特异性拟杆菌引物在珠江三角洲的适应性研究

在珠江三角洲地区采集人、猪、牛、狗以及家禽等33个粪便样品,高效提取基因组DNA,选取14种特异性拟杆菌引物进行检验分析,并进一步采用已知污染源类型的水样对其进行验证.结果表明:采用粪便样品DNA专属提取试剂盒和水体样品DNA专属提取试剂盒提取的基因组模版纯度和提取率均符合后续实验要求.拟杆菌通用引物2#(Bac32F/Bac708R)检出率为85%(28/33),特别是对哺乳类动物和鸡的粪便具有更高的检出率.人的拟杆菌特异性引物3#(HF134F/ Bac708R)和4#(HF183F/Bac708R)、反刍动物的拟杆菌特异性引物8#(CF128F/Bac708R)以及猪的拟杆菌特异性引物10#(PF163F/Bac708R)在珠江三角洲地区同时具有较高的灵敏度和较强的特异性.水体样品验证实验与实际污染类型相符,说明拟杆菌通用引物2#、人的特异性引物3# 和4# 以及猪的特异性引物10# 在珠江三角洲地区具有很好的适用性.     

Human-, Ovine-, and Bovine-Specific Viral Source Tracking Tools to Discriminate Between the Major Fecal Sources in Agricultural Waters

This study evaluated the sources of fecal contamination in different river catchments, using a combination of microbial source tracking tools, for human, ruminant, ovine and bovine livestock, in order to define appropriate water management strategies. Every source of waterway pollution was evaluated in river water samples from one urban river catchment and two important farming regions in New Zealand. Fecal pollution was initially measured by testing Escherichia coli and evaluating the presence of human- and ruminant-associated DNA markers of Bacteroidales (BiAdo, BacHum-UCD, BacH, and BacR) and human and ruminant fecal sterols/stanols ratios. Then specific fecal pollution sources were assessed with previously reported quantitative PCR assays targeting human-, bovine-, and ovine-specific viruses: human adenoviruses (HAdV), human JC polyomaviruses, bovine polyomaviruses (BPyV), and ovine polyomaviruses (OPyV). High level of ruminant fecal contamination was detected all over the farming areas, whereas no ruminant sources were identified in the urban river sampling sites. BacR was the most frequently observed ruminant marker and OPyV and BPyV allowed the identification of ovine and bovine fecal sources. The human fecal viral marker (HAdV) was the most frequently observed human marker, highly abundant in the urban sites, and also present in farming areas. This is the first study using simultaneously the ovine and the bovine viral markers to identify and quantify both bovine and ovine fecal pollution.     

城市污泥厌氧消化和脱水工艺对肠道病原菌的杀灭效应

运用最大或然数(MPN)培养法和定量PCR(qPCR)技术对不同城市污水处理厂污泥厌氧消化处理和脱水处理前后,污泥中埃希氏大肠杆菌(E.coli)、沙门氏菌(Salmonellaspp.)和志贺氏菌(Shigellaspp.)含量的变化进行了研究.MPN检测结果表明,污泥中大肠杆菌、沙门氏菌和志贺氏菌数量经厌氧消化处理后明显下降,平均下降2～5个数量级,但qPCR检测结果显示,种病原菌平均下降1～2个数量级.脱水处理3后,污泥中的肠道病原菌含量基本没有变化,没有发生脱水后再生长现象.大肠杆菌、沙门氏菌和志贺氏菌含量分别在104～107、104～105和103～105MPN·g-1(以污泥干重计)之间.MPN和qPCR检测结果之间的差异性显示厌氧消化会造成肠道病原菌的有活性但不可培养状态(VBNC),同时也提示应用传统的培养检测方法检测病原菌含量和评定污泥排放的生物安全性时需慎重.     

基于拟杆菌16S rRNA基因进行微生物溯源的研究进展

微生物溯源方法可利用粪便中的微生物区分来自人或动物的粪便污染.其中,拟杆菌以其丰度高、不能体外繁殖和宿主特异性强等优势被广泛应用于微生物溯源研究中.本文以拟杆菌16S rRNA基因为标记物,总结了拟杆菌及其标记物在环境中的衰减、拟杆菌引物的敏感性和特异性以及分子生物学技术在微生物溯源中的运用,可为粪便污染源解析提供一定的科学参考依据.     

北运河粪源微生物分布特征及健康风险评价

河流微生物污染研究对流域水环境保护和公共卫生管理至关重要.本研究以海河流域北运河水系典型河流断面为研究对象,系统评估了北运河水系微生物污染特征及其健康风险.结果表明,北运河水系丰水期的粪源微生物污染严重,大部分断面菌群量超过105MPN·L~(-1),平水期和枯水期粪源微生物菌群量约为103～105MPN·L~(-1),变化不显著;流域内的农业河段粪源微生物最为严重,呈劣五类水体概率超60%,城市河段降雨前后粪源微生物含量变化更明显,部分河流断面的粪源微生物菌群量从103MPN·L~(-1)增长为106MPN·L~(-1); 2017年丰水期,北运河水系粪大肠菌群的单次暴露风险主要在0. 015～0. 035之间,莲花河、马草河、清河下、温榆河下为人体健康风险的关键敏感断面,需重点关注.     

One-year survey of opportunistic premise plumbing pathogens and free-living amoebae in the tap-water of one northern city of China

In this study, qPCR was used to quantify opportunistic premise plumbing pathogens (OPPPs) and free-living amoebae in 11 tap water samples collected over four seasons from a city in northern China. Results demonstrated that the average numbers of gene copies of Legionella spp. and Mycobacterium spp. were significantly higher than those of Aeromonas spp. (p?<?0.05). Legionella spp. and Mycobacterium spp. were 100% (44/44) positively detected while P. aeruginosa and Aeromonas spp. were 79.54% (35/44) and 77.27% (34/44) positively detected. Legionella pneumophila was only detected in 4 samples (4/44), demonstrating its occasional occurrence. No Mycobacterium avium or Naegleria fowleri was detected in any of the samples. The average gene copy numbers of target OPPPs were the highest in summer, suggesting seasonal prevalence of OPPPs. Average gene copy numbers of OPPPs in the taps of low-use-frequency were higher than in taps of high-use-frequency, but the difference was not significant for some OPPPs (p?>?0.05). Moderate negative correlations between the chlorine concentration and the gene copy numbers of OPPPs were observed by Spearman analysis (r_s ranged from ? 0.311 to ? 0.710, p?<?0.05). However, no significant correlations existed between OPPPs and AOC, BDOC, or turbidity. Moderate positive correlations were observed between the target microorganisms, especially for Acanthamoeba spp., through Spearman analysis (p?<?0.05). Based on our studies, it is proposed that disinfectant concentration, season, taps with different-use frequency, OPPP species, and potential microbial correlations should be considered for control of OPPPs in tap water.     

有氧和厌氧环境下指示微生物的稳定性特征

建立有氧和厌氧水环境模拟反应器,利用荧光定量PCR和高通量测序技术探究了猪源拟杆菌标记物、部分指示微生物和潜在致病菌在有氧及厌氧水环境中的变化特征.结果表明,指示微生物 Streptococcaceae、Lactobacillaceae、Carnobacteriaceae、Ruminococcaceae、Lachnos...     

应用FCM-qPCR方法定量检测水中常见病原体

以往对水体病原体的研究主要是通过监测粪大肠杆菌作为指示,然而研究表明粪大肠杆菌与水中病毒和细菌病原体呈现出较差的相关性.因此,选取水中典型病原体并对其进行定量检测是当前需要解决的技术问题.为此本研究建立了流式细胞术和定量PCR联合使用方法,用于快速获取水环境中总病毒、细菌以及几种典型病原体(大肠杆菌、军团菌、腺病毒、贾第虫和隐孢子虫等)的浓度水平,并将该方法应用到污水处理厂进出水及受纳河流上下游的病原体检测中.结果表明,该污水处理厂对总细菌和总病毒以及几种典型病原体都具有较高的去除率(93%);污水处理厂排水对受纳水体病原体浓度水平基本没有负面影响.研究为评估污水处理厂处理效果及排水对受纳水体的生态影响提供了技术支持.     

Performance Evaluation of Human-Specific Viral Markers and Application of Pepper Mild Mottle Virus and CrAssphage to Environmental Water Samples as Fecal Pollution Markers in the Kathmandu Valley,Nepal

Monitoring of environmental water is crucial to protecting humans and animals from possible health risks. Although numerous human-specific viral markers have been designed to track the presence of human fecal contamination in water, they lack adequate sensitivity and specificity in different geographical regions. We evaluated the performances of six human-specific viral markers [Aichi virus 1 (AiV-1), human adenoviruses (HAdVs), BK and JC polyomaviruses (BKPyVs and JCPyVs), pepper mild mottle virus (PMMoV), and crAssphage] using 122 fecal-source samples collected from humans and five animal hosts in the Kathmandu Valley, Nepal. PMMoV and crAssphage showed high sensitivity (90–100%) with concentrations of 4.5–9.1 and 6.2–7.0 log₁₀ copies/g wet feces (n = 10), respectively, whereas BKPyVs, JCPyVs, HAdVs, and AiV-1 showed poor performances with sensitivities of 30–40%. PMMoV and crAssphage were detected in 40–100% and 8–90%, respectively, of all types of animal fecal sources and showed no significantly different concentrations among most of the fecal sources (Kruskal–Wallis test, P > 0.05), suggesting their applicability as general fecal pollution markers. Furthermore, a total of 115 environmental water samples were tested for PMMoV and crAssphage to identify fecal pollution. PMMoV and crAssphage were successfully detected in 62% (71/115) and 73% (84/115) of water samples, respectively. The greater abundance and higher mean concentration of crAssphage (4.1 ± 0.9 log₁₀ copies/L) compared with PMMoV (3.3 ± 1.4 log₁₀ copies/L) indicated greater chance of detection of crAssphage in water samples, suggesting that crAssphage could be preferred to PMMoV as a marker of fecal pollution.

     

城市地表水中肠道病原微生物与粪便污染指示菌的关系研究

本研究选择肠道病毒、伤寒沙门氏菌、志贺氏菌、大肠埃希氏菌作为城市水体中典型的肠道病原微生物,分别建立起相应的实时荧光定量PCR检测方法.对水源水、景观娱乐用水以及城市河流样品进行长期监测,考察典型肠道病原微生物的分布特征和变化规律.肠道病毒在各地表水体中都有检出,浓度大都在103copy·L-1以下.大肠埃希氏菌的含量在保护良好的水体和接纳城市污水的水体中有明显差别,而伤寒沙门氏菌和志贺氏菌通常出现在受粪便污染较重的水体中.统计学的分析结果显示各种水体中的肠道病原微生物检出浓度均符合对数正态分布规律.粪便污染指示菌与肠道病毒之间不存在显著的相关性,但却能在一定程度上反映典型肠道病原菌的存在情况.在大肠菌群或粪大肠菌群浓度为104CFU·L-1以上的样品中,伤寒沙门氏菌、志贺氏菌具有较高的阳性率.     

Evaluation of bacterial pathogen diversity, abundance and health risks in urban recreational water by amplicon next-generation sequencing and quantitative PCR

The microbial quality of urban recreational water is of great concern to public health.The monitoring of indicator organisms and several pathogens alone is not sufficient to accurately and comprehensively identify microbial risks.To assess the levels of bacterial pathogens and health risks in urban recreational water,we analyzed pathogen diversity and quantified four pathogens in 46 water samples collected from waterbodies in Beijing Olympic Forest Park in one year.The pathogen diversity revealed by 16 S r RNA gene targeted next-generation sequencing(NGS) showed that 16 of 40 genera and 13 of 76 reference species were present.The most abundant species were Acinetobacter johnsonii,Mycobacterium avium and Aeromonas spp.Quantitative polymerase chain reaction(q PCR) of Escherichia coli(uid A),Aeromonas(aer A),M.avium(16S r RNA),Pseudomonas aeruginosa(oaa) and Salmonella(inv A) showed that the aer A genes were the most abundant,occurring in all samples with concentrations of 10~(4–6) genome copies/100 m L,followed by oaa,inv A and M.avium.In total,34.8% of the samples harbored all genes,indicating the prevalence of these pathogens in this recreational waterbody.Based on the q PCR results,a quantitative microbial risk assessment(QMRA) showed that the annual infection risks of Salmonella,M.avium and P.aeruginosa in five activities were mostly greater than the U.S.EPA risk limit for recreational contacts,and children playing with water may be exposed to the greatest infection risk.Our findings provide a comprehensive understanding of bacterial pathogen diversity and pathogen abundance in urban recreational water by applying both NGS and q PCR.     

黄河支流非点源污染物(N、P)排放量的估算

为了估算黄河支流域非点源污染物的排放量,为今后黄河流域的治理提供科学的依据,对黄河流域的6个子流域(渭河流域、泾河流域、洛河流域、无定河流域、窟野河流域、黄甫川流域)进行了水样、土样和泥沙样的采集分析;结合6个子流域的多年水文资料,利用已经被证明了的平均浓度法对黄河6个子流域的氮磷污染物进行了定量描述,估算出各个流域的氮磷排放量.结果表明,95%的全磷、大于53%的全氮来自于非点源污染,非点源污染是造成黄河污染的主要原因.各流域非点源污染的发生具有明显的地域分异特征.硝态氮主要来自于渭河,铵态氮主要来自于泾河,其次是渭河.从全氮看,绝大部分氮来自于渭河和泾河.全磷则主要来自于泾河,其次是渭河.泾河和渭河是6条河流中对黄河污染物排放量贡献最大的2条河流.     

长江三峡库区蓄水后水质状况分析

2003年5—6月和10月对三峡库区蓄水后的水污染状况进行了监测分析.结果显示,蓄水后库区以Ⅲ类水质为主.若考虑粪大肠菌群指标,总体水质以Ⅳ类和劣Ⅴ类为主.三峡库区重金属含量符合《渔业水质标准》(GB11607-89)与《地表水环境质量标准》(GB3838-2002)Ⅰ类标准的要求.但各监测断面Pb含量较高,两期采样Hg含量均超过了《地表水环境质量标准》(GB3838-2002)Ⅲ类标准.三峡库区共检出19种挥发性有机污染物,其中国家《地表水环境质量标准》(GB3838-2002)控制的特定化学物质5种,分别为1,2-二氯乙烷,1,1,2-三氯乙烷,1,4-二氯苯,1,2-二氯苯,六氯丁二烯,但均不超标.挥发性有机污染物以卤代烃为主,苯系物较少.与国内其他水体相比,三峡库区水体受到挥发性有机物的污染较轻.      

基于模糊信息熵的流域水污染系统建模与控制

我国水污染局部得到了改善,但呈现流域污染蔓延的趋势。运用系统控制论构建流域水污染控制系统,突破了传统的"就水论水、见污治污"的还原论水污染控制方法,综合考虑总体控制战略决策(S)反馈系统以及社会发展布局(L)、经济发展模式(E)、居民生活方式(C)和治理工程措施(T)控制系统等多种控制措施及因素,通过输出量与参考给定输入的比较,采用模糊信息熵方法进行了定量分析方法的探讨。     

西江流域地表水微生物污染定量源解析

水体微生物污染标记物的源强特征是定量解析微生物污染来源的关键.应用实时荧光定量PCR（qPCR）技术分别对人源（qHS601F/qBac725R）、牛源（BacB2-590F/Bac708Rm）、猪源（Bac41F/Bac163R）及禽类（qC160F-HU/qBac265R-HU）特异性拟杆菌引物的源强特征进行了探索,并进一步对西江流域广东段进行微生物污染来源定量解析,结果表明：针对不同目标宿主,不同特异性拟杆菌引物的源强特征为人源（qHB） > 猪源（qPB） > 牛源（qRB） > 禽类（qCB）,DNA模板浓度对引物的源强特征影响较小.选取的目标研究区域水体普遍受到人、猪、牛及禽类粪便污染.干流中qHB、qCB及qPB自上游至下游缓慢升高.各支流中qHB、qCB及qPB的平均浓度较干流均上升了1个数量级,qRB在不同河段变化较小.说明下游河段的生活污水、猪源及禽类粪便污染较为严重,牛源污染的影响较小.统计分析显示在污染水平较高的各支流中,人源特异性拟杆菌引物与传统指示菌具有一定的相关性（P<0.05）,说明生活污水是传统指示菌浓度较高的主要原因,且为持续排放源.     

基于流域分析方法的湖泊水污染综合防治研究

鉴于流域思想在水污染防治中应用的重要性和必要性,提出了以流域保护方法、流域分析和景观生态学相关理论为基础的流域分析方法该方法将湖泊-流域水污染综合防治的主要内容界定为4部分:子流域划分与污染负荷预测,"源-途径-末端-汇"的污染防治工程和管理方案体系设计,备选技术方案的提出和方案优选,污染负荷的削减率计算和综合方案设计.以四川省邛海流域为例,在将流域划分为6个子流域的基础上,对TP入湖污染负荷进行了计算和预测,并识别出各子流域内污染物的贡献率,分区提出了以水土流失防治、点源和面源控制、河道生态修复、河流入湖口治理和内源疏浚为主体的污染防治方案,该方案可削减TP 22.5 t·a-1,达到了预期目标.     

基于FIB的北京温榆河流域粪便污染物分布特征和源解析

目前,北京市温榆河流域存在着较为严重的粪源微生物污染问题,对流域周边暴露人群的健康带来了潜在的威胁.为了明确温榆河粪便污染的分布特征及宿主来源,本研究采用传统培养法与定量聚合酶链式反应(qPCR)分别对常规粪便指示细菌(Fecal indicator bacteria, FIB),包括粪大肠菌群(Fecal Coliform, FC)、大肠杆菌(Escherichia coli, EC)与肠球菌(Enterococcus,ENT)和溯源FIB,包括不同宿主来源的特异性生物标记进行检测,并探究其相关性.研究结果表明,流域内粪源微生物污染较为严重,常规FIB浓度介于0～4.38×10~7 CFU·L~(-1)之间,溯源FIB处于1.37×10~6～4.29×10~(10) copies·L~(-1)之间.从时空分布来看,由于来水中各大支流及污水处理厂的贡献,从温榆河起点沙河闸至终点北关闸,所有的指示菌浓度呈现出大幅度的升高趋势.其中,龙道河为干流来水中粪便污染的首要地域,污水处理厂的尾水可能是清河河口及坝河处微生物污染的主要来源;降雨径流对粪源微生物含量的时空变化存在着非常显著的影响,使得FIB最高可达2个数量级的增长.微生物溯源结果显示温榆河流域内的粪源微生物来源主要为人类粪便污染,而非畜禽粪便污染,这同北京市近年来有关畜禽养殖管控密切相关.相关性分析表明,虽然FC与EC、ENT之间皆存在着统计学意义上的显著正相关性,但传统的FIB指标与拟杆菌及人源拟杆菌指标之间不存在显著相关性.本研究通过传统FIB培养和微生物溯源方法结合,明确了北京温榆河粪源污染程度及其主要来源,为北京温榆河污染防控提供了新的思路.     

水文水质模型联合应用于水库水质预测研究

联合应用流域水文非点源AnnAGNPS模型和水库水质CE-QUAL-W2模型,AnnAGNPS模型输出黑河流域非点源污染负荷,将其转化为金盆水库入库浓度作为CE-QUAL-W2模型的输入,对黑河金盆水质进行预测,研究非点源污染对陕西金盆水库水质影响.结果表明,在对水库水质进行预测时,应对洪水期和非洪水期的非点源污染区别对待.非点源污染在洪水期时对金盆水库水质有较大影响,而在非洪水期时非点源污染对水库水质影响不显著.非点源污染对水库纵向和垂向水质影响存在差异性.林地对流域非点源污染削减起到很大作用.     

滇池流域城市面源污染控制区划研究

滇池流域点源污染已逐步得到控制,面源污染成为水污染治理的重点。随着流域内城市迅速发展,城市面源污染控制将成为水环境改善的重要瓶颈。结合滇池流域城市发展规划,依据城市下垫面特征,降雨径流污染程度和距离入滇河道远近,将流域内城区划分为重点防控区、中等防控区和一般防控区,其面积百分比分别是17.9%、39.1%和43.0%;重点控制区为昆明市主城区,其次是呈贡新区,并在此基础上提出相应的防治措施。城市面源污染控制区划,对滇池流域面源污染治理具有重要意义。     

九龙江下游水源水中新发病原微生物和抗生素抗性基因的定量PCR检测

新发水传病原微生物和抗生素抗性基因对于饮用水的生物安全性有重要意义.以7种典型的水传病原微生物和4种抗生素抗性基因作为检测指标,对闽西南地区的重要水源九龙江下游水源水进行了检测.从九龙江下游4个采样点和附近某水厂进出水采集水样,用0.22μm的滤膜过滤后,进行DNA提取及荧光定量PCR检测.结果表明,九龙江下游水源水中Salmonella enterica(Salmonella spp.)、Legionella pneumophila(L.pneumophila)和Pseudomonas aeruginosa(P.aeruginosa)等个别指标可达到103、104、105copies.mL-1的水平.有机物浓度可能对病原微生物和抗生素抗性基因的水平有较大影响.水厂常规工艺对于大多数病原微生物和抗生素抗性基因可以实现有效去除,但对Salmonella spp.的去除效果不理想.因此为保证饮用水安全,需要在清理污染源、提高处理工艺水平以及末端控制方面加强管理.