bovine peripheral lymphocytes

The potential genotoxic effect of thiacloprid formulation on bovine peripheral lymphocytes was evaluated using the comet assay and the cytogenetic endpoints: chromosome aberrations (CAs), sister chromatid exchanges (SCEs) and micronuclei (MNi). Whole blood cultures were treated with the insecticide at concentrations of 30, 60, 120, 240 and 480 μg mL^?1 for 24, 48 h and/or 2 h of incubation. A statistically significant increase in the frequency of DNA damage, as well as in unstable chromosome aberrations (% breaks) were found after exposure to the insecticide at concentrations ranging from 120 to 480 μg mL^?1 (P < 0.05, P < 0.01, P < 0.001). For the detection of stable structural chromosome aberrations (e.g., translocations) and numerical aberrations by the FISH method, three whole chromosome painting probes for bovine chromosomes 1, 5 and 7 (BTA1, BTA5 and BTA7) were used in our experiments. We observed numerical aberrations, but without any statistical significance. Regarding the sister chromatid exchanges, no significant elevation in the SCE frequencies was found after 24-h exposure to the insecticide. A dose-related response in the SCE induction was obtained in bovine cultures after the prolonged time of exposure (48 h) to thiacloprid formulation at concentrations ranging from 120 to 480 μg mL^?1 in each donor (P < 0.05, P < 0.01), which was associated with a reduction of the PI (P < 0.05, P < 0.01). The insecticide failed to produce MNi; however, a significant reduction of CBPI was observed. Using real-time PCR, a decrease in the expression of bovine glutathione S-transferase M3 (GSTM3) was detected at the lowest dose. The higher concentrations of thiacloprid formulation caused an increase in the mRNA expression.     

城市生活污水中志贺氏菌ipaH毒力基因的定量PCR检测

基于ipaH毒力基因的实时荧光定量PCR检测,建立适合城市污水中志贺氏菌的定量检测方法。使用从临床分离出的志贺氏菌构建重组质粒作为实时荧光定量PCR的标准品。在ipaH基因模板量2.58×100～2.58×106copy范围内具有良好的线性关系,每100 mL水样中含有2.58×101copy以上的ipaH基因即可被检出。从西安市生活污水分离得到2株野生型志贺氏菌,分析ipaH基因数量和菌体数量的关系,从而确定ipaH基因定量检测志贺氏菌的可行性。该方法灵敏、快速、特异性好,适用于城市生活污水中志贺氏菌的检测。     

Non-biological inhibition-based sensing (NIBS) demonstrated for the detection of toxic arsenic compounds

This work demonstrates the success of a recently developed technique in chemical amplification, non-biological inhibition-based sensing (NIBS), for the detection of toxic arsenic compounds. Screening for toxic arsenic compounds is especially important due to their prevalence in wastewater and water sources. The detection method presented in this work amplifies the chemical response of toxic arsenic compounds by developing a sensor chemistry where the analyte inhibits, rather than enhances, the rate of a catalytic reaction. This technique mimics the work done with enzyme inhibition; however, using non-biological molecules allows for selective detection without the shelf-life issue associated with biological molecules. Using NIBS we find that we can enhance the sensitivity of the system by two orders of magnitude with no apparent loss in selectivity. This work demonstrates the versatility of NIBS, showing that the technique can be of general use for the detection of toxic compounds.     

一种新的检测土壤中壬基酚的荧光定量PCR方法

建立了一种新的外切酶保护-荧光定量PCR方法检测环境激素壬基酚,并将该方法应用于土壤样品中壬基酚的检测.壬基酚可以活化雌激素受体使之与包含特定序列的双链DNA结合,结合的DNA因受到蛋白质的保护可抵抗核酸外切酶Exo Ⅲ的消解而被保留,痕量的保留DNA可通过PCR扩增定量.根据此原理,首先从鱼肝脏中提取含雌激素受体的细胞溶质,与不同浓度的壬基酚结合使雌激素受体活化,形成配体-受体复合物;再设计合成特异性引物序列,采用常规PCR方法扩增制备双链结合DNA;使双链结合DNA与配体-受体复合物反应结合后,用核酸外切酶ExoⅢ和S_1核酸酶消解,去除未受到蛋白质保护的结合DNA.将消解后产物作为模板,进行荧光定量PCR扩增反应,从而建立C_t值与壬基酚质量浓度N(g/L)的标准曲线C_t=-1.828lgN+11.447.将该方法应用于土壤中壬基酚的检测,最低检测限达到2 pg/g,可用于检测大批量环境样品中壬基酚.     

荒漠绿洲土壤抗生素抗性基因分布特征及驱动机制

荒漠绿洲农田生态系统是干旱区环境下人类活动显著的复合生态系统.土壤微生物抗生素抗性与人类健康和生态平衡关系密切.研究荒漠绿洲环境不同土地利用类型模式下土壤抗生素抗性基因的多样性、分布特征和影响因素,对于评估干旱区土壤环境健康风险,促进绿洲农业生态的发展具有重要意义.采用高通量测序和高通量定量PCR技术对荒漠绿洲土壤微生物的群落结构和抗生素抗性基因多样性开展了研究,旨在探究干旱区土壤抗性基因的分布特征及其驱动机制.结果表明,从沙漠边缘到绿洲,荒漠沙生植物土壤、棉花地土壤、玉米地土壤、芦苇地土壤和湖泊沉积物中抗生素抗性的种类和丰度显著增加,与土地利用变化关系密切,农田土壤是抗性基因的重要存储库;荒漠绿洲土壤微生物群落与抗生素抗性基因显著相关,硫杆菌属(Thiobacillus)、沙漠细菌属(Pontibacter)、诺卡氏菌属(Nocardioides)、耐盐微杆菌属(Salinimicrobium)、土壤红杆菌属(Solirubrobacter)和链霉菌属(Streptomyces)等是各类抗性基因重要的潜在携带者;干旱区土壤中重(类)金属元素和可移动基因元件,与微生物群落共同塑造了抗生...     

PCR—DGGE技术在废水生物处理中的应用研究

聚合酶链式反应一变形梯度凝胶电泳（PCR—DGGE）作为一种分子指纹图谱能够较准确地反应污水处理过程中微生物群落结构多样性及其动态变化而广泛应用于废水生物处理技术的研究中。PCR—DGGE技术不仅能对样品中微生物群落结构多样性及种群演替进行分析,还能用于污水处理中优势菌群的分离与鉴定,克服了传统方法费时费力、培养条件苛刻等局限性,进而运用该技术构建高效的污染物降解工程菌,提高难降解有机物的去除效率,调节实际工艺参数,提高污水处理效率。     

Abundance and diversity of ammonia-oxidizing archaea in response to various habitats in Pearl River Delta of China, a subtropical maritime zone

Ammonia-oxidizing archaea (AOA) are widely considered key to ammonia oxidation in various environments. However, little work has been conducted to simultaneously investigate the abundance and diversity of AOA as well as correlations between archaeal amoA genotypes and environmental parameters of different ecosystems at one district. To understand the abundance, diversity, and distribution of AOA in Pearl River Delta of China in response to various habitats, the archaeal amoA genes in soil, marine, river, lake, hot spring and wastewater treatment plant (WWTP) samples were investigated using real-time fluorescent quantitative PCR and clone libraries. Our analyses indicated that the diversity of AOA in various habitats was different and could be clustered into five major clades, i.e., estuary sediment, marine water/sediment, soil, hot spring and Cluster 1. Phylogenetic analyses revealed that the structure of AOA communities in similar ecological habitats exhibited strong relation. The canonical correspondence method indicated that the AOA community structure was strongly correlated to temperature, pH, total organic carbon, total nitrogen and dissolved oxygen variables. Assessing AOA amoA gene copy numbers, ranging from 6.84×10⁶ to 9.45×10⁷ copies/g in dry soil/sediment, and 6.06×10⁶ to 2.41×10⁷ copies/L in water samples, were higher than ammonia-oxidizing bacteria (AOB) by 1-2 orders of magnitude. However, AOA amoA copy numbers were much lower than AOB in WWTP activated sludge samples. Overall, these studies suggested that AOA may be a major contributor to ammonia oxidation in natural habitats but play a minor role in highly aerated activated sludge. The result also showed the ratio of AOA to AOB amoA gene abundance was positively correlated with temperature and less correlated with other environmental parameters. New data from our study provide increasing evidence for the relative abundance and diversity of ammonia-oxidizing archaea in the global nitrogen cycle.     

氯/氯胺和紫外顺序消毒对管网抗性基因的去除

为探究供水系统中氯/氯胺与低压紫外顺序消毒对抗生素抗性基因(ARGs)分布特征的影响,采用生物膜反应器模拟供水管网,对管网出水和生物膜进行60mJ/cm²低压紫外线(254nm)消毒,并利用高通量定量PCR技术检测模拟管网进、出水及生物膜内的典型ARGs和遗传原件(MGEs).结果表明,管网反应器运行150d,氯和氯胺管网出水ARGs总相对丰度分别为0.13和0.137,生物膜ARGs分别为2.45和0.277,表明供水管网中低剂量的氯或氯胺可有效降低水相和生物膜相中ARGs的相对丰度达90%,且氯胺消毒对生物膜中的ARGs控制作用更显著.氯和氯胺消毒后管网出水再经低压紫外线照射后,ARGs相对丰度分别为0.0682和0.0537,管网生物膜中ARGs的相对丰度分别为2.01和0.194.ARGs与MGEs间的相关性发生显著变化,转座子与strB和mepA的相关性增强,与ermX和tetM相关性减弱,而整合子与acrF、cmlA1-01、oprJ及tolC-01的相关性增强.研究表明,将紫外线消毒工艺设置在用水终端可以显著降低氯和氯胺管网水中ARGs丰度,但对管网生物膜中的ARGs影响较小.