实时荧光定量聚合酶链反应快速检测产毒铜绿微囊藻

以铜绿微囊藻(Microcystis aeruginosa)产毒株微囊藻毒素合成酶基因(mcyA)为靶序列设计了一对特异性引物,运用实时荧光定量聚合酶链反应(qPCR)方法,对铜绿微囊藻进行了定性定量检测。结果表明,仅含铜绿微囊藻脱氧核糖核酸(DNA)模板的样品有扩增,对照组小球藻(Chlorella)没有检测到扩增信号;扩增产物熔解曲线平稳,峰尖且窄,熔解温度为(83±1)℃,证明该聚合酶链反应扩增产物极为特异。以重组质粒pMD-18T-mcyA为标准品,所得标准曲线具有高度线性相关性,重复性好,符合制备实时定量聚合酶链反应标准曲线的要求。利用该方法建立的标准曲线对实验室培养获得的铜绿微囊藻DNA样品进行定量检测,与显微镜计数结果基本一致。     

基于流式细胞法的铜绿微囊藻快速定量方法

采用超声波细胞破碎仪处理地表水样品,可使群体藻细胞分散开,用浮游植物流式细胞仪实现水样的快速测定,最适测定时间为180 s,藻细胞密度测定限值为5.0×106L-1,当样品中藻细胞密度低于该限值时,可适当浓缩样品。以铜绿微囊藻特征指标判断,当该方法测得铜绿微囊藻密度在5.0×106L-1以下时,可判定样品中不含有铜绿微囊藻。     

不同藻密度条件下铜绿微囊藻与大型溞的相互影响

通过设置模拟摄食试验,使用铜绿微囊藻藻液及滤液对大型溞同时进行急性毒理试验,探讨了不同藻密度条件下铜绿微囊藻与大型溞之间的相互影响。结果表明,大型溞的摄食行为对铜绿微囊藻的生长有抑制,抑制作用随藻密度升高而下降,中、低藻密度(1.01×108mL~(-1)、1.01×107mL~(-1))下的抑制率分别为54.6%、65.7%,高密度(1.01×109mL~(-1))下的抑制率为29.7%。同时,铜绿微囊藻对大型溞有毒性作用,在毒理试验中,藻液组24 h和48 h的LC50值分别为0.455×107mL~(-1)和0.036×107mL~(-1),滤液组24 h和48 h的LC50值分别为1.299×107mL~(-1)和0.179×107mL~(-1)。藻液组的LC50值明显低于滤液组,结合镜检表明,大型溞摄食铜绿微囊藻,铜绿微囊藻对大型溞的毒性影响以胞内毒素为主。在藻-溞微生态系统中,当藻密度低时,大型溞种群对铜绿微囊藻的去除效果显著,藻细胞被摄食殆尽后大型溞迅速死亡;当藻密度适中时,大型溞种群对铜绿微囊藻的去除效果良好,存活时间最长;当藻密度高时,大型溞种群受藻毒素强烈影响,短时间内死亡殆尽,对铜绿微囊藻的去除效果差。     

水环境中致病菌分子生物学检测技术研究进展

总结了水环境中主要致病菌及其导致的疾病；介绍了检测水环境样品中致病菌的聚合酶链式反应（PCR）、实时定量PCR、等温扩增技术、生物传感器和高通量测序等分子生物学技术研究进展；分析了不同检测技术的优缺点和应用特点；提出了水环境中致病菌分子生物学检测技术的未来发展方向，为水环境中致病菌分析检测和风险控制提供研究思路和技术支撑。     

鱼类样品中有机磷阻燃剂分析方法的优化研究

建立了鱼类组织样品中有机磷阻燃剂（OPFRs）的分析方法，优化了萃取条件、净化条件、液相色谱条件和质谱检测参数。确定了生物样品经冷冻干燥后，以1∶1（V/V）的正己烷/二氯甲烷混合溶液进行加速溶剂萃取，利用氨基固相萃取柱进行净化，以1∶1（V/V）的正己烷/二氯甲烷、二氯甲烷和9∶1（V/V）的二氯甲烷/甲醇为洗脱液，最后用超高效液相色谱-串联质谱同时对9种OPFRs进行定性定量检测。结果表明，各物质的基质加标回收率均为56.5%~108%，方法的测定下限为0.016~0.104 ng/g （以脂重计），满足了生物样品中OPFRs的分析检测要求。利用该方法测定了北京和广州市养殖和野生鲫鱼肌肉组织样品，主要的OPFRs同族体为磷酸三正丁酯（TNBP）、磷酸三乙基己基酯（TEHP）、磷酸三（1-氯-2-丙基）酯（TCIPP）和磷酸三氯乙基酯（TCEP），质量比为5.94~33.7 ng/g（以脂重计），显示出良好的适用性。     

微波消解-氢化物发生原子荧光法测定植物中汞和砷

采用微波消解-氢化物发生原子荧光法测定植物中汞和砷，优化了试验条件。汞在0μg／L-1．00μg／L、砷在0μg／L～20．0μg/L范围内线性良好，方法检出限汞为0．005mg／kg（以取0．1g样品消解定容至10mL计），砷为0．010mg／kg（以取0．1g样品消解定容至25mL计），植物样品测定的RSD≤4．5％，加标回收率为90．0％～107％。     

微波消解-石墨炉原子吸收法测定小麦中钴

利用微波消解-石墨炉原子吸收法检测小麦样品中的低含量钴，方法简单、操作便捷，测定结果精确度和准确度好。钴的线性回归方程为A=0．003303c＋0．004218，测定结果的相对标准偏差为4．1％（标准样品ESP-1）和1．1％（6#小麦样品），检出限为0．005mdkg。     

5 A分子筛气相色谱法测定痕量 CFC12

采用5A分子筛气相色谱电子捕获检测器测定气体样品中的CFC12,介绍了填充柱的制备方法。方法线性良好,用峰高定量略优于用峰面积,检出限为27μg/m^3（进样体积以1 mL计）,标准样品测定的RSD≤6.7%,加标回收率为92.3%～103%。     

标准曲线与工作曲线在不同分析方法中的使用

校准曲线是描述待测物质浓度或量与相应测量仪器响应值或其他指示量之间的定量关系曲线。校准曲线包括工作曲线（绘制标准曲线的溶液需与样品分析步骤完全相同）和标准曲线（标液的分析步骤有所省略，如不经过前处理等）。至于何时用标准曲线，何时用工作曲线，须经实验确定。若标液的某些前处理步骤省略后，     

废线路板粉末中二噁英含量的测定

采用加速溶剂萃取-柱层析-高分辨质谱法检测废线路板粉末样品中17种2,3,7,8-PCDD/Fs的含量。采用TRDIOXIN-5MS毛细管色谱柱(60 m×0.25 mm×0.25μm)进行分离和多离子检测(MID)模式进行检测,以保留时间和同位素特征离子丰度比进行定性,用13C标记同位素稀释内标法进行定量。结果表明,17种2,3,7,8-PCDD/Fs化合物的标准曲线的线性相关性良好,线性相关系数均大于0.999 9,该方法分析的提取内标的平均回收率范围为37.25%~82.75%,RSD8%,样品中各异构体的加标回收率为112.66%~124.26%,RSD7%。应用该方法检测废线路板粉末样品,回收率在58.67%~123.00%范围内,1,2,3,4,6,7,8-Hp CDF、1,2,3,4,6,7,8-Hp CDD、OCDF、OCDD的质量比分别为2.74、2.50、5.57、16.37 pg/g,TEQ分别为0.027 4、0.025 0、0.005 6、0.016 4 ng/kg。     

Detection and quantification of major toxigenic Microcystis genotypes in Moo-Tan reservoir and associated water treatment plant

Two molecular methods, denaturing gradient gel electrophoresis (DGGE) and quantitative real-time polymerase chain reaction (qPCR) with the Universal ProbeLibrary (UPL) probe, were developed and used for the characterization and quantification of several microcystin producers in Moo-Tan Reservoir (MTR), Taiwan and its associated water treatment plant (Shih-Men Water Treatment Plant, SMWTP). Internal transcribed spacer (ITS) sequence, a highly diversified region between the 16S rRNA and 23S rRNA genes, was used to further identify the isolated strains from MTR and also used in DGGE for the detection of the specific DNA fragments and biomarkers for 11 strains observed in MTR. These ITS-DGGE biomarkers were successfully applied to monitor the community changes of potential toxigenic Microcystis sp. over a period of five years. Two highly specific primers were combined with UPL probes to measure microcystins synthesis gene (mcyB) and phycocyanin intergenic spacer region (cpcB) concentrations in water samples. The copy concentrations of UPL-mcyB and UPL-cpcB correlated well with MC-RR concentrations/water temperature and Microcystis sp. cell numbers in the water samples, respectively. For SMWTP, toxin concentrations were low, but the DGGE bands clearly demonstrated the presence of potential microcystin producers in both water treatment plants and finished water samples. It was demonstrated that toxigenic Microcystis sp. may penetrate through the treatment processes and pose a potential risk to human health in the drinking water systems.     

Evaluation of interference to conventional and real-time PCR for detection and quantification of fungi in dust

Advances in polymerase chain reaction (PCR) have permitted accurate, rapid and quantitative identification of microorganisms in pure cultures regardless of viability or culturability. In this study, a simple sample processing method was investigated for rapid identification and quantification of fungal spores from dust samples using both conventional and real-time PCR. The proposed method was evaluated for susceptibility to interference from environmental dust samples. Stachybotrys chartarum and Aspergillus fumigatus were used as test organisms. The sensitivity of detection in pure culture was 0.1 spore DNA equivalents per PCR reaction corresponding to 20 spores ml(-1) in the sample. However, 1 spore DNA equivalent per PCR reaction corresponding to 200 spores ml(-1) in the sample was the lowest amount of spores tested without interference in dust samples spiked with spores of either fungal species. The extent of inhibition was calculated using conventional and real-time PCR reactions containing fungal spores, specific primers, specific probes (for real-time PCR) and various amounts of dust. The results indicate that the extent of inhibition by dust on PCR varies with the type and amount of dust, and number of spores. No interference in the analysis of spiked samples was detected from 0.2 mg ml(-1) of four real-life dust samples at p-value >0.05 using 2 x 10(4) spores for conventional PCR and 2 x 10(5) spores for real-time PCR. However, samples containing >0.2 mg ml(-1) real-life dust compromised the PCR assay. These results suggest the potential usefulness of a simple sample processing method in conjunction with PCR for monitoring the fungal content of aerosols collected from indoor environments.     

Rapid quantitative monitoring method for the fish spoilage bacteria Pseudomonas

Pseudomonas spp. is a group of microorganisms commonly found in fish and other fresh foods and is involved in their spoilage process. The aim of this study was to develop a rapid and accurate quantitative assay for Pseudomonas spp. in fish using real-time PCR. The assay targets the carbamoyl phosphate synthase gene (carA) with SYBR green based real-time PCR. The selectivity of the assay was confirmed using 24 Pseudomonas strains and 55 non-pseudomonad strains. A linear quantification was established over seven orders of magnitude, from 40 - 4(7) copies reaction(-1). The assay was validated on cod samples collected during two shelf life trials and showed a high degree of correlation to the plate count method (rP = 0.891) where the difference between the methods was 0.04 log(10) CFU g(-1) on average. The study shows that it is possible to quantify accurately the specific spoilage organisms belonging to the genus Pseudomonas in fish using real-time PCR. The method takes less than 5 h from sampling to results. The short detection time of the method can provide the fish industry with an important tool for quality control and processing management.     

Multiplex-PCR and PCR-RFLP assays to monitor water quality against pathogenic bacteria

In this work we developed and optimized two molecular-based approaches to monitor rapidly, sensitively and specifically bacterial pathogens from three different genera, Escherichia coli, Pseudomonas aeruginosa, and Salmonella spp., directly in waters. To achieve this aim, firstly a multiplex-PCR assay (M-PCR) was optimized using a primer pair specific for each pathogen. Secondly, as a molecular confirmatory test after isolation of the pathogens by classical microbiological methods, PCR-RFLP of their amplified 16S rDNA genes was performed. It was observed from the results that the developed M-PCR assay has significant impact on the ability to detect sensitively, rapidly and specifically the three pathogens directly in water within a short time (5 h from sampling to obtain final results), therefore it represents a considerable advancement over other known more time-consuming and less-sensitive methods for identification and characterization of these kinds of pathogens.     

火焰原子吸收分光光度法测定饮用水中铁和锰的不确定度评定

分析了测试过程中不确定度影响因素,其主要来源为标准溶液、标准曲线拟合、样品重复测定、分光光度计4部分。本次测量结果为:铁(0.232±0.012)mg/L;锰(0.092±0.004)mg/L,k=2(约95%置信水平)。     

氮、磷等环境因子对太湖微囊藻与水华鱼腥藻生长的影响

为探索太湖主要水华藻类（微囊藻与水华鱼腥藻）在多种环境因子作用下的生长变化机理，在实验室内对部分水华藻类（微囊藻、鱼腥藻）进行分离培养，研究氮、磷、温度等环境因子对水华藻类生长增殖的影响。研究表明，高水温（30℃）是微囊藻的最适生长温度；随着氮、磷浓度的提高，微囊藻的生长速率加快；低磷是鱼腥藻生长的限制因子。同时，通过野外测定的各项指标发现，当藻类密度较低时，其与总氮、总磷呈正相关。     

气质联用法测定环境空气中有机硫化物

采用 SUMMA罐采集空气样品，在预浓缩系统中经3级冷阱捕集后，用气相色谱-质谱联用技术测定环境空气中7种痕量有机硫化物。对试验条件进行优化，使得甲硫醇、乙硫醇、甲硫醚、二硫化碳、噻吩、乙硫醚和二甲二硫醚等7种有机硫化物在21．47μg/m3～336．43μg/m3范围内线性良好。试验表明，7种有机硫化物的方法检出限为0．004μg/m3～0．036μg/m3；标准气体平行测定6次结果的 RSD为2．7％～6．2％，加标回收率为92．2％～97．5％。用该方法测定实际空气样品，并与傅立叶红外光谱法测定的结果进行比对，结果令人满意。     

Monitoring toxic cyanobacteria and cyanotoxins (microcystins and cylindrospermopsins) in four recreational reservoirs (Khon Kaen, Thailand)

The toxic cyanobacterial communities of four recreational reservoirs (Bueng Kaen Nakhon, Bueng Thung Sang, Bueng Nong Khot, and Bueng See Than) in Amphur Muang, Khon Kaen Province, Thailand, were investigated. Water samples were collected via monthly sampling from June to October 2011 for the study on the diversity and density of toxic cyanobacteria and toxin quantification. The main toxic cyanobacteria present in these reservoirs were Aphanocapsa sp., Cylindrospermopsis sp., Leptolyngbya sp., Limnothrix sp., Microcystis sp., Oscillatoria sp., Planktolyngbya sp., Planktotrix sp., and Pseudanabaena sp. The dominant bloom-forming genera in the water samples from Bueng Nong Khot and Bueng See Than were Microcystis sp. and Cylindrospermopsis sp., respectively. Enzyme-linked immunosorbent assays specific for cyanotoxins were performed to detect and quantify microcystins and cylindrospermopsins, with the highest average microcystins content (0.913 μgL^?1) being found in the sample collected from Bueng Nong Khot and the highest average cylindrospermopsins content (0.463 μgL^?1) being found in the sample collected from Bueng See Than. The application of 16S rRNA analyses to cyanobacterial isolates BKN2, BNK1, BNK2, and BST1 indicated that these isolates are most closely related to Limnothrix planctonica (JQ004026) (98 % similarity), Leptolyngbya sp. (FM177494) (99 % similarity), Microcystis aeruginosa (DQ887510) (99 % similarity), and Limnothrix redekei (FM177493) (99 % similarity), respectively.     

Molecular approaches to microbiological monitoring: fecal source detection

Molecular methods are useful both to monitor natural communities of bacteria, and to track specific bacterial markers in complex environments. Length-heterogeneity polymerase chain reaction (LH-PCR) and terminal restriction fragment length polymorphism (T-RFLP) of 16S rDNAs discriminate among 16S rRNA genes based on length polymorphisms of their PCR products. With these methods, we developed an alternative indicator that distinguishes the source of fecal pollution in water. We amplify 16S rRNA gene fragments from the fecal anaerobic genus Bacteroides with specific primers. Because Bacteroides normally resides in gut habitats, its presence in water indicates fecal pollution. Molecular detection circumvents the complexities of growing anaerobic bacteria. We identified Bacteroides LH-PCR and T-RFLP ribosomal DNA markers unique to either ruminant or human feces. The same unique fecal markers were recovered from polluted natural waters. We cloned and sequenced the unique markers; marker sequences were used to design specific PCR primers that reliably distinguish human from ruminant sources of fecal contamination. Primers for more species are under development. This approach is more sensitive than fecal coliform assays, is comparable in complexity to standard food safety and public health diagnostic tests, and lends itself to automation and high-throughput. Thus molecular genetic markers for fecal anaerobic bacteria hold promise for monitoring bacterial pollution and water quality.     

固相萃取-GC/MS法测定水中16种有机氯农药

采用HLB固相萃取柱富集水样,乙酸乙酯溶剂洗脱,加入氘代菲作为内标,利用气相色谱/质谱联用法选择离子模式测定水中16种有机氯农药,优化了固相萃取条件。16种有机氯农药在5.00μg/L~250μg/L范围内线性良好,按1 L水样计算,方法最低检出限为1.4 ng/L~19.4 ng/L,相对标准偏差为3.5%~20.0%,平均加标回收率为44.7%~119%。