荧光定量PCR快速诊断21三体综合征

目的　建立荧光定量PCR技术检测 2 1三体综合征。方法　采用PCR方法同时扩增位于 2 1号染色体上的人肝型磷酸果糖激酶基因 (humanliver typephosphofructokinasegene ,PFKL CH 2 1)和位于 1号染色体上的人肌型磷酸果糖激酶基因 (humanmuscle typephosphofructokinasegene ,PFKM CH1) ,使用SYBRGreenⅠ荧光染料处理产物、琼脂糖电泳后在凝胶成像系统进行分析 ,得出扩增产物的荧光强度对比值。用此方法检测 2 6例 2 1三体综合征患儿及 2 0名正常人。结果　 2 6例 2 1三体综合征患儿PFKL CH2 1/PFKM CH 1扩增产物的荧光强度对比值为 1.5 8± 0 .17,而正常人为 1 0 0± 0 .0 5 ,两者差异有显著性。结论　SYBRGreenⅠ荧光定量PCR技术检测 2 1三体综合征具有准确、快速、安全、实用等特点 ,有较高的临床使用价值。     

Systemic and mucosal antibody responses specific to SARS-CoV-2 during mild versus severe COVID-19

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维生素A阻抑大鼠实验性肝纤维化的病理研究

利用光镜、电镜、免疫组化和形态学定量技术动态研究维生素A对大鼠四氯化碳肝纤维化的抑制作用。结果表明,维生素A可减少四氯化碳中毒大鼠肝内纤维连接蛋白和Ⅰ、Ⅲ型胶原沉积,抑制贮脂细胞向成纤维细胞转化,并可明显地减轻肝纤维化程度。本文还对维生素A抑制肝纤维化的机理及意义作了初步探讨。     

家兔实验性动脉粥样硬化血管重塑的形态定量分析

目的 :探讨AS血管重塑的几何形态变化规律。方法 :将 40只新西兰雄性家兔随机等分为正常对照组 (C组 )和动脉粥样硬化模型组 (AS组 ) ,分别于实验的第 4、8、12周末取各组家兔的胸主动脉进行定性观察及定量分析。结果 :随时间延长 ,AS组斑块面积逐渐增大 ,斑块检出率逐渐增加。管腔面积在早期斑块形成时并无改变 ,晚期则明显缩小。斑块面积及管腔面积均分别与内弹性膜包围面积 (IELA)、外弹性膜包围面积 (EELA)呈正相关关系 (P <0 .0 5 ) ,IELA和EELA间也呈显著正相关关系 (P <0 .0 1) ,但斑块面积与管腔面积间并无直线相关关系 (P >0 .0 5 )。结论 :家兔AS病变随时间延长逐渐加重 ,且在斑块形成时伴内弹性膜和外弹性膜的同时扩张 ,管腔狭窄与否主要与IELA和EELA相关。IELA和EELA可作为判断管腔狭窄及评价血管重塑的指标。     

Setting out the frame conditions for feasible use of FFPE derived RNA

Introduction

The usage of formalin-fixed paraffin embedded (FFPE) tissue is characterized by its long shelf-life and simple handling. Therefore it is the most commonly available tissue specimen in routine diagnostics and histological studies. Formaldehyde fixation may result in RNA degradation and cross linking with proteins, while storage conditions also affect RNA integrity. The present study was designed to investigate the influence of these factors on RNA analysis.

荧光定量PCR法在重组逆转录病毒pLXSN-BDNF滴度测定中的应用

目的制备含大鼠脑源性神经营养因子（BDNF）基因的逆转录病毒，建立荧光定量PCR测定滴度的方法。方法扩增BDNF基因，构建重组质粒pLXSN-BDNF，将其转染包装细胞，经G418筛选、病毒浓缩后用荧光定量PCR法和克隆形成法测定滴度。结果经PCR、酶切和测序鉴定，BDNF基因成功插入逆转录病毒载体中，筛选得到稳定的分泌重组病毒细胞系。重组病毒100倍浓缩后经荧光定量PCR法和克隆形成法测定的滴度分别为6．92×10^6拷贝/ml和3．2×10^5CFU/ml，两种方法测得的结果差异有统计学意义（P〈0．05）。结论成功扩增BDNF基因，制备了重组病毒pLX—SN-BDNF，建立了荧光定量PCR检测滴度的方法，为基因治疗青光眼性视神经损伤的实验研究提供重要参考指标。     

Reliable and reproducible LightCycler qPCR for HIV-1 DNA 2-LTR circles

Highly active anti-retroviral therapy (HAART) has reduced the plasma load of HIV-1 to undetectable levels. It has however failed to eliminate the virus from other body compartments. Current methods for monitoring persistent viral replication in HIV-1+ patients require a large amount of blood and/or repeated tissue biopsies. Furthermore, some of the viral reservoirs, such as brain and eye, are inaccessible for sampling. The detection of episomal HIV-1 DNA 2-LTR circles in CD4+ cells is indicative of recent, acute infection events. This paper describes a reliable and reproducible LightCycler-based assay for the quantitative measurement of HIV-1 DNA 2-LTR circles in human peripheral blood mononuclear (PBMN) cells. It details the modifications to the DNA extraction procedure and to the LightCycler PCR procedure that were required to achieve this. This new surrogate marker of persistent viral replication can now be reliably, reproducibly and robustly used to study the clinical progress of large numbers of patients whose plasma HIV-1 RNA has been reduced to undetectable levels by anti-retroviral drugs.     

Chromosome aberrations in B-cell chronic lymphocytic leukemia: reassessment based on molecular cytogenetic analysis

In B-cell chronic lymphocytic leukemia (B-CLL) clonal chromosome aberrations are detected in approximately 40–50% of tumors when using conventional chromosome banding analysis. Most studies find trisomy 12 to be the most frequent chromosome aberration, followed by structural aberrations of the long arm of chromosomes 13 and 14. Trisomy 12 and the ”14q+” marker are associated with shorter survival times, while the patients with 13q abnormalities have a favorable outcome, similar to those with a normal karyotype. The development of molecular cytogenetic techniques has greatly improved our ability to detect chromosome aberrations in tumor cells. Using fluorescence in situ hybridization, chromosome aberrations can be detected not only in dividing cells but also in interphase nuclei, an approach referred to as interphase cytogenetics. The prevalence of specific aberrations in B-CLL is currently being reassessed by interphase cytogenetics. By far the most frequent abnormality are deletions involving chromosome band 13q14, followed by deletions of the genomic region 11q22.3-q23.1, trisomy 12, deletions of 6q21-q23, and deletions/mutations of the TP53 tumor suppressor gene at 17p13. The evaluation of the true incidence of these aberrations now provides the basis for more accurate correlations with clinical characteristics and outcome. Deletions/mutations of the TP53 gene have been shown to be associated with resistance to treatment and to be an independent marker for poor survival. 11q deletions have been associated with extensive nodal involvement, rapid disease progression, and short survival times. Whether trisomy 12, 13q14, and 6q deletions have a prognostic impact awaits further study. The application of these molecular cytogenetic techniques will also contribute to the identification of the pathogenetically relevant genes that are affected by the chromosome aberrations in B-CLL. Received: 2 February 1998 / Accepted: 31 March 1998     

Detection of numerical chromosomal abnormalities in malignant cells on body fluids by fluorescence in situ hybridization of interphase cell nuclei with chromosome-specific probes.

To detect numerical chromosomal abnormalities (NCA) in malignant cells on body fluids, Fluorescence in situ hybridization (FISH) technique was tested in clinical specimens from patients with metastatic disease. Directly labeled DNA probes specific for chromosomes 8, 12, X, and Y (Imagenetics, Naperville, IL) were used for in situ hybridization to interphase cell nuclei. Fifteen body fluids (BF) from various sites were studied. Based initially on the Papanicolaou-stained slides, there were seven malignant and eight benign samples. Blind analysis (200 cells/sample) showed that all benign samples had a normal number of chromosomes, whereas six of seven malignant samples showed different NCA comprising 5-60% of the cell population ranging from three to 10 chromosome signals per cell. We conclude that interphase cytogenetic cell analysis of BF by FISH is: (1) feasible and gives superior signals for detection of NCA, (2) helpful in detecting malignant cells, (3) relatively simple with a turnaround time of less than 24 hr. This method may have diagnostic and prognostic application in the study of the biologic behavior of malignant neoplasms.     

Development of a quantitative monoclonal antibody‐based immunoassay for humicola ianuginosa on rice grains and comparison with conventional assays

A sensitive, specific, quantitative enzyme‐linked immunosorbent assay (ELISA) has been developed that can be used to determine the extent of mycelial growth of a sporulating thermophilic fungus, Humicola lanuginosa on the surface of rice grains. The assay employs a monoclonal antibody EC6, developed in a previous study, which does not recognize spores of the fungus. Using antigen‐coated wells, a direct linear relationship was established between dilutions of extracts from freeze‐dried mycelium (0.5 to 3 μg/ml) and absorbance values but to eliminate day‐to‐day variations it was found to be necessary to run a dilution series, prepared from stock freeze‐dried mycelium, with every test sample. The ELISA method was compared with conventional quantitative methods. Estimates of total mycelial length in freeze‐dried material by ELISA were found to be in the same order of magnitude as those determined by ergosterol and a theoretical calculation. The ELISA method also compared favourably with direct linear measurements (by photomicrography) of live mycelium present in aliquots from homogenates of a 1 cm² plug taken from a plate but estimates of the latter by the dilution plate count method were much lower. In assays with inoculated rice grains, the quantitative ELISA method proved more sensitive than either the ergosterol method or direct plating of surface‐sterilized grains. The ELISA method also has the advantage of being highly specific and quick to conduct.