小麦高分子量谷蛋白亚基基因的PCR及AS-PCR标记

选用高分子量麦谷蛋白亚基(HMW-GS)在Glu-D1位点已知的 26个普通小麦品种,包括 15个 (TriticumaestivumL. )川育系列品种及其 11个亲本,根据其亚基Dx5和Dy10基因的特异序列设计引物.通过对Dx5基因的PCR扩增及对Dy10基因的AS-PCR扩增,结果表明,所设计的引物对Dx5和Dy10都能扩增出特异条带,而携有Dx5基因的材料同时也携有Dy10基因.说明用所设计的引物可快速有效的鉴定出普通小麦中是否带有优质的 5 10亚基.图 4参 15     

豆豉纤溶酶在枯草杆菌WB800中的高水平表达

一个来源于豆豉产生菌DC12的新的纤溶酶基因序列被克隆测序.为了实现在枯草杆菌中高水平表达豆豉纤溶酶,将来源于枯草杆菌168的重叠启动子P43序列以及转录终止信号序列通过重叠延伸融合,将融合序列克隆到大肠杆菌-枯草杆菌穿梭载体pBE3中,构建了pBEP43载体.将豆豉纤溶酶基因插入到载体pBEP43后转化枯草杆菌WB800.结果表明,在P43启动子驱动下,豆豉纤溶酶基因在重组菌中的对数生长期和平衡期均获得了表达且分泌到培养基中.重组菌经培养后上清液中的纤溶酶活性最高达1270U/mL,是豆豉纤溶酶基因在自身启动子驱动下表达量的1.87倍和野生菌株枯草杆菌DC12表达量的4.1倍.图5参16     

葡枝根霉(Rhizopus stolonifer)YF6脂肪酶基因的克隆及其序列分析

筛选并鉴定了1株高产脂肪酶菌株葡枝根霉YF6,并采用简并PCR等分子技术从中克隆到1个新的脂肪酶基因lipRs及其对应的全长cDNA,序列分析表明,该基因编码区全长1173bp,不含内含子序列,编码1段由26个氨基酸残基组成的信号肽和1个由365个氨基酸残基组成的成熟蛋白,该成熟蛋白计算分子量(Mr)为39268.27,等电点为pH7.66,有5个可能的N-糖基化位点,含有脂肪酶特征序列GHSLGGA,与来自米根霉(Rhizopus oryzae)的脂肪酶(AAF32408)同源性最高,一致性为83%,相似性为89%.该基因序列已提交GenBank,登录号为DQ139862.     

PCR技术对水中病原体的检测

水中有病毒、细菌、原生动物、蠕虫等多种病原体。常规的检测方法是用大肠菌作为病原体的指示生物,但是大肠菌的生存特性与病毒、原生动物差异较大,难以指示病毒、原生动物的存在与否。对每一种病原体单独进行检测,费时、费力。由于PCR技术具有快速、灵敏、特异的特点,使得它对于水中病原体的检测具有较高的应用价值。     

Verification of Bacteroidales 16S rRNA markers as a complementary tool for detecting swine fecal pollution in the Yangtze Delta

To correctly assess and properly manage the public health risks associated with exposure to contaminated water, it is necessary to identify the source of fecal pollution in a watershed. In this study, we evaluated the efficacy of our two previously developed real time-quantitative PCR (qPCR) assays for the detection of swine-associated Bacteroidales genetic markers (gene 1–38, gene 3–53) in the Yangtze Delta watershed of southeastern China. The results indicated that the gene 1–38 and 3-53 markers exhibited high accuracy (92.5%, 91.7% conditional probability, respectively) in detecting Bacteroidales spp. in water samples. According to binary logistic regression (BLR), these two swine-associated markers were well correlated (P < 0.05) with fecal indicators (Escherichia coli and Enterococci spp.) and zoonotic pathogens (E. coli O157: H7, Salmonella spp. and Campylobacter spp.) in water samples. In contrast, concentrations of conventional fecal indicator bacteria (FIB) were not correlated with zoonotic pathogens, suggesting that they are noneffective at detecting fecal pollution events. Collectively, the results obtained in this study demonstrated that a swine-targeted qPCR assay based on two Bacteroidales genes markers (gene 1–38, gene 3–53) could be a useful tool in determining the swine-associated impacts of fecal contamination in a watershed.     

林可霉素菌渣堆肥抗生素抗性基因变化分析

为了研究抗生素菌渣堆肥过程中抗生素抗性基因（ARGs）的变化情况,以林可霉素菌渣-糠醛渣堆肥为研究对象,以污泥-糠醛渣堆肥为对照.运用荧光定量PCR技术检测到了堆肥过程中lnuA-01、sul1、ermA、ermB、ermC等5种林可霉素抗性基因和整合子基因intI1的变化情况.结果表明,堆肥化处理可以降解99%的林可霉素残留,两者堆肥ARGs总量绝对丰度均有较大增加,而相对丰度降低5%~22%.同时发现林可霉素菌渣堆肥有助于intI1的富集,表明林可霉素菌渣堆肥存在生态风险.冗余分析显示,ARGs变化受环境因子影响严重,影响顺序为pH值 > 林可霉素残留 > 温度 > C/N.     

升级A/O工艺污水处理系统中抗生素抗性基因的分布与去除研究

针对北方某采用升级A/O工艺的生活污水处理厂,使用实时荧光定量PCR技术,探究污水厂中ARGs的分布及各处理工艺段对ARGs的去除效果.结果表明：四环素抗性基因（tetA、tetC和tetM）、磺胺抗性基因（sul1和sul2）、大环内酯抗性基因（ermA和ermF）和喹诺酮抗性基因（parC和gyrA）在污水和污泥中均被检出.污水厂进水中ARGs的绝对丰度为2.65×10³~1.01×10⁶ copies·mL^-1,升级A/O工艺未能有效削减ARGs,出水中ARGs的绝对丰度为9.22×10³~1.15×10⁶ copies·mL^-1,污泥中ARGs的绝对丰度为8.07×10⁷~2.65×10¹¹ copies·g^-1.深度处理工艺对ARGs的去除效率对比结果显示,生物活性炭工艺对ARGs的削减效果优于紫外消毒.     

产毒微囊藻mcyA基因荧光定量PCR方法的建立

针对微囊藻毒素合成酶基因mcyA探索一种适用于自然水样中微囊藻毒素产毒潜能检测的TaqMan实时荧光定量PCR方法.该方法从铜绿微囊藻FACHB-1279中的mcyA基因中扩增出目的基因片段,用TA克隆方法将基因片段插入Allel TA vector (ABP-CETAVEC020)中来制备质粒标准晶.通过不同连续稀释...     

基于PMA-定量PCR选择性检测技术的病原菌消毒特性研究

建立了一种核酸染料propidium monoazide(PMA)与定量PCR技术联合选择性检测活性病原菌的技术(PMA-qPCR),以大肠杆菌作为模式菌,研究了氯和一氯胺消毒对病原菌的灭活特性.结果表明,PMA染料能够分别去除99.94%和99.99%的来自非活性大肠杆菌和沙门氏菌的DNA,PMA-qPCR技术能够有效区分活性菌与非活性菌;PMA-qPCR技术得到的氯和一氯胺消毒对大肠杆菌的灭活曲线符合一级动力学方程,灭活速率常数分别为 2.24 L·(mg·min)^-1和0.0175 L·(mg·min)^-1,低于平板培养法得到的灭活速率常数;当大肠杆菌的去除率达到99%时,采用PMA-qPCR技术检测需要的ct值相比于平板培养法从0.6 mg·L^-1·min上升到0.9 mg·L^-1·min(氯消毒)和从20 mg·L^-1·min上升到超过100 mg·L^-1·min(一氯胺消毒);随着ct值的升高,常规qPCR的检测结果基本不变,因此常规qPCR不能够反映氯和一氯胺消毒对病原菌的灭活效果.作为一种新的表征消毒特性的检测技术,PMA-qPCR技术有助于更为准确地评价氯和一氯胺消毒对病原菌的灭活效果.     

Purification of total DNA extracted from activated sludge

Purification of the total DNA extracted from activated sludge samples was studied. The effects of extraction buffers and lysis treatments (lysozyme, sodium dodecyl sulfate (SDS), sonication, mechanical mill and thermal shock) on yield and purity of the total DNA extracted from activated sludge were investigated. It was found that SDS and mechanical mill were the most effective ways for cell lysis, and both gave the highest DNA yields, while by SDS and thermal shock, the purest DNA extract could be obtained. The combination of SDS with other lysis treatment, such as sonication and thermal shock, could apparently increase the DNA yields but also result in severe shearing. For the purification of the crude DNA extract, polyvinyl polypyrrolidone was used for the removal of humic contaminants. Cetyltrimethyl ammonium bromide, potassium acetate and phenol/chloroform were used to remove proteins and polysaccharides from crude DNA. Crude DNA was further purified by isopropanol precipitation. Thus, a suitable protocol was proposed for DNA extraction, yielding about 49.9 mg (total DNA)/g volatile suspended solids, and the DNA extracts were successfully used in PCR amplifications for 16S rDNA and 16S rDNA V3 region. The PCR products of 16S rDNA V3 region allowed the DGGE analysis (denatured gradient gel electrophoresis) to be possible.