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.     

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。     

PCR-DGGE技术在城市污水化学生物絮凝处理中的特点

通过PCR-DGGE等分子生物学技术可以不经过常规培养直接从活性污泥和生物膜样品中提取DNA,对16Sr DNA V3区进行PCR扩增,结合DGGE(变性梯度凝胶电泳),从而分析活性污泥与生物膜中微生物种群结构.研究证实,活性污泥培养前后微生物种群结构发生很大的改变.同时对2种污水处理工艺中微生物种群结构进行了对比研究,对同一反应器不同位置微生物分布以及不同工况下的微生物种群结构进行了初步探讨.测定了活性污泥中部分菌种的16S rDNA V3区片段序列,通过NCBI(美国国立生物技术信息中心)基因库比对,初步确定细菌的属.结果显示,PCR-DGGE结合测序技术是一种完全可行的快速进行环境学样品微生物研究的分析方法.     

Analysis of the effects diclofenac has on Japanese medaka (Oryzias latipes) using real-time PCR

The expression levels of cytochrome P450 1A, p53 and vitellogenin were investigated in three different tissues of male medaka fish after exposure to diclofenac that is one of the main concerns among pharmaceuticals frequently found in sewage treatment plant (STP) effluents. The results showed that cytochrome P450 1A, p53 and vitellogenin were highly expressed in tissue-specific gene expression patterns after exposure to 8 mg/l and 1 μg/l of diclofenac. These elevated expression levels of three biomarkers suggested that diclofenac has potential to cause cellular toxicity, p53-related genotoxicity and estrogenic effects. It is also noteworthy that diclofenac has the potential to cause these effects even at an environmentally relevant concentration of diclofenac, 1 μg/l.     

Autonomous real-time adaptive management of soil salinity using a receding horizon control algorithm: a pilot-scale demonstration

Soil salinization is a potentially negative side effect of irrigation with reclaimed water. While optimization schemes have been applied to soil salinity control, these have typically failed to take advantage of real-time sensor feedback. This study incorporates current soil observation technologies into the optimal feedback-control scheme known as Receding Horizon Control (RHC) to enable successful autonomous control of soil salinization. RHC uses real-time sensor measurements, physically-based state prediction models, and optimization algorithms to drive field conditions to a desired environmental state by manipulating application rate or irrigation duration/frequency. A simulation model including the Richards equation coupled to energy and solute transport equations is employed as a state estimator. Vertical multi-sensor arrays installed in the soil provide initial conditions and continuous feedback to the control scheme. An optimization algorithm determines the optimal irrigation rate or frequency subject to imposed constraints protective of soil salinization. A small-scale field test demonstrates that the RHC scheme is capable of autonomously maintaining specified salt levels at a prescribed soil depth. This finding suggests that, given an adequately structured and trained simulation model, sensor networks, and optimization algorithms can be integrated using RHC to autonomously achieve water reuse and agricultural objectives while managing soil salinization.     

甘蔗蔗糖磷酸合成酶SPSⅡ cDNA片段克隆与表达分析

蔗糖磷酸合成酶(Sucrose phosphate synthase,SPS)是蔗糖合成途径的关键限速酶,在蔗糖积累和碳分配中具有重要作用.在茎组织中SPSⅡ表达量占SPS转录总量的40%,表明SPSⅡ cDNA的克隆与表达对蔗茎中蔗糖的积累有着重要的影响.通过RT-PCR技术克隆分离SPSⅡ基因cDNA片段.序列分析表明该cDNA片段包含1个3 183 bp的开放读码框,可编码1 060个氨基酸.GenBank登录号为EU269038.通过实时荧光定量PCR检测SPSⅡ基因在甘蔗糖分积累的不同时期、不同组织部位的相对表达量.结果表明,在糖分积累初期蔗茎中的SPSⅡ相对表达量最大,在糖分积累中期蔗叶中的SPSⅡ相对表达量达到最高峰;同组织部位中,糖分积累初期SPSⅡ相对表达量高于糖分积累中期、后期.图6参11     

Potential dissemination mechanism of the tetC gene in Aeromonas media from the aerobic biofilm reactor under oxytetracycline stresses

The tetC gene has been found to be one of the most widely distributed tetracycline resistance (tet) genes in various environmental niches, but the detailed dissemination mechanisms are still largely unknown. In the present study, 11 tetC-containing Aeromonas media strains were isolated from an aerobic biofilm reactor under oxytetracycline stresses, and the genome of one strain was sequenced using the PacBio RSII sequencing approach to reveal the genetic environment of tetC. The tetC gene was carried by an IS26 composite transposon, named Tn6434. The tetC-carrying Tn6434 structure was detected in all of the A. media strains either in a novel plasmid pAeme2 (n=9) or other DNA molecules (n=2) by PCR screening. The NCBI database searching result shows that this structure was also present in the plasmids or chromosomes of other 13 genera, indicating the transferability of Tn6434. Inverse PCR and sequencing confirmed that Tn6434 can form a circular intermediate and is able to incorporate into a preexisting IS26 element, suggesting that Tn6434 might be responsible for the dissemination of tetC between different DNA molecules. This study will be helpful in uncovering the spread mechanism of tet genes in water environments.