人类特异性粪厌氧菌基因标记物实时荧光定量PCR检测方法的建立和应用

针对人类粪便污染,选择来源于拟杆菌和双歧杆菌16S rRNA的基因片段作为标记物,分别建立了相应的实时荧光定量PCR检测方法.选取不同来源的粪便样品进行检测,证实了引物的特异性.回收纯化从污水中扩增所得的目标PCR片段,经过连接转化,筛选阳性克隆提取其重组质粒作为实时荧光定量PCR的标准品.通过检测一系列梯度稀释的标准品,确定了拟杆菌基因标记物和双歧杆菌基因标记物定量PCR检测方法分别在1.98×102～1.98×107 copy/μL和2.12×101～2.12×107 copy/μL范围内具有良好的线性关系.     

实时荧光定量PCR对A2/O短程硝化系统内氨氧化菌的定量分析

通过控制好氧区低DO浓度以及缩短好氧实际水力停留时间(actual hydraulic retention time,AHRT),在处理低C/N比实际生活污水的A2/O工艺中,成功启动并维持了短程硝化反硝化;系统亚硝酸盐积累率稳定维持在90%左右,氨氮去除率在95%以上。通过提取富集氨氧化菌(ammonia oxidizing bacteria,AOB)的基因组DNA,经两次常规PCR扩增和琼脂糖凝胶电泳,以纯化回收的DNA扩增片段作为实时荧光定量PCR检测AOB数量的DNA标准品,建立了检测AOB数量的实时荧光定量PCR标准曲线。利用实时荧光定量PCR技术比较了A2/O系统在不同运行条件及亚硝酸盐积累率情况下AOB菌群数量。结果表明,随着系统亚硝酸盐积累率的上升,系统内AOB菌群数量也大幅上升。全程硝化和短程硝化时,系统内的AOB菌群数量分别为5.28×109cells/g MLVSS和3.95×1010cells/g MLVSS。此外,亚硝酸盐积累率的下降相对于AOB菌群数量的下降有一定的滞后性。     

城市污水处理工艺对宿主特异性标记物和肠道病原菌的去除效果

采用自制小型反应器模拟实验和实时荧光定量PCR检测方法,针对指示人类、犬类粪便污染的宿主特异性标记物,以及病原性大肠埃希氏菌、沙门氏菌和志贺氏菌等典型的肠道病原菌,分别考察了序批式活性污泥法(SBR)、混凝、过滤工艺对它们的去除效果。研究结果表明,SBR工艺的去除效果优于混凝和过滤工艺,对标记物和病原菌毒力基因的去除率都在93.5%以上。SBR工艺中初期短时间的搅拌即可去除大部分标记物和病原菌。在这3种处理工艺中,宿主特异性标记物与肠道病原菌毒力基因的去除率具有显著相关性,这说明宿主特异性标记物能够在一定程度上反映污水处理过程中肠道病原菌的含量。     

水中抑制物对定量PCR与膜过滤法检测E.coli的影响

针对水中病原微生物的污染,选择大肠埃希氏菌E.coli作为病原示踪剂,以E.coli染色体上β-葡萄糖醛酸酶uid A目的基因建立了SYBR Green实时荧光定量PCR的检测方法。该定量PCR方法灵敏度高,检测限可达104CFU/L,线性关系良好,相关系数为R2=0.999。研究表明,定量PCR与菌液浓度呈显著正相关R2=0.935。通过人工投加腐殖酸、COD,研究了水中抑制物对定量PCR和膜过滤MF培养法的影响。结果显示,腐殖酸可使E.coli在培养皿上的菌落变小聚集,且显色不明显,当腐殖酸量为20 mg/L时,浓度为50 CFU/100 m L的E.coli完全受到抑制,没有菌落出现。腐殖酸对PCR扩增的抑制作用明显,当腐殖酸浓度增加到10 mg/L,定量PCR检测结果基因拷贝数减少约1 log,增加到20 mg/L定性PCR检测结果为阴性。     

采用RT-PCR法解析餐厨垃圾厌氧消化微生物群落多样性

在中温条件下进行实验室规模的连续式餐厨垃圾厌氧消化试验。采用实时荧光定量PCR(RT-PCR)技术对餐厨垃圾单相厌氧消化失衡条件下系统内古菌和产甲烷菌的数量变化规律进行探析。结果表明,在厌氧消化系统从稳定期到抑制期的过程中,古菌和产甲烷菌的基因浓度都明显降低,其降幅达到70%~80%。2次冲击负荷试验过程中,产甲烷菌基因浓度降幅都大于古菌,说明在系统抑制过程中产甲烷菌相对于古菌来说受到的影响更大。当厌氧消化系统出现抑制状态时,产甲烷菌不能适应环境变化而大量死亡,导致系统平衡被破坏。     

水中肠道病毒检测方法的分析评价

采用Hep-2细胞分离培养水中的肠道病毒,根据肠道病毒5’-UTR核酸的保守区,建立了一种细胞培养与实时荧光定量PCR(ICC-RT-q PCR)联合检测感染性肠道病毒的方法。对RT-q PCR、TCID50和ICC-RT-q PCR 3种检测方法进行灵敏度、相关性分析,评价RT-q PCR、ICC-RT-q PCR用于估计水中感染性病毒含量的可行性。结果表明,RT-q PCR方法高估了水中病毒的感染性。肠道病毒低浓度(4.4×10-1~4.4×103TCID50/m L)时TCID50与ICC-RT-q PCR线性关系良好,相关系数R2=0.99。水样经浓缩,ICC-RT-q PCR检测病毒含量为1.0×103~3.2×104copies/L,估计含量为3~30 TCID50/L,与病毒感染性11~29 TCID50/L结果相近,检出率为83.3%。高于TCID50的检测结果(33.3%)。因此,ICC-RT-q PCR方法快速、灵敏,可对水中感染性肠道病毒进行准确的定量分析。     

紫外与次氯酸钠消毒效果及影响因素研究

选取大肠埃希氏菌(Escherichia coli)为示踪菌种,研究了紫外消毒和次氯酸钠消毒的灭活效果(用对数灭活率来衡量),进行了实时荧光定量聚合酶链式反应(PCR)检测,同时考察了浊度、Fe3+浓度、有机物对紫外消毒的灭活效果影响,以及pH、氨氮浓度对次氯酸钠消毒的灭活效果影响。结果表明:(1)紫外消毒和次氯酸钠消毒对大肠埃希氏菌均有较好的灭活效果。紫外辐射剂量为15mJ/cm2时即可达到4.55的对数灭活率;次氯酸钠投加量为2.5mg/L,消毒时间30min即可100%灭活。(2)当紫外辐射剂量为15mJ/cm2时,浊度、Fe3+浓度增加或投加腐殖酸均可使紫外消毒的灭活效果变差。(3)pH升高或者氨氮浓度增大均会导致次氯酸钠消毒的灭活效果变差。     

土霉素生产废水生物处理装置中微生物群落结构特征解析

抗生素对细菌具有强抑制作用,从而会影响废水生物处理系统中的微生物群落结构。在用定量PCR方法对土霉素生产废水处理装置(进水中土霉素浓度为1 662.1±248.6μg/L)中的细菌16S rRNA基因和真菌18S rRNA基因的含量进行比较的基础上,利用16S rRNA基因克隆文库方法对污泥中的细菌群落结构进行了详细解析。定量PCR结果显示,土霉素废水活性污泥中真菌(18S rRNA)/细菌(16S rRNA)基因的拷贝数比例高达1.2,明显较高于非抗生素肌苷废水活性污泥中的比例1.52×10-6,表明真菌对于土霉素废水中有机物的去除可能发挥重要作用。细菌克隆文库分析结果显示,Alpha变形菌和Beta变形菌是主要的优势菌,比例分别为23.7%和22.0%,其次是酸杆菌(17.0%)和拟杆菌(11.9%)。     

A/O工艺膜生物反应器处理生活污水的脱氮特性及硝化菌群的分子检测

采用A/O工艺膜生物反应器(MBR),以生活污水为处理对象,考察了系统的脱氮特性,并采用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)与荧光原位杂交(FISH)技术对系统中硝化菌群进行了分子检测.结果表明,A/O工艺MBR处理生活污水,TN去除率在85%左右,NH3-N去除率在95%以上;DGGE图谱显示,随着系统运行时间的延长,硝化菌群数量逐渐增加,并且不同菌种的丰度也发生了变化;FISH检测显示,系统中硝化菌的优势菌种为氨氧化菌和亚硝酸氧化菌.应用Motic Fluo 1.0软件对FISH结果进行分析,结果显示,系统运行初期到末期,氨氧化菌占硝化菌的比例一直保持在25%左右;亚硝酸氧化菌占硝化菌的比例由系统运行初期的35%逐渐增加到系统运行末期的55%左右.     

人工湿地中抗生素抗性大肠杆菌和抗性基因的去除与分布

抗生素的滥用导致抗生素抗性菌和抗性基因随生活污水和养殖废水的排放在环境中肆意散播,其去除及环境行为越来越受到关注。采用K-B纸片法测定了9套不同工艺构型模拟人工湿地中大肠杆菌对7种抗生素的抗性率,并应用多重PCR检测磺胺类sul1、2、3与四环素tetA、B、C、D抗性基因,探究人工湿地对抗性菌的去除效率及抗性菌、抗性基因的分布规律。结果显示,人工湿地能有效去除污水中70%左右的抗性大肠杆菌,有效降低了细菌抗性的传播风险;共计分离出535株大肠肝菌中有378株对一种以上抗生素有抗性性,以四环素、磺胺类和氨苄西林抗性率最高,达到25%以上,其他4种抗性率较低,不足20%;2种抗性基因的检出率都在70%以上;对不同采样点大肠杆菌的抗性性及抗性基因的比较发现,各部分大肠杆菌的抗性水平、多重抗性指数(MRI)以及抗性基因(sul、tet)检出率和组合数表现出:基质≥出水>进水,推测抗性菌被湿地基质截留,在基质生物膜上发生抗性基因的重组,并释放抗性菌,提高了出水中抗性水平和抗性基因检出率。     

Identification and quantitative detection of Legionella spp. in various aquatic environments by real-time PCR assay

In this study, a SYBR green quantitative real-time PCR was developed to quantify and detect the Legionella spp. in various environmental water samples. The water samples were taken from watershed, water treatment plant, and thermal spring area in Taiwan. Legionella was detected in 13.6 % (24/176), and the detection rate for river water, raw drinking water, and thermal spring water was 10, 21.4, and 16.6 %, respectively. Using real-time PCR, concentration of Legionella spp. in detected samples ranged between 9.75?×?10⁴ and 3.47?×?10⁵?cells/L in river water, 6.92?×?10⁴ and 4.29?×?10⁵?cells/L in raw drinking water, and 5.71?×?10⁴ and 2.12?×?10⁶?cells/L for thermal spring water samples. The identified species included Legionella pneumophila (20.8 %), Legionella jordanis (4.2 %), Legionella nautarum (4.2 %), Legionella sp. (4.2 %), and uncultured Legionella sp. (66.6 %). The presence of L. pneumophila in aquatic environments suggested a potential public health threat that must be further examined.     

The StepOne real-time polymerase chain reaction detection of Salmonella sp., Salmonella enterica ser. typhimurium and enteritidis in milk and meat

The aim of this study was to follow contamination of ready to eat milk and meat products with Salmonella spp. by using the StepOne real-time polymerase chain reaction (PCR). Classical microbiological methods for detection of foodborne bacteria involve the use of pre-enrichment and/or specific enrichment, following isolation of bacteria in solid media and the final confirmation by biochemical and/or serological tests. We used the PrepSEQ Rapid Spin Sample Preparation Kit for isolation of DNA and MicroSEQ® Salmonella spp. Detection Kit for pursuance of the real-time PCR (Applied Biosystems). In samples without incubation we detected strain of Salmonella sp. in 5 out of 25 samples (swabs), as well as in the internal positive control (IPC), which was positive in all samples. This StepOne real-time PCR assay is extremely useful for any laboratory equipped by real-time PCR. It is a fast, reproducible, simple, specific and sensitive way to detect nucleic acids, which could be used in clinical diagnostic tests in the future. Our results indicated that real-time PCR assay developed in this study could sensitively detect Salmonella spp. in ready-to-eat food. This could prevent infection caused by Salmonella, and also could benefit food manufacturing companies by extending their product's shelf-life as well as saving the cost of warehousing their food products while awaiting pathogen testing results.     

In vitro assessment of corticosteroid effects of eight chiral herbicides

Abstract

Information regarding the enantioselective endocrine disruption of chiral herbicides is scarce. This study assessed the disrupting effects of eight typical chiral herbicides on corticosteroids (including glucocorticoids and mineralocorticoids). Enantioselectivity of eight chiral herbicides were evaluated for their agonistic/antagonistic effects on glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) with CHOK1 cell line using reporter gene assay. Their influence on the production of corticosteroids were further investigated in H295R cell line using enzyme-linked immunosorbent assay (ELISA). None of the racemates or enantiomers of eight chiral herbicides exhibited GR or MR agonistic activity at non-cytotoxic concentrations. However, rac-propisochlor and S-imazamox antagonized cortisol-induced transactivation of GR by 21.79% and 38.73% at the concentration of 1.0?×?10^?7 M and 1.0?×?10^?6 M, respectively, and R-napropamide remarkably attenuated aldosterone-induced MR transactivation by 68.78% at 1.0?×?10^?6 M. The secretion of cortisol was significantly restrained after treated with 1.0?×?10^?6 M rac-propisochlor and rac-/R-napropamide at the concentration of 1.0?×?10^?6 M by 26.49%, 30.10% and 35.27%, respectively, while this glucocorticoid was remarkably induced by 1.0?×?10^?5 M rac-diclofop-methyl and its two enantiomers at the concentration of 1.0?×?10^?5 M by 75.60%, 100.1% and 68.78%, respectively. Exposure to rac-propisochlor (1.0?×?10^?6 M), S-diclofop-methyl (1.0?×?10^?5 M) or rac-/S-/R- acetochlor (1.0?×?10^?6 M) and rac-/S-/R-lactofen (1.0?×?10^?6 M) inhibited the secretion of aldosterone by approximately 40%. Our findings suggested that chiral herbicides disrupted corticosteroid homeostasis in an enantioselective way. Therefore, more comprehensive screening is required to better understand the ecological and health risks of chiral pesticides.     

Monitoring of genetically modified Escherichia coli in laboratory wastewater

Containment of genetically modified (GM) microorganisms such as Escherichia coli is a legal requirement to protect the environment from an unintended release and to avoid horizontal gene transfer (HGT) of recombinant DNA to native bacteria. In this study, we sampled the laboratory wastewater (LWW) at a large Swiss university from three sources over 2 years and cultured ampicillin-resistant, presumptive GM E. coli. From a total of 285 samples, 127 contained presumptive GM E. coli (45%) at a mean concentration of 2.8 × 10² CFU/ml. Plasmid DNA of 11 unique clones was partially or entirely sequenced. All consisted of cloning vectors harboring research-specific inserts. To estimate the chance of HGT between GM E. coli and native bacteria in LWW, we identified taxa representative for the bacterial community in LWW using 16S rRNA amplicon sequencing and measured conjugation frequencies of E. coli with five LWW isolates. At optimal conjugation conditions, frequencies were between 3.4 × 10⁻³ and 2.4 × 10⁻⁵. Given the absence of transferable broad-host range plasmids and suboptimal conjugation conditions in the LWW system, we conclude that the chance of HGT is relatively low. Still, this study shows that the implementation of robust containment measures is key to avoid the escape of GM microorganisms.

     

Effect of influent C/N ratio on N2O emissions from anaerobic/anoxic/oxic biological nitrogen removal processes

The problem of producing strong greenhouse gas of nitrous oxide (N₂O) from biological nitrogen removal (BNR) process in wastewater treatment plants (WWTP) has elicited great concern from various sectors. In this study, three laboratory-scale wastewater treatment systems, with influent C/N ratios of 3.4, 5.4, and 7.5, were set up to study the effect of influent C/N ratio on N₂O generation in anaerobic/anoxic/oxic (A²O) process. Results showed, with the increased influent C/N ratio, N₂O generation from both nitrification and denitrification process was decreased, and the N₂O-N conversion ratio of the process was obviously reduced from 2.23 to 0.05%. Nitrification rate in oxic section was reduced, while denitrification rate in anaerobic and anoxic section was elevated and the removal efficiency of COD, NH₄ ⁺-N, TN, and TP was enhanced in different extent. As the C/N ratio increased from 3.4 to 7.5, activities of three key denitrifying enzymes of nitrate reductase, nitrite reductase, and nitrous oxide reductase were increased. Moreover, microorganism analysis indicated that the relative abundance of ammonium-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were positively correlated with N₂O generation, which was reduced from (8.42 ± 3.65) to (3.61 ± 1.66)% and (10.38 ± 4.12) to (4.67 ± 1.62)%, respectively. NosZ gene copy numbers of the A²O system were increased from (1.19 ± 0.49) × 10⁷ to (2.84 ± 0.54) × 10⁸ copies/g MLSS with the influent C/N ratio elevated from 3.4 to 7.5. Hence, appropriate influent C/N condition of A²O process could optimize the microbial community structure that simultaneously improve treatment efficiency and decrease the N₂O generation.

     

Determination of Room Temperature OH Rate Constants for Acetylene,Ethylene Dichloride,Ethylene Dibromide,p-Dichlorobenzene and Carbon Disulfide

A relative rate procedure was used to measure hydroxyl rate constants at room temperature in the presence of oxygen. The photolysis of methyl nitrite in the presence of nitric oxide was used to generate OH radicals. The rate of loss of the test compounds was measured relative to that of ethane (k_OH = 2.74 × 10^-13 cm³ molec^-1 s^-1). The rates obtained at 297 ± 2 K are: acetylene = (7.8 ± 1.6) × 10^-13 cm³ molec^-1 s^-1,1,2-dichloroethane (2.8 ± Q.6) × 10^-13 cm³ molec^-1 s^-1, 1,2-dibromoethane (2.4 ± 0.5) × 10^-13 cm³ molec^-1 s^-1, p-dichlorobenzene (4.3 ± 0.9) × 10^-13 cm³ molec^-1 s^-1 and carbon disulfide (29 ± 6) × 10^-13 cm³ molec^-1 s^-1. Under a proposed EPA rule, this OH rate determination procedure could be used to determine if a given volatile organic will be subject to control for reduction of photochemical ozone.     

Biopesticide and formulation processes based on starch industrial wastewater fortified with soybean medium

Abstract

The aim of this study was to produce Bacillus thuringiensis-based biopesticide using starch-producing industry wastewater (SIW) fortified with soybean medium and optimize the formulated product using different adjuvants. This study was necessary as low endotoxin concentration is obtained in formulated biopesticide when SIW alone is used as fermentation medium. The fermentation runs were conducted using SIW alone and SIW fortified with 25% soybean (w/v) medium in 2000?L and 150?L bioreactor, respectively. SIW supplemented with soybean medium showed an increase in cell count (from 1.95?×?10⁸ to 1.65?×?10⁹ CFU mL^–1), spore synthesis (from 1.5?×?10⁸ to 1.35?×?10⁹ CFU mL^–1) and endotoxin concentration (from 436 to 1170?μg mL^–1) when compared to SIW medium alone. The fermented broth was concentrated using continuous centrifugation and adjuvants were added for biopesticide formulation in order to enhance its resistance against UV rays and rainfastness. Entomotoxicity of the formulation produced using fermented broth of SIW fortified with soybean (38,000?IU μL^–1) was higher than that obtained by SIW medium alone (21,000?IU μL^–1), commercial biopesticide Foray 76B (20,000?IU μL^–1) and Btk sander’s (12,500?IU μL^–1).