布鲁杆菌环介导等温扩增快速检测方法的建立

目的 建立一种环介导等温扩增(LAMP)快速检测动物布鲁杆菌的方法.方法 使用Primer 4.0,针对布鲁杆菌外膜蛋白(OMP31)基因保守区设计4条特异引物,在Bst大片段聚合酶的作用下,实现DNA的梯状等温扩增,并在此方法最适扩增条件优化的基础上,对其特异性、灵敏度进行实验,同时与普通PCR灵敏度进行比较,以及进行LAMP可视化实验.结果 本研究建立的检测方法对牛种布鲁杆菌(Brucella abortus,B.abortus)544A和104M、羊种布鲁杆菌(Brucella melitensis,B.melitensis)Rev-1和16M、猪种布鲁杆菌(Brucella suis,B.suis)S2和1330S、犬种布鲁杆菌(Brucellac anis,B.canis)RM6/66、绵羊附睾种布鲁杆菌(Brucela ovis,B.ovis)63/290、沙林鼠种布鲁杆菌(Brucella neotomae,B.neotomae)5K33检测阳性,而耶尔森氏菌(Yersinia enterocolitica,Y.enterocolitica)O∶9、大肠杆菌(Escherichia coli E.coli)O157∶H7和沙门氏菌(Salmonella typhimurium,S.typhimunum)47729检测阴性.LAMP法检出最低DNA浓度为8.5×10-8 mg/L,较普通PCR检测灵敏度高.检测结果既可以通过电泳判定也可以通过可视化判定.结论 本研究所建立的布鲁杆菌LAMP检测方法具有特异、灵敏、设备要求简单等特点,适用于基层兽医部门进行布鲁杆菌的快速检测.     

Mechanotransduction and strain amplification in osteocyte cell processes

A paradox in bone tissue is that tissue-level strains due to animal and human locomotion are too small to initiate intracellular chemical responses directly. A model recently was proposed to resolve this paradox, which predicts that the fluid flow through the pericellular matrix in the lacunar-canalicular porosity due to mechanical loading can induce strains in the actin filament bundles of the cytoskeleton that are more than an order of magnitude larger than tissue level strains. In this study, we greatly refine this model by using the latest ultrastructural data for the cell process cytoskeleton, the tethering elements that attach the process to the canalicular wall and their finite flexural rigidity EI. We construct a much more realistic 3D model for the osteocyte process and then use large-deformation "elastica" theory for finite EI to predict the deformed shape of the tethering elements and the hoop strain on the central actin bundle. Our model predicts a cell process that is 3 times stiffer than in a previous study but hoop strain of >0.5% for tissue-level strains of >1,000 microstrain at 1 Hz and >250 microstrain at frequencies >10 Hz. We propose that this strain-amplification model provides a more likely hypothesis for the excitation of osteocytes than the previously proposed fluid-shear hypothesis.     

环介导等温扩增技术用于寄生虫检测的研究进展

寄生虫病在我国分布广泛,严重危害着人们的身体健康.环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)是一种新的核酸扩增技术,具有敏感性高、特异性强和简便快速等优点,可用于寄生虫的快速诊断.该文就LAMP检测寄生虫的研究现状进行综述.     

Algorithmic amplification of politics on Twitter

Content on Twitter’s home timeline is selected and ordered by personalization algorithms. By consistently ranking certain content higher, these algorithms may amplify some messages while reducing the visibility of others. There’s been intense public and scholarly debate about the possibility that some political groups benefit more from algorithmic amplification than others. We provide quantitative evidence from a long-running, massive-scale randomized experiment on the Twitter platform that committed a randomized control group including nearly 2 million daily active accounts to a reverse-chronological content feed free of algorithmic personalization. We present two sets of findings. First, we studied tweets by elected legislators from major political parties in seven countries. Our results reveal a remarkably consistent trend: In six out of seven countries studied, the mainstream political right enjoys higher algorithmic amplification than the mainstream political left. Consistent with this overall trend, our second set of findings studying the US media landscape revealed that algorithmic amplification favors right-leaning news sources. We further looked at whether algorithms amplify far-left and far-right political groups more than moderate ones; contrary to prevailing public belief, we did not find evidence to support this hypothesis. We hope our findings will contribute to an evidence-based debate on the role personalization algorithms play in shaping political content consumption.

Political content is a major part of the public conversation on Twitter. Politicians, political organizations, and news outlets engage large audiences on Twitter. At the same time, Twitter employs algorithms that learn from data to sort content on the platform. This interplay of algorithmic content curation and political discourse has been the subject of intense scholarly debate and public scrutiny (1–15). When first established as a service, Twitter used to present individuals with content from accounts they followed, arranged in a reverse chronological feed. In 2016, Twitter introduced machine learning algorithms to render tweets on this feed called Home timeline based on a personalized relevance model (16). Individuals would now see older tweets deemed relevant to them, as well as some tweets from accounts they did not directly follow.Personalized ranking prioritizes some tweets over others on the basis of content features, social connectivity, and user activity. There is evidence that different political groups use Twitter differently to achieve political goals (17–20). What has remained a matter of debate, however, is whether or not any ranking advantage falls along established political contours, such as the left or right (2, 7), the center or the extremes (1, 3), specific parties (2, 7), or news sources of a certain political inclination (21). In this work, we provide systematic quantitative insights into this question based on a massive-scale randomized experiment on the Twitter platform.     

Gene amplification in non-Hodgkin's lymphoma

Summary. Among 426 consecutively ascertained and karyotypically abnormal non-Hodgkin's lymphoma (NHL) tumours, cytological evidence for gene amplification in the form of homogeneously staining regions (HSRs) was encountered in nine cases of large cell diffuse lymphoma (LC-DL). The mean age of patients with HSRs was 62·9 years and four died within a year of diagnosis. To identify candidate gene(s) amplified in these tumours, we performed a Southern blot analysis of tumour DNA using probes for 23 known protooncogenes and the multidrug resistance gene, PGY1 , Besides a two-fold amplification of the BCL2 gene in two cases, no evidence for overt amplification of any of the genes assayed was found. To confirm DNA amplification in these specimens we performed the DNA in-gel renaturation assay. Evidence for presence of amplified DNA fragments was obtained in four of seven specimens. These results suggest amplification of a novel gene(s). To our knowledge, this is the first formal study of gene amplification in a large consecutively ascertained series of fresh lymphoma biopsies.     

环介导等温扩增技术检测蓝氏贾第鞭毛虫

目的 建立应用环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)检测蓝氏贾第鞭毛虫的方法. 方法 体外培养蓝氏贾第鞭毛虫滋养体,提取DNA.根据GenBank显示的贾第虫序列及环介导等温扩增技术的原理,设计4条贾第虫特异引物,利用LAMP检测蓝氏贾第鞭毛虫DNA,以隐孢子虫卵囊DNA、疟原虫DNA为对照,并将不含病原体DNA的纯水作为阴性对照.LAMP产物经SYBR green I显色后观察结果,绿色为阳性,棕色为阴性;对LAMP产物进行琼脂糖凝胶电泳分析,观察其特征条带的情况. 结果 蓝氏贾第鞭毛虫DNA检测管经显色后呈绿色,隐孢子虫卵囊DNA、疟原虫DNA及水阴性对照管呈棕色.含有蓝氏贾第鞭毛虫DNA的LAMP产物经电泳后呈LAMP特征性梯状条带,安氏隐孢子虫DNA、恶性疟原虫DNA及阴性对照水无扩增产物. 结论 成功建立了检测蓝氏贾第鞭毛虫的LAMP方法.     

核酸扩增技术在淋球菌检测中的研究进展

淋病是一种性传播疾病,其病原体是淋球菌。淋球菌主要寄生于人体尿道粘膜,导致人类泌尿生殖系感染。及时、准确地诊断淋球菌感染是控制淋病传播的关键,核酸扩增检测技术对淋球菌的临床确诊及治疗有重大意义。本文综述了核酸扩增技术在淋球菌检测中的应用进展。     

Uniform and accurate single-cell sequencing based on emulsion whole-genome amplification

Whole-genome amplification (WGA) for next-generation sequencing has seen wide applications in biology and medicine when characterization of the genome of a single cell is required. High uniformity and fidelity of WGA is needed to accurately determine genomic variations, such as copy number variations (CNVs) and single-nucleotide variations (SNVs). Prevailing WGA methods have been limited by fluctuation of the amplification yield along the genome, as well as false-positive and -negative errors for SNV identification. Here, we report emulsion WGA (eWGA) to overcome these problems. We divide single-cell genomic DNA into a large number (10⁵) of picoliter aqueous droplets in oil. Containing only a few DNA fragments, each droplet is led to reach saturation of DNA amplification before demulsification such that the differences in amplification gain among the fragments are minimized. We demonstrate the proof-of-principle of eWGA with multiple displacement amplification (MDA), a popular WGA method. This easy-to-operate approach enables simultaneous detection of CNVs and SNVs in an individual human cell, exhibiting significantly improved amplification evenness and accuracy.Single-cell sequencing, characterization the genome of individual cells, is highly needed for studying scarce and/or precious cells, which are inaccessible for conventional bulk genome characterization, and for probing genomic variations of a heterogeneous population of cells (1–3). Recently single-cell genomics has unveiled unprecedented details of various biological processes, such as tumor evolution (4–6), embryonic development (7), and neural somatic mosaicism (8). Single-cell whole-genome amplification (WGA) is required to generate enough replicates of genomic DNAs for library preparation in conjunction with current sequencing protocols. Single-cell WGA has been increasingly used in cutting-edge clinical diagnostic applications such as molecular subtyping of single tumor cells (4, 9) and preimplantation genetic screening of in vitro fertilized embryos (10).An ideal single-cell WGA method should have high uniformity and accuracy across the whole genome. The WGA uniformity is critical for copy number variation (CNV) detection, whereas the WGA accuracy is essential for avoiding single-nucleotide variation (SNV) detection errors, either false positives or false negatives. The false positives arise from misincorporation of wrong bases in the first few cycles of WGA. In a diploid human cell, the false negatives primarily arise from the allelic dropout (ADO), i.e., heterozygous mutations are mistaken as homozygous ones because of the lack of amplification in one of the two alleles (11).Existing WGA chemistry includes degenerate oligonucleotide-primed PCR (DOP-PCR) (12), multiple displacement amplification (MDA) (13–17), and multiple annealing and looping-based amplification cycles (MALBAC) (4, 18, 19), which have successively achieved genome analysis at the single-cell level. DOP-PCR is based on PCR amplification of the fragments flanked by universal priming sites, and provides high accuracy for detecting CNVs in single cells but has low coverage and high false-positive and false-negative rates for calling SNVs (5). MDA has a much improved coverage but tends to have lower precision/sensitivity in CNV determination due to its variation of the amplification gain along the genome, not reproducible from cell to cell (20). By virtue of quasilinear amplification, MALBAC suppresses the random bias of amplification and exhibits reduced ADO rates, yielding low false negatives for SNV detection (2, 11, 18, 19). Notwithstanding its drawbacks, MDA still offers comparable or higher genome coverage than MALBAC, at least for single diploid cells, possibly taking advantage of the randomness (2). In fact, even higher coverage has been obtained for cells with aneuploidy, such as dividing cells (21), and cancer cells (22). MDA’s main advantage is its lower false-positive rate for SNV detection on account of the use of Phi-29, a highly processive polymerase with high fidelity.Microfluidic devices have been carried out for single-cell WGA (16, 20, 23, 24), allowing avoidance of contaminations and high-throughput analyses of multiple single cells in parallel. The small total reaction volumes (microliters to nanoliters- or picoliters) of the microfluidic devices not only facilitate the efficiency of reactions but also allow significant cost reduction for enzymes and regents used. It was reported that the nanoliter volume of a microfluidic device improved uniformity of the amplification compared with microliter devices in the WGA of single bacterial cells (20).Here, we report a method, emulsion whole-genome amplification (eWGA), to use the small volume of aqueous droplets in oil to better the WGA chemistry for uniform amplification of a single cell’s genome. By distributing single-cell genomic DNA fragments into a large number (10⁵) of picoliter droplets, a few DNA fragments in each droplet is allowed to reach saturation of DNA amplification. After merging the droplets by demulsification, the differences in amplification gain among the DNA fragments are significantly minimized.Although this approach can be used for any chemistry of WGA, we take MDA as an example to greatly reduce the random bias of amplification by separating the reactions into a large amount of emulsion droplets. We carried out detailed comparison with MDA, MALBAC, and DOP-PCR performed in tube using single cells from normal diploid human cells and a monoclonal human cancer cell line with inherited CNVs. Our results indicate that eWGA not only offers higher coverage but also enables simultaneous detection of SNVs and CNVs with higher accuracy and finer resolution, outperforming the prevailing single-cell amplification methods in many aspects.     

环介导等温扩增技术及其在病原生物检测中的应用

环介导等温扩增(LAMP)技术是近年来发展起来的一种新型核酸扩增技术,具有操作简便、快速、特异性高、灵敏性高等特点,自2000年开发以来,在短短的十几年内被广泛应用于细菌、病毒和寄生虫等病原生物的检测,在即时检验和基层实验室推广中有重要意义。     

环介导等温扩增技术检测钩端螺旋体的研究

目的 利用环介导等温扩增技术,建立检测钩端螺旋体的方法。方法 选取钩体LipL32基因,设计了LAMP引物。对27株钩体、25株非钩体样本予以检测,然后进行灵敏度和模拟样本的研究。结果 扩增27株钩体结果为阳性,扩增25株非钩体结果为阴性。灵敏度为200拷贝/μL,模拟样本检测下限为200条钩体/μL(煮沸法)和20条钩体/μL(试剂盒提取法)。结论 LAMP实验操作简单,对设备要求不高,可以作为钩体的快速检测方法。     

显微成像系统在寄生虫学实验教学中的应用

将显微成像系统应用于寄生虫学实验教学,不仅可以提高教师的工作效率,而且有利于激发学生的学习兴趣,提高动手能力,给学生提供一个互相学习,发现问题,共同进步的平台。     

环介导等温扩增技术检测恶性疟原虫的研究

目的环介导等温扩增(LAMP)技术检测恶性疟原虫。方法酚氯仿法提取恶性疟原虫基因组DNA,设计四条扩增恶性疟原虫环子孢子蛋白基因的LAMP引物,以间日疟原虫、弓形虫、卡氏肺孢子虫、人全血DNA为对照,进行LAMP反应。LAMP产物经显色、电泳鉴定。将原虫血症为1.5%的恶性疟原虫血样用正常人血按1∶10倍比稀释为1.5×10-3、1.5×10-4、1.5×10-5、1.5×10-6、1.5×10-7、1.5×10-86个浓度后进行LAMP,检测其敏感性。结果恶性疟原虫检测管经显色后呈绿色(阳性),对照组均呈棕色(阴性)。恶性疟原虫LAMP产物经电泳后呈LAMP特征性梯状条带,对照组均无扩增产物。LAMP可检测恶性疟原虫的敏感度为1.5个疟原虫/107RBC。结论检测恶性疟原虫的LAMP方法特异、敏感及简便。     

环介导等温扩增法在结核病诊断中的应用

目的研究环介导等温扩增法在不同临床样本中的结核病诊断价值。方法使用环介导等温扩增法检测肺泡灌洗液、局部穿刺液、腹水和脑脊液中的结核分枝杆菌复合群的特异性基因IS1081,并与实时荧光定量PCR进行比较。结果在荷菌量较高的肺泡灌洗液和局部穿刺液中,环介导等温扩增法敏感性比实时荧光定量PCR低(80%vs 92%);在荷菌量极低的腹水和脑脊液中,环介导等温扩增法敏感性比实时荧光定量PCR高(34.6%vs 15.4%),但特异性略有下降(80%vs 93.3%);综合计算所有样本的敏感性,环介导等温扩增法与实时荧光定量PCR无显著差别(56.9%vs 52.9%)。结论环介导等温扩增法具有较高敏感性和特异性,可以用于结核病诊断。     

环介导等温扩增技术在嗜水气单胞菌检测中的应用

目的 建立环介导等温扩增技术(Loop-mediated isothermal amplification, LAMP)对嗜水气单胞菌检测的方法,并用临床标本对该方法进行评价。方法 根据嗜水气单胞菌desA 基因的保守序列设计特异性引物,利用参考质粒评价该检测体系的灵敏度;用30种其他肠道致病菌及院内感染中常见的致病菌评价该检测体系的特异性;用粪便模拟样本以及临床样本评价该检测体系的临床检测的可行性。结果 LAMP检测体系对嗜水气单胞菌重组质粒的检测灵敏度为1×10²拷贝/反应体系;LAMP检测体系在检测30种其他肠道致病菌及院内感染中常见的致病菌时未出现假阳性;LAMP检测体系在粪便模拟标本中的检测下限为5 × 10³ CFU/g;通过与qPCR技术相比较,LAMP具有更高的灵敏度以及更优秀的临床标本的检测能力。结论 本研究建立的LAMP方法是首次将LAMP技术运用到了嗜水气单胞菌的检测,所需仪器设备简单,可作为一种快速的筛查方法在临床检验和基层实验室应用。     

环媒恒温基因扩增法检测藤黄微球菌的研究

目的建立藤黄微球菌的环媒恒温基因扩增检测方法。方法基于藤黄微球菌23SrRNA基因部分序列设计1套（共4条）能够识别6个区域的特异性引物；优化扩增条件；评估该方法的特异性与敏感性。结果用7个参考株和大肠埃希菌等8种其他临床病原菌检验本检测法，成功地建立了环媒恒温基因扩增（LAMP）检测法。结论环媒恒温基因扩增法检测藤黄微球菌快速、灵敏高、特异性强。     

RNA恒温扩增实时检测技术快速鉴定偶发分枝杆菌的研究

目的建立RNA恒温扩增实时检测技术快速鉴定偶发分枝杆菌(RIARD-MF)的方法,评估其在鉴定分枝杆菌临床分离株中的应用效果。方法将偶发分枝杆菌的16SrRNA特异序列作为目的靶标进行检测,设计含T7启动子逆转录扩增引物和RNA探针,分别对5种非分枝杆菌细菌、20种分枝杆菌标准株和259株临床分枝杆菌分离株进行42℃恒温扩增实时检测,参考标准为PCR基因测序结果。结果RIARD-MF方法学检测灵敏度可达60CFU/mL。在25种细菌的RIARD-MF特异性检测中:只有偶发分枝杆菌检测结果为阳性,其余24种细菌均为阴性,与测序检测结果一致。在检测分枝杆菌临床分离菌株时,RIARD-MF鉴定偶发分枝杆菌5株,其余为非偶发分枝杆菌,与测序结果一致,检测灵敏度和特异度均为100%。结论 RIARD-MF鉴定偶发分枝杆菌具有较高的特异度、灵敏度和准确性,且检测快速,有望成为一种新的偶发分枝杆菌临床分离菌株鉴定方法。     

环介导恒温扩增(LAMP)技术及其在寄生虫检测中的应用

近年来开发出一种新的恒温核酸扩增方法,即环介导恒温扩增(loop-mediated isithermal amplification,LAMP)法.LAMP技术具有简便、快速、高效、特异性强等优点,尤其适用于人类和动物疾病的检测.该文简要介绍了LAMP技术的原理,并对其在检测寄生虫如锥虫、疟原虫、巴贝虫、隐孢子虫和水生动物孢子虫等方面的应用进行综述.     

环介导等温扩增技术检测旋毛虫的研究

目的 用环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术检测旋毛虫DNA. 方法 用酚-氯仿法提取旋毛虫基因组DNA,设计两对扩增旋毛虫18S rRNA基因的LAMP引物,以日本血吸虫DNA作对照,进行LAMP反应.将旋毛虫基因组DNA经梯度稀释,进行LAMP反应,验证该方法的敏感性.产物经SYBR Green I显色后观察结果,绿色判为阳性,棕色判为阴性. 结果 LAMP反应结果显示,旋毛虫基因检测管经显色后呈绿色,对照组均呈棕色.LAMP技术可检测到的旋毛虫最低浓度为415fg/μl. 结论 建立了检测旋毛虫DNA的LAMP技术.