运用数字PCR检测乳腺癌组织FFPE样品中人表皮生长因子受体2拷贝数的变化

目的:建立数字PCR(dd PCR)法检测乳腺癌福尔马林固定石蜡包埋(FFPE)切片样品中人表皮生长因子受体2(HER2)的拷贝数变化(CNV),并与免疫组化(IHC)、荧光原位杂交(FISH)结果比较,以寻求更客观、准确、通量的肿瘤标志物检测方法。方法:以细胞系293T、HOEC、SKOV3基因组DNA(g DNA)为模板,对靶基因HER2及校正基因CEP-17建立双通道dd PCR检测法,并对21例IHC检测HER2阳性(++/+++)的乳腺癌FFPE样品进行CNV(HER2/CEP-17)检测,与IHC、FISH结果比较。结果:单、双通道法对HER2 CNV检测结果一致;细胞及临床样品检测结果表明,dd PCR检测HER2 CNV的阴性、阳性Cut-off值分别为1.2、2.1,中间值为1.2~2.1。14例FISH阴性标本中,dd PCR的一致性为93%(13/14);3例IHC"+++"的样品中,FISH检测均为阳性,2例dd PCR检测为阳性,1例(17#)检测临界阳性。2例IHC"++"的样品,FISH检测阳性,但dd PCR检测为阴性。结论:dd PCR能检测样本HER2 CNV,且与FISH结果判读部分一致,具有临床应用前景,但仍需大量样品来进行验证及建立结果判断标准。     

羊源性基因组DNA标准物质研制

近年来,羊肉及其肉制品的掺假等食品安全事件层出不穷,为了完善市场执法检查法律依据,利用数字PCR定值羊HELZ基因,研制了羊基因组DNA标准物质。由于标准物质可以用于衡量检测方法的准确性,因此可以判定食品及相关制品中羊肉的掺假情况。利用数字PCR对研制的标准物质进行均匀性和稳定性评估,结果表明该批标准物质均匀性良好,在4℃、25℃可以稳定保存14 d,在-20℃可以稳定保存6个月。来自全国9个不同实验室羊源性基因组DNA标准物质(高浓度)和羊源性基因组DNA标准物质(低浓度)的联合定值结果显示,标准值及其扩展不确定度分别为(5.44±0.45)×10³ copies·μL^-1和(5.68±0.54)×10² copies·μL^-1。该羊源性基因组DNA标准物质为动物源性标准物质的市场应用提供了技术基础,完善了羊源性基因组DNA标准物质的制备、定量检测、质量控制和量值溯源技术平台。     

牛绝对端粒长度测定及DNA提取造成的影响

目的：端粒是真核生物染色体末端的一种高度保守的负责维持染色体稳定的特殊结构,其DNA序列长度即端粒长度,会随着年龄增长或疾病发生发展而逐渐缩短,检测端粒长度可以为评估机体衰老和健康状况提供参考,但目前缺乏测定微量牛DNA样本绝对端粒长度的方法;通过实时荧光定量PCR(real-time quantitative PCR, qPCR)实现微量牛DNA样本绝对端粒长度的测定并评估DNA提取方法对牛绝对端粒长度测定结果的影响,为进行端粒长度研究时选择合适的DNA提取方法和端粒长度分析方法提供参考。方法：利用标准曲线对检测样本的端粒和内参Ct值进行转换,通过qPCR测定牛端粒长度绝对值;采用膜吸附法、苯酚-氯仿法和磁珠法3种方法分别提取相同样本的DNA,分别用端粒末端限制性片段(terminal restriction fragment, TRF)分析法和qPCR法分析端粒长度,比较不同DNA提取方法对牛绝对端粒长度测定的影响。结果：(1)qPCR可以测定纳克级别DNA样本的绝对端粒长度,检测结果重复性良好,并且和“金标准”TRF测定结果的相关性良好。(2)不同方法提取的DNA用TRF分析法和...     

基于国产微滴数字PCR的SNP快速检测技术研究

目的 在法医学领域,现有的SNP检测主要依赖进口,检测工作量大、耗时长且成本较高。微滴数字PCR (droplet digital PCR,ddPCR)作为新一代的PCR技术,可以快速检测低浓度样本DNA,且有较强的抗干扰能力。本研究旨在国产ddPCR平台建立SNP分型检测体系并对其性能进行评估,以探讨ddPCR技术在法医学检验领域的应用价值。方法 在ddPCR平台建立高原适应性EPAS1单倍型(rs115321619、rs73926263、rs73926264、rs73926265和rs55981512)检测体系,测试各位点引物探针特异性,对体系的准确性、稳定性、灵敏度、检材适应性进行评估,并比较了ddPCR和SNaPshot微测序检测体系的抗抑制性,最后对样本地区来源进行测试。结果 ddPCR在2.5 h内即可快速获取检测结果,体系准确性和稳定性好,检测灵敏度为0.312 5 ng,且抗抑制性能力突出。70份测试样本检测结果与背景信息一致。结论 基于ddPCR的SNP检测体系具有准确可靠、简便快速、抗抑制能力强等优势,在法医学快速检验领域有较强的应用潜力,适合法医现场检验需求。     

DNA提取过程中混入的实验室空气微生物DNA定量

元基因组测序方法为微生物研究提供了有力的工具。但其中的DNA提取过程,会不可避免地混入实验室中的空气微生物。这些微生物DNA,是否会对一些极微量的元基因组检测 (如皮肤样本等) 结果造成影响,有多大影响,仍没有明确结论。本研究首先收集了实验室空气样品,用16S rRNA引物建立了基于qPCR的标准曲线,并检测了在开放环境下提取DNA过程中可掺杂的环境微生物DNA量。然后在开放环境下提取纯水DNA样品并进行元基因组分析,以确定掺杂环境微生物的种类。最后分别在生物安全柜和实验室开放环境下提取皮肤样本,并用鸟枪测序方法对样本的微生物组成进行分析,以评估掺杂环境微生物对元基因组检测结果的影响。结果显示,在实验室开放环境的DNA提取过程中,环境微生物的DNA残留可达28.9 pg,可达某些极微量样本DNA总量的30%。元基因组分析显示,样品中掺杂的环境微生物主要是痤疮杆菌Cutibacterium acnes、大肠杆菌Escherichia coli等皮肤常见细菌。与洁净皮肤样本的信息相比,开放环境下提取掺杂了数十种环境微生物,并导致主要菌种的丰度大幅降低,从而影响结果的真实性。因此,微量样品的DNA提取应在洁净环境下执行。     

基于微滴式数字PCR方法的鱼类环境DNA样本处理与保存技术优化

以实验室内的鲫(Carassius auratus)为研究对象,利用微滴式数字PCR(Droplet Digital PCR,ddPCR)定量技术,优化了鱼类环境DNA(Environmental DNA,eDNA)样本的捕获、提取和保存方法,并对免DNA提取的PCR直扩技术进行了探索.研究结果如下:(1)在同一孔径、...     

木糖葡萄球菌实时荧光定量PCR检测方法的建立及其应用

目的 本研究拟建立一种灵敏快速的实时荧光定量PCR(real-time quantitative PCR, qPCR)方法,用于检测大、小鼠木糖葡萄球菌(Staphylococcus xylosus,S.xylosus)。方法 本研究选择特异性gehM基因片段作为靶标合成了一套引物,建立了木糖葡萄球菌检测的qPCR方法。对木糖葡萄球菌标准菌株和其他非目标菌进行特异性分析。将木糖葡萄球菌的DNA进行10倍稀释测定其灵敏度。用送检的样本进行了临床应用并测序验证,同时与培养法进行比较。结果 仅木糖葡萄球菌出现特异性扩增曲线,而其他非目标菌未出现,表明设计的引物对木糖葡萄球菌具有特异性,灵敏度为100 fg/μL,组内和组间重复性均小于3%。共检测60份临床样品,有5份样品扩增曲线为典型的S曲线,将该qPCR产物克隆测序并进行同源性比对,该序列与木糖葡萄球菌的同源性为99.63%,表明该样本木糖葡萄球菌核酸阳性,所检测样本阳性率为8.3%,而培养法的阳性率为6.7%,qPCR方法阳性检出率比培养法略高。结论 建立的木糖葡萄球菌qPCR方法,具有快速、灵敏度高、特异性强和重复性好的优点,可用于实...     

基于CRISPR/Cas13a的金黄色葡萄球菌mecA耐药基因检测方法的建立

【目的】 针对目前耐药基因检测通常需要依赖专业检测设施和设备,仍缺乏耐药基因快速检测方法这一问题,旨在建立一种基于成簇规律间隔的短回文重复序列(clustered regularly interspaced short palindromic repeats, CRISPR)的金黄色葡萄球菌(Staphylococcus aureus) mecA耐药基因快速检测方法。【方法】 首先在mecA基因序列的保守区中设计筛选出灵敏度较高的重组酶介导链替换核酸扩增(recombinase aided amplification, RAA)引物和CRISPR RNA (CRISPR RNA, crRNA),通过结合消线法核酸检测试纸技术(easy-readout and sensitive enhanced, ERASE)建立针对mecA基因的检测方法,最后利用模拟样本及临床分离样本对建立的新方法与传统方法进行比较。【结果】 成功筛选出了1组针对mecA耐药基因的高效扩增引物和crRNA,并建立了基于CRISPR-ERASE的mecA耐药基因高灵敏核酸检测方法,最低检出限为10 copies/μL,在32株临床分离的金黄色葡萄球菌中,该方法共检出24株mecA耐药基因阳性菌株,与药敏试验及荧光定量PCR (quantitative real-time PCR, qPCR)检测结果符合率为100%。【结论】 建立了一种基于CRISPR-ERASE核酸检测试纸技术的简单、高灵敏的mecA耐药基因检测方法。     

标准品制备方法对FQ-PCR定量基因表达水平的影响

实时荧光定量PCR (FQ-PCR)标准曲线法准确定量基因表达的关键在于标准品与待检样本的扩增效率是否一致. 为检测DNA标准品与样本cDNA扩增效率的一致性,探讨定量用标准品的最佳制备方法,本研究以脂肪酸结合蛋白5(Fabp5)、过氧化物酶体增殖活化受体α (Ppar-α)及β肌动蛋白（β-Actin）的3个基因为对象,分别采用质粒纯化法、PCR产物直接纯化法、PCR产物凝胶回收法制备DNA标准品,10倍梯度稀释后用FQ PCR制作标准曲线. 并以10倍梯度稀释的样本cDNA标准曲线的参数为对照,进行比较分析. 结果表明,不同方法制备的DNA标准品的扩增效率差异较大,并且与cDNA的扩增效率不一致,不能对cDNA样本进行准确定量. 另外,虽然目的基因在cDNA样本中的拷贝未知,不能对基因表达水平进行绝对定量,但因不同cDNA样本的同一基因的扩增效率一致, 可对基因的表达进行准确的相对定量.     

应用RAPD技术鉴定地霉的实验研究

目的探讨RAPD技术在快速鉴定地霉中应用。方法用E.Z.N.A.yeastDNAkit提取地霉菌基因组DNA,采用随机引物AP3(5'-TCGTAGCCAA-3')、ATG(5'-ATGGATCGGC-3')、RP2(5'-AAGGATCAGA-3')、OPA-10(5'-GTGATCGCAG-3')对临床上致病性白地霉、林生地霉皮损株和血液株的基因组DNA进行扩增,对各病原菌的DNA指纹的特征进行分析。结果成功提取了地霉的基因组DNA,其纯度和浓度均能满足PCR反应的要求。利用4种引物对基因组DNA进行扩增,不同种真菌的DNA显示不同的DNA带型,分离自不同感染部位的同种不同株真菌的DNA显示的主要DNA带型基本一致。结论采用E.Z.N.A.yeastDNAkit提取的地霉基因组DNA可以用于PCR反应。RAPD法鉴定地霉菌简单、快速、特异,可用于临床诊断。     

Foreign exploration for Scirtothrips perseae Nakahara (Thysanoptera: Thripidae) and associated natural enemies on avocado (Persea americana Miller)

Scirtothrips perseae Nakahara was discovered attacking avocados in California, USA, in 1996. Host plant surveys in California indicated that S. perseae has a highly restricted host range with larvae being found only on avocados, while adults were collected from 11 different plant species. As part of a management program for this pest, a “classical” biological control program was initiated and foreign exploration was conducted to delineate the home range of S. perseae, to survey for associated natural enemies and inventory other species of phytophagous thrips on avocados grown in Mexico, Guatemala, Costa Rica, the Dominican Republic, Trinidad, and Brazil. Foreign exploration efforts indicate that S. perseae occurs on avocados grown at high altitudes (>1500 m) from Uruapan in Mexico south to areas around Guatemala City in Guatemala. In Costa Rica, S. perseae is replaced by an undescribed congener as the dominant phytophagous thrips on avocados grown at high altitudes (>1300 m). No species of Scirtothrips were found on avocados in the Dominican Republic, Trinidad, or Brazil. In total, 2136 phytophagous thrips were collected and identified, representing over 47 identified species from at least 19 genera. The significance of these species records is discussed. Of collected material 4% were potential thrips biological control agents. Natural enemies were dominated by six genera of predatory thrips (Aeolothrips, Aleurodothrips, Franklinothrips, Leptothrips, Scolothrips, and Karnyothrips). One genus each of parasitoid (Ceranisus) and predatory mite (Balaustium) were found. Based on the results of our sampling techniques, prospects for the importation of thrips natural enemies for use in a “classical” biological control program in California against S. perseae are not promising.     

Characterization of SSEA-1 glycolipids from the brain of a patient with fucosidosis

Neutral glycolipids from the brain of a patient with Fucosidosis were analyzed and two complex glycolipids containing five and eight sugars were isolated from the cortical grey matter. These two glycolipids reacted with antibodies recognizing the SSEA-1 [Le^x(X)] carbohydrate determinant. SSEA-1 glycolipids are normally expressed in human embryonic brain but are found in only small amounts in postnatal human brain. The accumulation of the two SSEA-1 glycolipids in Fucosidosis brain thus represents a defect which affects the normal developmentally regulated decrease in postnatal, expression of these glycolipids, and may be a contributing factor in the abnormal brain development associated with the disease. Chemical characterization of the two isolated glycolipids by gas chromatographic and mass spectrometric analyses has identified the two glycolipids as lacto-N-fucopentaosylceramide (III) and difucosyl-neolactonorhexaosylceramide.Abbreviations DCl direct chemical ionization - FAB tastatiom bombardment - GC gas chromatography - GSLs glycosphingolipids - MS mass spectrometry - SSEA-1 stage specific embryonic antigen-1 - TLC thin layer chromatographys     

Genome Analysis inNeurospora crassa;Cloning of Four Lociarginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) and Associated Chromosome Walking

We have cloned fourNeurospora crassagenes by complementation analysis. Cloned genes include thearginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) loci. Chromosome walks were initiated in ordered cosmid libraries from the cloned loci. A total of about 700 kb of theNeurosporagenome is covered in these walks.     

1H and13C-NMR analysis of four pentasaccharides from urine of blood-group A and B,Leb adults. Confirmation of the structure of two new oligosaccharides: Fuc(α1–2)[GalNAc(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc and Fuc(α1–2)[Gal(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc

The major pentasaccharides Fuc(1-2)[GalNAc(1-3)]Gal(1-4)[Fuc(1-3)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-4)[Fuc(1-3)]Glc, which are normally present in the urine of bloodgroup A Le^b and B Le^b healthy subjects, were each found to be contaminated by a minor component when analysed by¹H-NMR. The determination of these structures, Fuc(1-2) [GalNAc(1-3)]Gal(1-3)[Fuc(1-4)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-3)[Fuc(1-4)]Glc, was based on the results of methylation analysis and¹H/¹³C-NMR spectroscopy.Abbreviations HPLC high performance liquid chromatography - GLC gas liquid chromatography - NMR nuclear magnetic resonance - COSY correlation spectroscopy - Gal d-galactopyranose - GalNAc 2-acetamido-2-deoxy-d-galactopyranose - Glc d-glucopyranose - Fuc l-fucopyranose - LNDFH I lacto-N-difucohexaose I (Le^b determinant     

“HAIRY CANOLA” – Arabidopsis GL3 Induces a Dense Covering of Trichomes on Brassica napus Seedlings

Transformation with the Arabidopsis bHLH gene 35S:GLABRA3 (GL3) produced novel B. napus plants with an extremely dense coverage of trichomes on seedling tissues (stems and young leaves). In contrast, trichomes were strongly induced in seedling stems and moderately induced in leaves of a hairy, purple phenotype transformed with a 2.2 kb allele of the maize anthocyanin regulator LEAF COLOUR (Lc), but only weakly induced by BOOSTER (B-Peru), the maize Lc 2.4 kb allele, or the Arabidopsis trichome MYB gene GLABRA1 (GL1). B. napus plants containing only the GL3 transgene had a greater proportion of trichomes on the adaxial leaf surface, whereas all other plant types had a greater proportion on the abaxial surface. Progeny of crosses between GL3⁺ and GL1⁺ plants resulted in trichome densities intermediate between a single-insertion GL3⁺ plant and a double-insertion GL3⁺ plant. None of the transformations stimulated trichomes on Brassica cotyledons or on non-seedling tissues. A small portion of bHLH gene-induced trichomes had a swollen terminal structure. The results suggest that trichome development in B. napus may be regulated differently from Arabidopsis. They also imply that insertion of GL3 into Brassica species under a tissue-specific promoter has strong potential for developing insect-resistant crop plants. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Validation of the <Emphasis Type="Italic">VRN-H2</Emphasis>/<Emphasis Type="Italic">VRN-H1</Emphasis> epistatic model in barley reveals that intron length variation in <Emphasis Type="Italic">VRN-H1</Emphasis> may account for a continuum of vernalization sensitivity

The epistatic interaction of alleles at the VRN-H1 and VRN-H2 loci determines vernalization sensitivity in barley. To validate the current molecular model for the two-locus epistasis, we crossed homozygous vernalization-insensitive plants harboring a predicted “winter type” allele at either VRN-H1 (Dicktoo) or VRN-H2 (Oregon Wolfe Barley Dominant), or at both VRN-H (Calicuchima-sib) loci and measured the flowering time of unvernalized F₂ progeny under long-day photoperiod. We assessed whether the spring growth habit of Calicuchima-sib is an exception to the two-locus epistatic model or contains novel “spring” alleles at VRN-H1 (HvBM5A) and/or VRN-H2 (ZCCT-H) by determining allele sequence variants at these loci and their effects relative to growth habit. We found that (a) progeny with predicted “winter type” alleles at both VRN-H1 and VRN-H2 alleles exhibited an extremely delayed flowering (i.e. vernalization-sensitive) phenotype in two out of the three F₂ populations, (b) sequence flanking the vernalization critical region of HvBM5A intron 1 likely influences degree of vernalization sensitivity, (c) a winter habit is retained when ZCCT-Ha has been deleted, and (d) the ZCCT-H genes have higher levels of allelic polymorphism than other winterhardiness regulatory genes. Our results validate the model explaining the epistatic interaction of VRN-H2 and VRN-H1 under long-day conditions, demonstrate recovery of vernalization-sensitive progeny from crosses of vernalization-insensitive genotypes, show that intron length variation in VRN-H1 may account for a continuum of vernalization sensitivity, and provide molecular markers that are accurate predictors of “winter vs spring type” alleles at the VRN-H loci.     

N-andO-glycosylation of glycoprotein C synthesized by Herpes simplex virus type 1-infected ricin resistant cells

The progeny of Herpes simplex virus type 1 (HSV-1) grown in ricin-resistant 14 cells (Ric^R14) lackingN-acetylglucosaminyltransferase I was released in the extracellular medium at a very low rate. By using a monoclonal antibody immobilized on Sepharose we purified from HSV-1-infected Ric^R14 cells a viral glycoprotein (gC), which carries bothN-andO-linked oligosaccharides. Glycopeptides obtained from [³H]mannoselabeled gC by Pronase digestion were entirely susceptible to endo--N-acetylglucosaminidase H, and the major oligosaccharide released was Man₄GlcNAc. The accumulation of this high-mannose species was related to the enzymic defect of the host cells and to the long retention of the viral glycoprotein within the cells. The extent ofO-glycosylation evaluated in [¹⁴C]glucosamine-labeled gC from Ric^R14 cells as compared to that of gC from wild type cells did not appear to be significantly modified.Abbreviations Con A concanavalin A - BHK cells baby hamster kidney cells - HSV Herpes simplex virus     

Structural Peculiarities of Na+,K+-ATPase Isozymes from the Calf Brain

Functionally active preparations of Na⁺,K⁺-ATPase isozymes from calf brain that contain catalytic subunits of three types (1, 2, and 3) were obtained using two approaches: a selective removal of contaminating proteins by the Jorgensen method and a selective solubilization of the enzyme with subsequent reconstitution of their membrane structure by the Esmann method. The ouabain inhibition constants were determined for the isozymes. The real isozyme composition of the Na⁺ pump from the grey matter containing glial cells and the brain stem containing neurons was determined. The plasma membranes of glial cells were shown to contain mainly Na⁺,K⁺-ATPase of the 11 type and minor amounts of isozymes of the 22(1) and the 31(2) type. The axolemma contains 21 and 31 isozymes. A carbohydrate analysis indicated that 11 enzyme preparations from the brain grey matter substantially differ from the renal enzymes of the same composition in the glycosylation of the 1 isoform. An enhanced sensitivity of the 3 catalytic subunit of Na⁺,K⁺-ATPase from neurons to endogenous proteolysis was found. A point of specific proteolysis in the amino acid sequence PNDNR⁴⁹² Y⁴⁹³ was localized (residue numbering is that of the human 3 subunit). This sequence corresponds to one of the regions of the greatest variability in 1-, 2-, 3-, and 4-subunits, but at the same time, it is characteristic of the 3 isoforms of various species. The presence of the 3 isoform of tubulin (cytoskeletal protein) was found for the first time in the high-molecular-mass Na⁺,K⁺-ATPase 31 isozyme complex isolated from the axolemma of brain stem neurons, and its binding to the 3 catalytic subunit was shown.