高通量测序技术在五类疾病分子诊断中的应用及质量管理策略

高通量测序技术(high-throughput sequencing)又称下一代测序(next generation sequencing, NGS)能一次并行对成千上万个基因进行测序,极大地提高了DNA测序效率并降低了成本。近年来,NGS在医学研究和临床诊断中的应用日益广泛,在无创产前筛查,肿瘤筛查、诊断和治疗,微生物病原体检测,遗传性疾病的筛查与诊断及器官移植排斥筛查等多个领域具有广泛的应用前景。NGS技术作为一项新兴技术,在相关临床应用中也存在诸多问题,其质量管理体系尚不完善,还面临许多挑战,需要医学实验室和相关部门共同努力,以促进NGS在临床诊断中规范开展。     

Molecular epidemiology of bluetongue virus serotype 1 circulating in Italy and its connection with northern Africa

Western BTV-1 emerged in the Mediterranean basin in 2006 and it has since been isolated in southern and northern European countries. Six BTV-1 strains isolated from infected sheep in Italy between 2006 and 2013 and a BTV-1 strain isolated from an infected sheep in Tunisia in 2011 were fully sequenced. The seven strains were shown to be nearly identical in each gene segment. The Seg-2 sequences of the BTV-1 strains group according to the year of isolation reflecting the time of BTV incursions in Italy. Combined results suggest that BTV-1 strains isolated in Sardinia, Sicily and mainland Italy in 2012 and 2013 have a direct northern African origin. The Italian strains originated from a strain closely related to a BTV-1 strain isolated in Tunisia in 2011. Better surveillance programs with northern and sub-Saharan African countries should be implemented making the control of spread of BTV easier and effective.     

Characterisation of a diverse range of circular replication-associated protein encoding DNA viruses recovered from a sewage treatment oxidation pond

Our knowledge of circular replication-associated protein encoding single-stranded (CRESS) DNA virus diversity has increased dramatically in recent years, largely due to advances in high-throughput sequencing technologies. These viruses are apparently major virome components in most terrestrial and aquatic environments and it is therefore of interest to determine their diversity at the interfaces between these environments. Treated sewage water is a particularly interesting interface between terrestrial and aquatic viromes in that it is directly pumped into waterways and is likely to contain virus populations that have been strongly impacted by humans. We used a combination of high-throughput sequencing, full genome PCR amplification, cloning and Sanger sequencing to investigate the diversity of CRESS DNA viruses present in a sewage oxidation pond. Using this approach, we recovered 50 putatively complete novel CRESS viral genomes (it remains possible that some are components of multipartite viral genomes) and 11 putatively sub-genome-length circular DNA molecules which may be either defective genomes or components of multipartite genomes. Thirteen of the genomes have bidirectional genome organisations and share similar conserved replication-associated protein (Rep) motifs to those of the gemycircularviruses: a group that in turn is most closely related to the geminiviruses. The remaining 37 viral genomes share very low degrees of Rep similarity to those of all other known CRESS DNA viruses. This number of highly divergent CRESS DNA virus genomes within a single sewage treatment pond further reinforces the notion that there likely exist hundreds of completely unknown genus/family level CRESS DNA virus groupings.     

Presence of enteric viruses in freshwater and their removal by the conventional drinking water treatment process.

A review of results published in English or French between 1980 and 1990 was carried out to determine the levels of indigenous human enteric viruses in untreated surface and subsurface freshwaters, as well as in drinking water that had undergone the complete conventional treatment process. For this purpose, the conventional treatment process was defined as an operation that included coagulation followed by sedimentation, filtration, and disinfection. Also assessed was the stepwise efficiency of the conventional treatment process, as practised at full-scale facilities, for removing indigenous viruses from naturally occurring freshwaters. A list was compiled of statistical correlations relating to the occurrence of indigenous viruses in water.     

孕妇血浆中胎儿游离DNA片段浓度和长度均值的计算方法研究

目的:建立计算孕妇外周血中胎儿游离DNA片段(cffDNA)浓度和长度均值的新的数学模型。方法:根据孕妇外周血中母亲和胎儿的DNA片段的长度分布不同的现象,利用K-means算法和EM算法开发新的算法。用提出的新方法分析孕妇外周血样本的DNA片段长度分布,cffDNA浓度和片段长度均值。结果:(1)三份样本中来源于母亲的DNA片段长度均值,分别为167bp,166bp和165bp;cffDNA的长度均值为160bp,163bp和158bp;(2)孕妇血浆中cffDNA的浓度分别为10.4%,5.5%,17.4%。此结果与基于Y染色体的百分含量的方法计算所得的结果相近。结论:用本课题组提出的新方法分析孕妇外周血的cffDNA的浓度和长度均值是有效的、准确的。     

对混凝沉淀法分散式饮水除砷的研究

采用混凝沉淀法进行分散式饮水除砷试验。结果表明,当水样中五价砷［Ａｓ（Ⅴ）］含量为１．０ｍ ｇ／Ｌ时,不调节ｐＨ值（ｐＨ７．８２）,直接投加５０ｍ ｇ／Ｌ硫酸铁,室温下沉淀静置１２ｈ,可使倾析液残留砷含量低于０．０５ｍ ｇ／Ｌ。如沉淀反应后静置３０～４０ ｍ ｉｎ 即过滤,则只需投加３０ｍ ｇ／Ｌ的硫酸铁或４０ｍ ｇ／Ｌ的硫酸铝即可达到同样的除砷效果。随着投加量的增加,２种混凝剂对砷的去除率均升高,当水样Ａｓ（Ⅴ）≤１．０ｍ ｇ／Ｌ和≤０．５ｍ ｇ／Ｌ时,分别投加３０ｍ ｇ／Ｌ 硫酸铁和硫酸铝,过滤后可使残留砷含量达到现行饮水卫生标准（＜０．０５ｍ ｇ／Ｌ）。在水样处于不同ｐＨ 值、水温、浊度、硬度等条件下硫酸铁的除砷性能较硫酸铝稳定。一般情况下,沉渣中的砷不会再次进入水中     

Applicability of quantitative PCR for determination of removal efficacy of enteric viruses and Cryptosporidium by water treatment processes

This study aims to assess the applicability of quantitative PCR (qPCR) for removal studies of adenovirus, coxsackievirus, echovirus and Cryptosporidium by water treatment processes. Bench-scale coagulation jar tests were performed using the enteric viruses and Cryptosporidium. Standard methods (conventional cell-culture methods for the viruses and an immunofluorescence assay (IFA) for Cryptosporidium) were used to compare to qPCR. A significant correlation between microbial removals determined by qPCR and the standard detection methods and an approximate 1:1 correlation were observed for the challenge microorganisms. The results indicated that qPCR could be a satisfactory alternate for microbial removal studies using a relative quantification approach.     

Isolation of Burkholderia cenocepacia and Burkholderia vietnamiensis from human sewage

Fresh human sewage was examined from a sewage treatment plant for the presence of members of the Burkholderia cepacia complex (BCC) of bacterial organisms and confirmed the presence of viable B. cenocepacia and B. vietnamiensis, by a combination of cultural, phenotypic and genotypic techniques. Both these organisms are important respiratory pathogens for patients with cystic fibrosis (CF). Presently, the survival dynamics of these organisms in sewage effluent and sludge is, as yet, unknown. Therefore, as this study represents the first report of these CF pathogens in sewage and until such survival data is available, careful risk assessment needs to be undertaken in relation to the end use application of potentially contaminated sewage and where such material comes into association with non-colonised patients with cystic fibrosis, so that any potential transmission of these pathogens from sewage to patient is assessed and minimised/eliminated.     

In-depth genome analyses of viruses from vaccine-derived rabies cases and corresponding live-attenuated oral rabies vaccines

Live-attenuated rabies virus strains such as those derived from the field isolate Street Alabama Dufferin (SAD) have been used extensively and very effectively as oral rabies vaccines for the control of fox rabies in both Europe and Canada. Although these vaccines are safe, some cases of vaccine-derived rabies have been detected during rabies surveillance accompanying these campaigns. In recent analysis it was shown that some commercial SAD vaccines consist of diverse viral populations, rather than clonal genotypes. For cases of vaccine-derived rabies, only consensus sequence data have been available to date and information concerning their population diversity was thus lacking.In our study, we used high-throughput sequencing to analyze 11 cases of vaccine-derived rabies, and compared their viral population diversity to the related oral rabies vaccines using pairwise Manhattan distances. This extensive deep sequencing analysis of vaccine-derived rabies cases observed during oral vaccination programs provided deeper insights into the effect of accidental in vivo replication of genetically diverse vaccine strains in the central nervous system of target and non-target species under field conditions. The viral population in vaccine-derived cases appeared to be clonal in contrast to their parental vaccines. The change from a state of high population diversity present in the vaccine batches to a clonal genotype in the affected animal may indicate the presence of a strong bottleneck during infection. In conclusion, it is very likely that these few cases are the consequence of host factors and not the result of the selection of a more virulent genotype. Furthermore, this type of vaccine-derived rabies leads to the selection of clonal genotypes and the selected variants were genetically very similar to potent SAD vaccines that have undergone a history of in vitro selection.     

2011-2013年广西农村垃圾和污水处理监测分析

目的：了解广西农村垃圾和污水处理现状及其动态变化,为农村环境卫生治理提供科学依据。方法采用统一的调查表,通过访谈、现场观察等收集有关垃圾和污水处理的相关资料。结果从2011年至2013年,广西农村生活垃圾从以随意堆放为主（66.03%）转为以定点堆放为主(66.90%);生活垃圾处理方式以焚烧为主,从58.10%上升到67.93%;工业垃圾处理以填埋为主,从36.07%上升到53.85%,其次为焚烧,从21.15%上升到36.84%;养殖业垃圾处理以再利用为主,从36.39%上升到51.57%,其次为高温堆肥,从32.51%上升到40.36%。农村生活污水随意排放率从2011年的65.17%下降到2013年的31.21%;排放地点以坑塘为主,其次为河流和农田。3年间,工业污水处理后排放的比例从44.19%上升到53.33%;养殖业污水处理后排放的比例从28.80%上升到32.15%。结论广西农村垃圾和污水卫生处理现状有所改善,但处理设施仍匮乏,存在各种影响健康的危险因素,亟需采取措施予以改善。     

Inactivation of Nitrosomonas europaea and pathogenic Escherichia coli by chlorine and monochloramine

The purpose of this study was to measure the chlorine and monochloramine inactivation kinetics of Nitrosomonas europaea at 21°C in the presence and absence of particles. The inactivation kinetics rates were compared with those obtained with Escherichia coli O157:H7. The results show that, in pure water, the use of free chlorine produced 4 log₁₀ of N. europaea inactivation at a CT value of 0.8 mg.min l^-1, whereas monochloramine yielded 4 log₁₀ of inactivation at CT values of approximately 9.9–16.4 mg.min l^-1. With E. coli, chlorine produced approximately 4 log₁₀ of inactivation at a CT of 0.13 mg.min l^-1, whereas monochloramine resulted in 4 log₁₀ of inactivation at a CT of approximately 9.2 mg.min l^-1. These results suggest that N. europaea is more resistant to monochloramine and chlorine than E. coli. Corrosion debris, soil material and wastewater had no statistically significant (p < 0.05) impact on the inactivation of N. europaea by either chlorine or monochloramine. It seems likely that the CT values present in distribution systems would be sufficient to control suspended cells of these two organisms, especially under conditions of breakpoint chlorination, which could be used to control nitrification. Adequate disinfection should prevent the growth of these organisms in a distribution system.     

人感染高致病性H5N6禽流感病毒表面糖蛋白分子进化特征

目的  对19例人感染高致病性H5N6禽流感病毒的血凝素(hemagglutinin, HA)和神经氨酸酶(neuraminidase, NA)蛋白进行分子进化分析。 方法  运用下一代测序平台对病毒分离物进行全基因组测序, 从美国国家生物技术信息中心(national center for biotechnology information, NCBI)和全球流感序列数据库(global initiative on sharing avian influenza data, GISAID)下载参考序列, 利用Blasts、Mega 6.1及Clustal X 2.1等软件进行序列分析。 结果  2014-2018年中国共发生23例人感染H5N6禽流感病毒病例。对19个病例的H5N6病毒的HA和NA基因进行进化分析。HA进化分析显示病毒都属于Clade 2.3.4.4, 其中涉及17个病例的病毒属于Group C; 首例H5N6病例毒株(A/Sichuan/26221/2014)属于Group D; 福建一个病例(A/Fujian-Sanyuan/21099/2017)属于Group B。所有19个病例的病毒HA蛋白的裂解位点含有多个碱性氨基酸。所有病毒的HA蛋白的受体结合位点226~228位氨基酸是QS(R)G(氨基酸排序以H3-HA为准), 理论上对禽类受体α2-3半乳糖苷唾液酸(SAα2-3Gal)有嗜性。18病例病毒的HA蛋白发生了T160A的突变, 导致在158N位点失去糖基化。除了A/Sichuan/26221/2014外, 18个病例的病毒NA蛋白在58~68位缺失了10个氨基酸。9个病例的病毒PB2蛋白发生E627K突变。 结论  2014-2018年间中国人感染H5N6病毒进化活跃, 具有明显的基因多样性, 需要加强对病毒分子进化的监测。     

Ammonium removal from aqueous solutions by clinoptilolite: determination of isotherm and thermodynamic parameters and comparison of kinetics by the double exponential model and conventional kinetic models

The adsorption isotherm, the adsorption kinetics, and the thermodynamic parameters of ammonium removal from aqueous solution by using clinoptilolite in aqueous solution was investigated in this study. Experimental data obtained from batch equilibrium tests have been analyzed by four two-parameter (Freundlich, Langmuir, Tempkin and Dubinin-Radushkevich (D-R)) and four three-parameter (Redlich-Peterson (R-P), Sips, Toth and Khan) isotherm models. D-R and R-P isotherms were the models that best fitted to experimental data over the other two- and three-parameter models applied. The adsorption energy (E) from the D-R isotherm was found to be approximately 7 kJ/mol for the ammonium-clinoptilolite system, thereby indicating that ammonium is adsorbed on clinoptilolite by physisorption. Kinetic parameters were determined by analyzing the nth-order kinetic model, the modified second-order model and the double exponential model, and each model resulted in a coefficient of determination (R(2)) of above 0.989 with an average relative error lower than 5%. A Double Exponential Model (DEM) showed that the adsorption process develops in two stages as rapid and slow phase. Changes in standard free energy (?G°), enthalpy (?H°) and entropy (?S°) of ammonium-clinoptilolite system were estimated by using the thermodynamic equilibrium coefficients.     

Bacterial removal in the treatment of sewage sludge by composting,fixed‐bed and stirred anaerobic digesters

An assessment of the performance and hygienic evaluation of composting, fixed‐bed and stirred anaerobic digester systems for sewage sludge treatment was conducted. The digesters performance in terms of bacterial indicators and pathogenic bacterial form was assessed. The results indicated that an average removal percentage of Faecal coliform and Faecal streptococci for either the stirred and fixed‐bed digesters was 24–33% and 34–42% respectively. Composting of the sewage sludge increased its total solids from 19% to 93% after 6 weeks, while the reduction of organic matter was 41 % and nutrients were consumed. Complete removal of Salmonellae and Faecal coliform occurred, while the other tested parameters were more resistant to the treatment conditions.     

Detecting association of rare and common variants by testing an optimally weighted combination of variants

Next-generation sequencing technology will soon allow sequencing the whole genome of large groups of individuals, and thus will make directly testing rare variants possible. Currently, most of existing methods for rare variant association studies are essentially testing the effect of a weighted combination of variants with different weighting schemes. Performance of these methods depends on the weights being used and no optimal weights are available. By putting large weights on rare variants and small weights on common variants, these methods target at rare variants only, although increasing evidence shows that complex diseases are caused by both common and rare variants. In this paper, we analytically derive optimal weights under a certain criterion. Based on the optimal weights, we propose a Variable Weight Test for testing the effect of an Optimally Weighted combination of variants (VW-TOW). VW-TOW aims to test the effects of both rare and common variants. VW-TOW is applicable to both quantitative and qualitative traits, allows covariates, can control for population stratification, and is robust to directions of effects of causal variants. Extensive simulation studies and application to the Genetic Analysis Workshop 17 (GAW17) data show that VW-TOW is more powerful than existing ones either for testing effects of both rare and common variants or for testing effects of rare variants only.     

氢化物原子荧光法测定生活饮用水及其水源水中的铅

目的:建立简便、灵敏、准确的生活饮用水及其水源水中铅的测定方法。方法:采用氢化物原子荧光法测定生活饮用水及其水源水中的铅。结果:实验表明,方法的线性范围0~70μg/L,相关系数r优于0.9990,方法的检出限为0.084μg/L,用相对标准偏差考察方法的精密度,RSD均小于4.0%,用加标回收和标准物质考察方法的准确性,加标回收率均在85.0%~116.5%之间,测定了国家标准参考物GSBZ5009-88,测定值均在标准值范围内。结论:该方法简便、灵敏、准确,适用于生活饮用水及其水源水中铅的测定。     

Phytoremediation of fungicides by aquatic macrophytes: Toxicity and removal rate

The rate of removal of two fungicides (dimethomorph and pyrimethanil) from water by five macrophyte species (L. minor, S. polyrhiza, C. aquatica, C. palustris and E. canadensis) was assessed in laboratory tests. In order to assure that these studies were performed with healthy plants the effects of the fungicides on chlorophyll fluorescence were studied as well. At exposure concentrations of 600 μg L⁻¹ the effects of the fungicides on chlorophyll fluorescence were minor, so that this initial concentration level was selected for the fungicide removal rate tests. The removal yields during the 4-d test periods varied from 10% to 18% and 7% to 12% for dimethomorph and pyrimethanil, respectively. The maximum removal rate during the 4-d test period was 48 μg g⁻¹ fresh weight (FW) for dimethomorph and 33 μg g⁻¹ FW for pyrimethanil. L. minor and S. polyrhiza showed the highest removal efficiency for the two fungicides.     

2015-2017年盐城市乙型流感病毒HA1、NA基因分子特征

  目的   对盐城市2015-2017年流行的乙型流感病毒血凝素(hemagglutinin, HA)和神经氨酸酶(neuraminidase, NA)基因进行分子进化特征研究。   方法   将盐城市2015-2017年流感监测哨点医院以及流感暴发点采集的流感样病例咽拭子标本进行核酸、病毒分离检测定型, 对选取的18株乙型流感病毒分离株采用一步法RT-PCR方法扩增其HA1基因和NA基因, 扩增产物经纯化测序, 采用生物信息软件从核苷酸、氨基酸以及分子进化层面对毒株进行分子特征分析。   结果   盐城市2015-2017年分离的乙型流感病毒HA1基因和NA基因聚类的分支关系基本一致, 2015年Yamagata系毒株位于Yamagata Clade 3分支, 属Phuket/3073类毒株; 2016-2017年Victoria系毒株分布于Victoria Clade 1A分支, 属Brisbane/60类毒株。Yamagata系所有毒株在190-helix抗原表位均发生了D196N位点的变异; Victoria系毒株共涉及2个抗原表位, 120-loop抗原表位的117、129位点, 190-helix抗原表位的197、199位点。盐城株未发生系内、系间重配。18株分离株未发生NA蛋白酶活性位点以及耐药位点的变化。   结论   2015年Yamagata系毒株与疫苗株B/Phuket/3073/2013匹配性较好。2016-2017年Victoria系毒株HA1和NA抗原基因变异位点在积累, 这些变异位点的积累很可能会导致流感病毒发生实质性的抗原性的漂移, 降低与流感疫苗株的匹配度, 减弱流感疫苗的保护作用。     

Genetic,antigenic and pathogenic characterization of four infectious bursal disease virus isolates from China suggests continued evolution of very virulent viruses

Infectious bursal disease virus (IBDV) causes an economically significant disease of young chickens worldwide. The emergence of very virulent IBDV (vvIBDV) strains has brought more challenges for effective prevention and control of this disease. The aim of the present study was to characterize four IBDV isolates from various regions of China between late 1990s and recent years and to compare them with previously isolated European IBDV strains. In this study, one Chinese vvIBDV strain isolated in 1999 and three strains isolated between 2005 and 2011 were analyzed at the genetic, antigenic and pathogenic levels. Strain SH99 was closely related and clustered in the same genetic lineage as the typical vvIBDV based on the genomic sequences of segments A and B. However, the three more recent Chinese vvIBDV (HLJ0504, HeB10 and HuN11) showed several genetic changes in both segments and clustered in a distinct lineage from the typical vvIBDV and the previously known Chinese vvIBDV. Based on the binding to a panel of neutralizing monoclonal antibodies in antigen capture enzyme-linked immunosorbent assays, all Chinese vvIBDVs exhibited similar antigenicity with the European typical vvIBDV strains. Nonetheless, the pathogenicity caused by the recent Chinese vvIBDV was higher than that induced by the European typical vvIBDV. This study calls for a sustained surveillance of IBD situation in China in order to support a better prevention and control of the disease.