应用qPCR方法检测中国近海塔玛亚历山大藻复合种的研究

亚历山大藻属的部分藻种(Alexandrium spp.)能够产生麻痹性贝毒毒素(PST),是重要的有害藻华(HAB)原因种.由于亚历山大藻属中的有毒和无毒藻种形态相似,且海水中亚历山大藻的细胞密度通常很低,因此,高灵敏度、高特异性的分子生物学方法在亚历山大藻检测方面具有重要的应用前景.塔玛亚历山大藻和链状亚历山大藻是中国近海主要的有毒亚历山大藻藻种,大多属于塔玛亚历山大藻复合种第四类核糖体型(GroupⅣ,以往称为“亚洲温带核糖体型”).因此,本研究尝试将实时荧光定量PCR(qPCR)方法用于我国近海塔玛亚历山大藻复合种(GroupⅣ)的快速检测,并对方法的特异性和灵敏度进行了检验.研究表明,所建立的qPCR方法能够特异性地检测我国近海不同海域分离的塔玛亚历山大藻复合种(GroupⅣ),方法具有良好的线性响应关系和较高的灵敏度,最低可检出5个藻细胞,具有良好的应用前景.然而,实验也发现,自我国近海分离的塔玛亚历山大藻复合种的不同藻株之间,以及处于不同生长阶段的藻细胞之间,qPCR检测结果存在显著差异,反映了目标藻核糖体RNA基因拷贝数的差异和变化对qPCR检测结果的影响.因此,建议在将qPCR方法用于不同海域亚历山大藻样品检测时,应采用该海域分离的藻株专门构建标准工作曲线,以减小定量分析误差.     

Rapid prenatal diagnosis of Duchenne muscular dystrophy with gene duplications by ion-pair reversed-phase high-performance liquid chromatography coupled with competitive multiplex polymerase chain reaction strategy

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by various mutations in the dystrophin gene. Rapid prenatal diagnosis of DMD with gene duplications is difficult due to limitation in gene dosage determination and the requirement for a known disease-causing mutation in the pedigree to achieve a rapid and accurate diagnosis. We report, here, a case with rapid prenatal diagnosis of DMD-affected male with gene duplications in the absence of a known disease-causing variation in the pedigree by using ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) coupled with competitive multiplex polymerase chain reaction (PCR) protocol. In cases with clinical diagnosis of DMD/BMD, this test should identify greater than 92% of disease-causing DNA variants. The postnatal genetic diagnosis of this case and the same disease-causing mutations subsequently identified in other members of the pedigree confirmed the accuracy of competitive multiplex PCR/IP-RP-HPLC assay in direct prenatal diagnosis of DMD. Copyright © 2007 John Wiley & Sons, Ltd.     

Whole genome amplification from a single cell: a new era for preimplantation genetic diagnosis

Preimplantation genetic diagnosis (PGD) is a technique used for determining the genetic status of a single cell biopsied from embryos or oocytes. Genetic analysis from a single cell is both rewarding and challenging, especially in PGD. The starting material is very limited and not replaceable, and the diagnosis has to be made in a very short time. Different whole genome amplification (WGA) techniques have been developed to specifically increase the DNA quantities originating from clinical samples with limited DNA contents. In this review, currently available WGA techniques are introduced and, among them, multiple displacement amplification (MDA) is discussed in detail. MDA generates abundant assay-ready DNA to perform broad panels of genetic assays through its ability to rapidly amplify genomes from single cells. The utilization of MDA for single-cell molecular analysis is expanding at a high rate, and MDA is expected to soon become an integral part of PGD. Copyright © 2007 John Wiley & Sons, Ltd.     

First application of preimplantation genetic diagnosis to neurofibromatosis type 2 (NF2)

Neurofibromatosis type 2 (NF2) is a dominantly inherited cancer predisposition syndrome that is caused bymutations in the NF2 gene. We report here the first clinical preimplantation genetic diagnosis (PGD) forNF2. A protocol was developed to simultaneously amplify the mutation and a single nucleotide polymorphism (SNP) located within the gene. The mutation and polymorphism were analysed by simultaneous fluorescent single-strand conformation polymorphism (SSCP) on an automated DNA sequencer. The mutation, carried by the male partner, was a single base pair substitution affecting a splice site in intron 4 of the gene. The female partner was infertile due to polycystic ovary syndrome and would require IVF to conceive. The couple was found to be informative at a linked intragenic SNP situated in the 5′ untranslated region of the gene. The SNP was included in the assay to reduce the risk of misdiagnosis due to allele dropout (ADO). The couple underwent three cycles of treatment during which a total of 43 blastomeres were biopsied from 31 embryos. Amplification at both loci was obtained in 35 cells (81%). A total of five embryos were transferred, two in the first cycle, two in the second and one in the third. No pregnancy ensued. The results of the diagnoses indicated that, in this couple, the inheritance of the mutation may be non-Mendelian. Out of a total of 32 embryos tested only four were found not to carry the mutation. The reasons for this apparent skew remain unknown. Copyright © 2002 John Wiley & Sons, Ltd.     

废水处理系统中抗生素抗性基因分布特征

废水处理厂被认为是抗生素抗性基因(ARGs)的重要污染源.为探究抗性基因在进水状况复杂的废水处理厂沿程的分布变化特征,选取以生产抗生素为主导行业的某化工园区废水处理厂,使用实时荧光定量PCR对废水处理厂沿程ARGs的种类、丰度变化进行研究.结果表明,废水处理厂水体中检出16种ARGs,四环素类、磺胺类ARGs为废水处理厂中占主导的抗性基因,并检出可移动遗传元件int I1,其丰度与磺胺类抗性基因的丰度(P 0. 05,r 0. 95)存在相关性,表明可移动遗传元件int I1可能促进了磺胺类抗性基因的迁移和转化.园区医药企业以合成大环内酯类抗生素为主,由于选择性压力,园区废水中,erm B抗性基因的绝对丰度远远高于其他废水中erm B的绝对丰度.废水经过废水处理厂生物处理工艺,总ARGs绝对丰度下降了1. 16个数量级,经过芬顿工艺处理后,总ARGs绝对丰度下降了2. 46个数量级,表明该废水处理工艺中深度处理工艺对ARGs的去除效果优于生物处理.高浓度、可移动的ARGs已经存在于水体中,如果没有得到有效治理,从废水处理厂排出,将给环境带来高度风险.     

Functional relationship between ammonia-oxidizing bacteria and ammonia-oxidizing archaea populations in the secondary treatment system of a full-scale municipal wastewater treatment plant

The abundance of ammonia-oxidizing bacteria and archaea and their amoA genes from the aerobic activated sludge tanks, recycled sludge and anaerobic digesters of a full-scale wastewater treatment plant (WWTP) was determined. Polymerase chain reaction and denaturing gradient gel electrophoresis were used to generate diversity profiles, which showed that each population had a consistent profile although the abundance of individual members varied. In the aerobic tanks, the ammonia-oxidizing bacterial (AOB) population was more than 350 times more abundant than the ammonia-oxidizing archaeal (AOA) population, however in the digesters, the AOA population was more than 10 times more abundant. Measuring the activity of the amoA gene expression of the two populations using RT-PCR also showed that the AOA amoA gene was more active in the digesters than in the activated sludge tanks. Using batch reactors and ddPCR, amoA activity could be measured and it was found that when the AOB amoA activity was inhibited in the anoxic reactors, the expression of the AOA amoA gene increased fourfold. This suggests that these two populations may have a cooperative relationship for the oxidation of ammonia.     

Horizontal and vertical gene transfer drive sediment antibiotic resistome in an urban lagoon system

Rapid urbanization has resulted in pervasive occurrence of antibiotic resistance genes (ARGs) in urban aquatic ecosystems. However, limited information is available concerning the ARG profiles and the forces responsible for their assembly in urban landscape lagoon systems. Here, we employed high-throughput quantitative PCR (HT-qPCR) to characterize the spatial variations of ARGs in surface and core sediments of Yundang Lagoon, China. The results indicated that the average richness and absolute abundance of ARGs were 11 and 53 times higher in the lagoon sediments as compared to pristine reference Tibetan lake sediments, highlighting the role of anthropogenic activities in ARG pollution. Co-occurrence network analysis indicated that various anaerobic prokaryotic genera belonging to Alpha-, Deltaproteobacteria, Bacteroidetes, Euryarchaeota, Firmicutes and Synergistetes were the potential hosts of ARGs. The partial least squares-path modeling (PLS-PM) analysis revealed positive and negative indirect effects of physicochemical factors and heavy metals on the lagoon ARG profiles, via biotic factors, respectively. The horizontal (mediated by mobile genetic elements) and vertical (mediated by prokaryotic communities) gene transfer may directly contribute the most to drive the abundance and composition of ARGs, respectively. Furthermore, the neutral community model demonstrated that the assembly of sediment ARG communities was jointly governed by deterministic and stochastic processes. Overall, this study provides novel insights into the diversity and distribution of ARGs in the benthic habitat of urban lagoon systems and underlying mechanisms for the spread and proliferation of ARGs.     

制药废水厂抗性基因和微生物群落相关性研究

以2座制药废水厂的生物曝气阶段为例,结合Miseq测序分析技术和荧光定量PCR技术研究活性污泥中微生物群落和β-内酰胺类抗性基因的分布特征、扩增情况及其相关性.结果表明：β-内酰胺类抗性基因OXA-1、OXA-2和OXA-10在2个水厂（J厂和K厂）中均能被检出,OXA型基因丰度在K厂中为5.82×10⁵~3.94×10⁷copies/g（干重）,在J厂的丰度范围为4.84×10⁷~1.09×10¹⁰copies/g,3种基因丰度在曝气处理中显著扩增.Miseq测序结果表明：K厂中主要优势菌门为Proteobacteria,Planctomycetes,Bacteroidetes,Chloroflexi和Acidobacteria等,总平均相对丰度比例为82.13%;J厂中主要优势菌门Proteobacteria,Bacteroidetes,Verrucomicrobia,Gemmatimonadetes和Thermi,总平均相对丰度比例为85.76%.冗余分析显示：生物群落中Bdellovibrio、KD8-87和Paracoccus等菌属可能是OXA-1、OXA-2和OXA-10的主要携带菌属;Hyphomicrobium、Thermomonas和Comamonadaceae可能是OXA-1的主要携带菌属,Caldilineaceae、Myxococcales和Pirellulaceae可能是OXA-10的主要携带菌属.     

玉米—大豆轮作体系对黑土土壤固氮菌群落结构及其质量的影响

以大豆连作（CS）、玉米连作（CM）、玉米—大豆轮作（MS）、玉米—玉米—大豆（MMS）和玉米—大豆—大豆（MSS）为研究对象,利用荧光定量PCR和高通量测序分析种植制度对土壤固氮菌丰度和群落结构的影响。结果表明：轮作中土壤有机质（SOM）、全磷（TP）、有效氮（AN）和有效磷（AP）的含量显著高于连作;轮作固氮菌丰度显著高于CM,显著低于CS;MMS与MSS固氮菌多样性显著高于CM;轮作和连作土壤固氮菌群落结构差异明显,全氮（TN）是固氮菌群落结构变化的主要驱动因子,种植制度通过土壤化学性质间接影响固氮菌丰度和多样性。这说明,在吉林省西部半干旱区,MSS与MMS更有利于土壤固氮菌繁殖,可以从微生物学的角度为合理种植和氮素调控提供科学依据。