基于高通量定量PCR研究城市化小流域微生物污染特征

水体微生物污染(包括致病菌、病毒、寄生虫)会引起多种传染病和寄生虫病,对生产生活用水安全和人体健康造成重要威胁。本研究应用基于Taq Man探针的高通量荧光定量PCR技术对厦门市后溪流域冬季微生物污染进行检测,包含了5种粪便污染源(人源、反刍动物源、猪源、家禽源、狗源)微生物源示踪分子标记物与12种病原微生物。结果表明,该流域在上游及水库5个位点没有粪便污染,仅在其中一个水库位点检测出棘阿米巴,微生物污染极小;中下游检测出人类、反刍动物、猪、家禽、狗粪便污染,并且检测出产气荚膜梭菌、肠聚集性大肠杆菌、肠毒素型大肠杆菌、幽门螺杆菌、霍乱弧菌、副溶血弧菌、棘阿米巴、克雷伯氏肺炎杆菌等病原菌,其中流经旧城区居民生活生产区水样微生物污染严重,下游新城区微生物污染较小。这些结果暗示着城市人类活动是流域微生物污染主要来源,应从污染源头加强微生物污染控制。     

环境因素对不同工艺人工湿地反硝化功能基因丰度影响

反硝化细菌在人工湿地对N元素去除的过程中发挥着关键作用.为了解湿地类型、基质、植被和水力负荷及物理参数对人工湿地反硝化功能细菌丰度的影响,构建了9个不同工艺组合的人工湿地小试装置.利用实时荧光定量PCR技术检测湿地基质中反硝化功能基因nirS,nirK和nosZ的丰度.结果显示,湿地中nirS,nirK和nosZ丰度(以每ng总DNA中的拷贝数计)分别为4.88×103～2.13×105 copies/ng,1.2×101 ～ 2.14×103 copies/ng和1.98×103～ 2.09 × 105copies/ng,其中nirS显著高于nirK丰度,nirS丰度与湿地出水的pH显著正相关.分析湿地4种设计参数对反硝化功能基因丰度的影响程度,结果显示,对nirS丰度影响程度大小依次为湿地类型＞植被类型＞水力负荷＞基质类型,水平潜流和上行垂直流人工湿地中nirS丰度显著高于下行垂直流人工湿地,无植物人工湿地nirS丰度显著高于有植物人工湿地;对nirK丰度影响程度大小依次为湿地类型＞植被类型＞基质类型＞水力负荷,水平潜流人工湿地nirK丰度显著高于下行垂直流湿地;对nosZ丰度影响程度大小依次为湿地类型＞水力负荷＞植被类型＞基质类型,但影响都不显著.     

Application of an early warning indicator and CaO to maximize the time–space-yield of an completely mixed waste digester using rape seed oil as co-substrate

In order to increase the organic loading rate (OLR) and hereby the performance of biogas plants an early warning indicator (EWI-VFA/Ca) was applied in a laboratory-scale biogas digester to control process stability and to steer additive dosing. As soon as the EWI-VFA/Ca indicated the change from stable to instable process conditions, calcium oxide was charged as a countermeasure to raise the pH and to bind long-chain fatty acids (LCFAs) by formation of aggregates. An interval of eight days between two increases of the OLR, which corresponded to 38% of the hydraulic residence time (HRT), was sufficient for process adaptation. An OLR increase by a factor of three within six weeks was successfully used for biogas production. The OLR was increased to 9.5 kg volatile solids (VS) m^?3 d^?1 with up to 87% of fat. The high loading rates affected neither the microbial community negatively nor the biogas production process. Despite the increase of the organic load to high rates, methane production yielded almost its optimum, amounting to 0.9 m³ (kg VS)^?1. Beneath several uncharacterized members of the phylum Firmicutes mostly belonging to the family Clostridiaceae, a Syntrophomonas-like organism was identified that is known to live in a syntrophic relationship to methanogenic archaea. Within the methanogenic group, microorganisms affiliated to Methanosarcina, Methanoculleus and Methanobacterium dominated the community.     

Identification of potential biomarkers for diazinon exposure to Japanese Medaka (Oryzias latipes) using annealing control primers

A new differential display-polymerase chain reaction (PCR) method based on annealing control primers was used to screen and identify potential biomarkers from differentially expressed genes (DEGs) in medaka exposed to sub-lethal concentration of diazinon (100 ppb). Among the differentially expressed genes identified, the majority were in functional categories of protein biosynthesis, transport and metabolism according to the gene ontology classification. The differential expression of ribosomal protein genes was quantified by real time PCR. The genes encoding ribosomal proteins including L3 and S17 were selected as potential biomarkers for diazinon exposure in medaka fish.     

Detection of Escherichia coli in a cattle manure composting process by selective cultivation and colony polymerase chain reaction

Livestock manure is suitable for use as a composting material. However, various intestinal microbes, such as Escherichia coli, are significant components of such manures. Thus, it is desirable that the level of intestinal microbes, and particularly opportunistic pathogens, in compost is inspected and counted regularly. The sensitivity and specificity of detection of E. coli in compost have been improved by selective cultivation followed by colony polymerase chain reaction (PCR) using the ECO primer. Indeed, the sensitivity of this method is higher than that of DNA extraction from compost and PCR. In this study, changes in numbers of E. coli present in a field-scale composting process over time was assessed using selective cultivation and colony PCR. Numbers of ECO-positive colonies after 24 h decreased, with a concomitant rise in compost temperature. ECO-positive colonies were not detected from 33 to 48 h. However, ECO-positive colony numbers increased beginning on day 4 and continuing until day 42. Thus, it seems likely that the high temperatures reached during the composting process did not affect E. coli numbers in the final compost. Additionally, selective cultivation followed by colony PCR using specific primers is an appropriate method of determining levels of cultivable pathogens in composted materials.     

PCR技术在水体微生物检测中的应用

聚合酶链式反应(PCR)技术因其特异性强、灵敏度高以及快速简便等优点广泛应用于水体微生物的检测。综述了PCR技术的原理、方法及其在水体微生物检测中的研究进展。主要介绍了该技术应用于水环境中微生物病原体的检测、污水生物处理过程的监测与评价中的重要意义以及本实验室在该领域的研究进展。PCR技术为在分子水平上进行环境微生物的结构、多样性分析提供了有力的实验手段,有着广泛的应用前景。     

池塘水体氨氧化细菌季节变化及其与环境影响因子相关性分析

以氨氧化细菌(AOB)的氨单加氧酶amoA基因为分子标记,调查了湖北省公安县崇湖渔场草鱼池塘水体AOB季节变化,并分析了AOB丰度与主要环境因子的相关性.结果表明,池塘表、底层水体AOB amoA基因拷贝数均呈相似的季节变动规律,即夏季显著高于春、秋两季(p0.05),而春、秋两季间无显著差异(p0.05);相同季节池塘表、底层水体AOB amoA基因拷贝数无显著性差异(p0.05).表、底层水体AOB amoA基因拷贝数均与温度、总磷呈显著正相关;与溶解氧呈显著负相关.此外,表层AOB amoA基因拷贝数与总有机碳呈极显著负相关.主成分分析将8个环境因子划分为3个主成分.表层第一主成分为水温、溶解氧、氨氮与总磷,第二主成分为硝态氮与总有机碳,第三主成分为总氮与亚硝态氮;底层第一主成分为水温、溶解氧、氨氮、总磷与总有机碳,第二主成分为硝态氮与亚硝态氮,第三主成分为总氮.     

荧光定量PCR技术在环境领域的应用

实时荧光定量PCR技术作为一项新兴技术,越来越受到人们的重视,它以快速、准确定量、便捷等优点广泛应用于科学研究各个领域。近年来,实时荧光定量PCR技术不断发展,并应用于环境领域中,大大提高了环境监测水平。综述了实时荧光定量PCR技术的基本原理及其在国内外环境领域中的应用。     

日本地震实时监测系统中液化识别方法可行性研究

日本地震超高密度实时监测系统(SUPREME)建设中的核心技术之一是依据强震记录对液化场地进行反演识别,系统中目前使用的是Suzuki法,但实际应用效果并不理想。选取不同地震下多种类别场地上的实际地震记录,对Suzuki法的适用性进行了研究,讨论了其不同指标对不同类别场地识别结果的影响。分析表明:Suzuki法对不同类别非液化场地的识别能力不同,最显著的缺陷是易将软土场地与液化场地混淆;方法所使用的4个识别指标中,过零周期为控制参数,其它指标仅起到初判作用;方法出现误判的原因是液化场地的过零周期范围与非液化中软土场地上的过零周期范围存在明显交叉,而这一点理论上是无法避免的客观存在;改进的方法是应放弃现有进行绝对值对比的做法,改用地震动某些参数的相对变化作为新的识别指标。     

PCR-酶切技术在石油烃降解菌筛选鉴定中的应用

将以原油驯化所得的纯培养微生物菌株作为模板,采用PCR(聚合酶链式反应)法,用扩增细菌16S rDNA的一对通用引物(8 f,1492 r)对模板扩增,并用限制性内切酶R saI和M spI对PCR产物进行ARDRA多态性分析。聚类去除重复菌株后60株菌株可分为11种不同分类操作单元(OTU),同时得出每个分类操作单元的数量。实验结果表明,此方法可快速去除重复菌株并反映出菌株间的系统进化关系;同时实验数据可构建成库,使后续分离菌株的筛选工作只需比对数据即可完成。