Design and use of group-specific primers and probes for real-time quantitative PCR

Real-time quantitative polymerase chain reaction (qPCR) has gained popularity as a technique to detect and quantify a specific group of target microorganisms from various environmental samples including soil, water, sediments, and sludge. Although qPCR is a very useful technique for nucleic acid quantification, accurately quantifying the target microbial group strongly depends on the quality of the primer and probe used. Many aspects of conducting qPCR assays have become increasingly routine and automated; however, one of the most important aspects, designing and selecting primer and probe sets, is often a somewhat arcane process. In many cases, failed or non-specific amplification can be attributed to improperly designed primer-probe sets. This paper is intended to provide guidelines and general principles for designing group-specific primers and probes for qPCR assays. We demonstrate the effectiveness of these guidelines by reviewing the use of qPCR to study anaerobic processes and biologic nutrient removal processes. qPCR assays using group-specific primers and probes designed with this method, have been used to successfully quantify 16S ribosomal Ribonucleic Acid (16S rRNA) gene copy numbers from target methanogenic and ammonia-oxidizing bacteria in various laboratory- and full-scale biologic processes. Researchers with a good command of primer and probe design can use qPCR as a valuable tool to study biodiversity and to develop more efficient control strategies for biologic processes.     

Preliminary study of groundwater denitrification using a composite membrane bioreactor

A composite membrane bioreactor (CMBR) integrating the immobilized cell technique and the membrane separation technology was developed for groundwater denitrification. The CMBR had two well mixed compartments with one filled with the nitrate- containing influent and the other with a dilute ethanol solution; the compartments were separated by the composite membrane consisting of a microporous membrane facing the influent and an immobilized cell membrane facing the ethanol solution. Nitrate and ethanol molecules diffused from the respective compartments into the immobilized cell membrane where nitrate was reduced to gaseous nitrogen by the denitrifying bacteria present there with ethanol as the carbon source. The microporous membrane was attached to one side of the immobilized cell membrane for retention of the disaggregated bacteria. Relative to the single dose of external ethanol, the two-dose supplementation produced better treatment results as evidenced by the lower concentrations of NO3--N and ethanol (as measured by total organic carbon) of the effluent. The batch treatment in CMBR removed most of the nitrate in the influent and attained a stable denitrification rate of 0.1 g·m^-2·h^-1 for most of the 96-h cycles during the 30-cycle study. The effluent was essentially free of ethanol and nitrite nitrogen.     

Single particle analysis of ambient aerosols in Shanghai during the World Exposition, 2010: two case studies

ATSI Model 3800 aerosol time-of-flight mass spectrometer (ATOFMS) was deployed for single-particle analysis in Shanghai during the World Exposition (EXPO), 2010. Measurements on two extreme cases: polluted day (1st May) and clean day (25th September) were compared to show how meteorological conditions affected the concentration and composition of ambient aerosols. Mass spectra of 90496 and 50407 particles were analyzed respectively during the two sampling periods. The ART-2a neural network algorithm was applied to sort the collected particles. Seven major classes of particles were obtained: dust, sea salt, industrial, biomass burning, organic carbon (OC), elementary carbon (EC), and NH₄-rich particles. Number concentration of ambient aerosols showed a strong anti-correlation with the boundary layer height variation. The external mixing states of aerosols were quite different during two sampling periods because of different air parcel trajectories. Number fraction of biomass burning particles (43.3%) during polluted episode was much higher than that (21.6%) of clean time. Air parcels from the East China Sea on clean day diluted local pollutant concentration and increased the portion of sea salt particle dramatically (13.3%). The large contribution of biomass burning particles in both cases might be an indication of a constant regional background of biomass burning emission. Mass spectrum analysis showed that chemical compositions and internal mixing states of almost all the particle types were more complicate during polluted episode compared with those observed in clean time. Strong nitrate signals in the mass spectra suggested that most of the particles collected on polluted day had gone through some aging processes before reaching the sampling site.     

基于希瓦氏菌合成的荧光碳点及其用于Fe3+和对硝基苯酚(p-NP)的检测

本文以Shewanella oneidensis MR-1为原料,通过一步水热法合成了荧光碳点(CDs@MR-1).采用透射电子显微镜(TEM)、X射线光电子能谱(XPS)、傅里叶红外光谱(FT-IR)、X射线衍射(XRD)和荧光光谱(PL)等手段对CDs@MR-1的形貌、结构和组成、表面基团以及光学性能进行了表征.研...     

Effects of repeated pesticide exposure on the peripheral and central nervous systems

This study was designed to compare organic farmers (comparison group) and pesticide-using farmers (pesticide group) in terms of neurobehavioral performance and sensorimotor function. The subjects were recruited in October 2004 from Chungnam Province and the neighboring Jeolla Province in western South Korea. Psychological assessment was carried out using the Wechsler Adult Intelligence Scale, the Mini-Mental State Examination, and the Bender Gestalt test. Neuroselective sensory nerve conduction threshold was measured at both left and right middle fingers by using an electrodiagnostic device to determine current perception thresholds (CPTs). Three independent CPT measurements were obtained from each test site by using sinusoidal stimuli at three frequencies. CPT testing showed no difference between the two groups. No significant differences were found in the large myelinated (2000?Hz stimulation; cutaneous touch, pressure), small myelinated (250?Hz stimulation; mechanoreceptive, pressure, temperature, fast pain) and small unmyelinated (5?Hz stimulation; polymodal nociceptive, temperature, slow pain, postganglionic sympathetic) C-fibers. Furthermore, there was no difference found between the pesticide and comparison groups in psychological variables, including intelligence quotient, orientation, memory, calculation, similarities, visual sensitivity, and psychomotor speed. In conclusion, chronic pesticide exposure has no apparent effect on either the peripheral or central nervous system in Korean farmers.     

Gliding arc plasma processing for decomposition of chloroform

The decomposition of chloroform (CHCl₃) diluted in air was studied. The experiment was carried out by using a gliding arc plasma. Different values of initial concentrations of chloroform, total gas flow rates, and input power frequencies have been used to investigate this effects on the conversion reaction products both qualitatively and quantitatively. Experimental results indicate that the maximum conversion of chloroform was 97% at a total gas flow rate of 180?L?h^?1 containing 1% chloroform. Using air as carrier gas, decomposition of CHCl₃ produces CCl₄, CO₂, CO, and Cl₂ as the main products. Small amounts of HCl and COCl₂ are also detected. Liquid products were also produced.     

The estimation of bioconcentration factors of aromatic hydrocarbons by high‐performance liquid chromatography

The determination of Bioconcentration Factors (BCF) via HPLC capacity factors (k') has been studied, including the effect of column type and mobile phase composition on the correlation between log BCF and log k’. Values of BCF correlate well with the phenylsilica column capacity factors. The phenylsilica column followed by C18 column give better correlations than either C8 or C2 column. The use of HPLC with phenylsilica as stationary phase and methanol‐water as mobile phase in the prediction of BCF can be at least as good or better than the use of n‐octanol/water partitioning system. There are no significant differences in the correlations between log k’ and log BCF with the changes of methanol concentration in the mobile phase, and it seems that a high proportion of methanol in the eluent is required to obtain good results.     

Determination and QSPR analysis of log Kow and log Koc for fluorine containing benzene derivatives

Octanol‐water partition coefficients (Kow) and soil organic carbon sorption coefficients (Koc) were determined for 14 fluorinated benzene derivatives. Quantitative structure‐property relationships were developed using molecular connectivity indices and quantum chemical parameters to analyze the most significant factors influencing these physico‐chemical properties of the compounds. The substitution by F in benzene derivatives has greater influence on Koc than on Kow.     

利用甘油脱水酶基因构建产3-羟基丙醛工程菌及其表达比较

3-羟基丙醛(3-HPA)是一种重要的化工产品,可由甘油经甘油脱水酶作用后生成.为获得产3-HPA基因工程菌,在已构建含甘油脱水酶基因及其激活因子大亚基质粒pEtac-dhaB-gdrA的基础上,构建了包含小亚基gdrB激活因子的重组质粒pEtac-dhaB-gdrA-gdrB.利用大肠杆菌通用tac启动子将该质粒在不同Escherichia coli BL21、DH5a及JM109中进行表达.阳性转化子经IPTG诱导后,提取总RNA,以cDNA为模板进行RT-PCR发现,目标基因在不同宿主都能较好转录.SDS-PAGE、酶活测定和3-HPA浓度测定结果表明,目标蛋白表达存在差异;酶活分别为4.7(±0.44)、3.5(±0.95)、8.1(±0.66)U/mg;发酵液中3-HPA的含量分别为0.012(±0.0044)、0.014(±0.003)、0.375(±0.018)g L-1,重组E.coli JM109/pEtac-dhaB-gdrA-gdrB具有较好的甘油脱水酶基因表达和产3-HPA性能.该基因工程菌与克雷伯氏菌(Klebsiella pneumoniae)相比,发酵副产物明显较少,有利于后期提取,为生产3-HPA提供了一条新思路.