Development of sandwich-form biosensor to detect Mycobacterium tuberculosis complex in clinical sputum specimens

Mycobacterium tuberculosis, the causing agent of tuberculosis, comes second only after HIV on the list of infectious agents slaughtering many worldwide. Due to the limitations behind the conventional detection methods, it is therefore critical to develop new sensitive sensing systems capable of quick detection of the infectious agent. In the present study, the surface modified cadmium-telluride quantum dots and gold nanoparticles conjunct with two specific oligonucleotides against early secretory antigenic target 6 were used to develop a sandwich-form fluorescence resonance energy transfer-based biosensor to detect M. tuberculosis complex and differentiate M. tuberculosis and M. bovis Bacille Calmette–Guerin simultaneously. The sensitivity and specificity of the newly developed biosensor were 94.2% and 86.6%, respectively, while the sensitivity and specificity of polymerase chain reaction and nested polymerase chain reaction were considerably lower, 74.2%, 73.3% and 82.8%, 80%, respectively. The detection limits of the sandwich-form fluorescence resonance energy transfer-based biosensor were far lower (10 fg) than those of the polymerase chain reaction and nested polymerase chain reaction (100 fg). Although the cost of the developed nanobiosensor was slightly higher than those of the polymerase chain reaction-based techniques, its unique advantages in terms of turnaround time, higher sensitivity and specificity, as well as a 10-fold lower detection limit would clearly recommend this test as a more appropriate and cost-effective tool for large scale operations.     

Trisomy 7 in synovial fluid cells of patients with rheumatoid arthritis

Objective Recent studies revealed trisomy 7 as a chromosomal abnormality in non-neoplastic disorders such as rheumatoid arthritis (RA). In the present study, we investigated the presence of trisomy 7 in the synovial fluid cells of patients with RA using fluorescence in situ hybridisation (FISH) analysis.Methods Synovial fluid from 15 patients with RA was collected from knee joints. The control group consisted of seven patients with traumatic synovial effusion in their knee joints. The arthrocenteses were performed under aseptic conditions. Dual-colour FISH analysis was performed using chromosome-7-specific LSI D7S522 (7q31) and chromosome-5-specific LSI EGR1 (5q31)/D5S721 (5p15.2) probes on the slides prepared from synovial fluid of RA patients and controls.Results The slides of our cases were analysed using two different DNA probes. When the slides hybridised with chromosome-5-specific probes were analysed, no trisomic or monosomic cells were revealed in both patients and controls. However, in eight of 15 patients, trisomy 7 occurred in variable percentages of cells (23% to 48%) of synovial fluid. No monosomic 7 cells were detected in these specimens. All control cases were disomic for chromosome 7.Conclusion The results of the present investigation suggest that trisomy 7 may play a role in the pathogenesis of synovial hyperproliferation in RA.     

A method for the detection and quantification of bacteria in human carious dentine using fluorescent in situ hybridisation

Objective. Previous studies have evaluated bacterial numbers in carious dentine using conventional culturing methods, capable of detecting only a proportion of the total bacteria present within lesions. The aim of this study was to detect and enumerate the total bacterial population present in carious human dentine by means of fluorescent in situ hybridisation.

Method. Five freshly extracted carious molars were sequentially hand excavated under sterile conditions through four levels in the lesions. Replicate samples were probed with a rhodamine-tagged, 16S rRNA-directed probe (EUB338), specific for the bacterial domain. Two of the five original samples were examined using fluorescence microscopy and by using a systematic visual counting strategy; direct enumeration of the bacterial population in carious dentine was performed.

Results. In the superficial, middle and deep/excavation front zones, a mean of 7.34×10⁶ (standard error of the mean, SEM±0.44), 5.23×10⁶ (SEM±0.18), and 1.69×10⁶ (SEM±0.15) total bacteria/mg dentine were found, respectively. In the advancing front zone (beyond the conventional clinical excavation boundary) a mean of 0.34×10⁶ (SEM±0.05) total bacteria/mg dentine was recorded.

Conclusion. A bacterial enumeration strategy was developed and detected greater numbers of bacteria through the depth of carious lesions that had been reported previously. The technique could be further developed using species-specific probes to determine the distribution, abundance and viability of all bacteria in carious dentine. This new information in turn will lead to a better understanding of the pathological process of caries and ultimately, its clinical treatment.     

拓展眼底自身荧光临床研究的广度和深度,提升其应用研究水平

眼底自身荧光(FAF)主要来源于视网膜色素上皮(RPE)细胞内堆积的代谢产物脂褐质,是活体条件下评价RPE活性与形态学改变的重要检测下具.作为一种无损伤、可重复、简便快捷的检测手段,FAF结合其他眼底形态与功能检测方法,可从RPE结构和功能变化的角度为部分常见眼底疾病提供辅助诊断依据.在逐步认识各种眼底疾病FAF表现特...     

多发性一过性白点综合征的临床和眼底血管造影特征

目的 观察多发性一过性白点综合征(MEWDS)的临床表现和眼底血管造影特征.方法 经荧光素眼底血管造影(FFA)和吲哚青绿血管造影(ICGA)检查确诊的40例MEWDS患者40只眼的临床资料纳入研究.分析其临床特征、FFA和ICGA改变.结果 40例患者均为单眼发病,其中,男性12例,女性28例;年龄16～64岁,平均年龄29.4岁.初诊时最佳矫正视力0.1～1.0,平均最佳矫正视力0.82.眼底检查均可见多个灰白色圆形斑点状病灶,直径约100～500 μm,边界欠清晰;位于视网膜深层或视网膜色素上皮层.部分患者伴有轻度玻璃体混浊.FFA检查早期发现所有患者在白点状病灶对应部位可见有圆形或环形强荧光斑,晚期着染;18例患者出现视盘强荧光,并伴有视盘周围静脉渗漏;7例患者出现黄斑区强荧光斑.ICGA造影早期可见到视网膜后极部及周边部弱荧光斑,晚期持续存在,无明显渗漏;与FFA 检查比较,其弱荧光斑数目多且明显.所有患者的主诉症状均在6～8周内消失.结论 MEWDS患者眼底表现为多发性灰白色点状病灶;FFA早期表现为圆形强荧光和视网膜血管异常;ICGA检查可清晰显示视网膜后极部及中周部弱荧光斑,病灶数目明显多于FFA检查和检眼镜检查所见.

Abstract：

Objective To observe the clinical and fundus angiography characteristics of multiple evanescent white dot syndrome (MEWDS). Methods Forty eyes of 40 patients (12 males/28 females) with MEWDS, diagnosed by fundus fluorescein angiography (FFA) or indocyanine green angiography (ICGA)were enrolled. All cases were unilateral. The age was ranged from 16 to 64 years old, with a mean of 29.4years. The initial average corrected vision was ranged from 0.1 to 1.0, with a mean of 0.82. The characteristics of clinical manifestations, the features of FFA and ICGA were analyzed. Results Multiple gray-white dots (100-500 μm) were found throughout the posterior pole and the mid-periphery areas. The lesions were at the depth of outer retina and retinal pigment epithelium layers. Some patients presented with mild vitreous opacity. FFA showed round or ring hyper-fluorescence spots at the early stage and tissue staining at the late stage, corresponding to the gray-white dots. Hyper-fluorescence spots and leakages at the retinal veins near optic disk were seen in 18 patients. The hyper-fluorescence spots near macular area were found in 7 patients. ICGA showed that numerous dark hypo-fluorescent dots in the mid-periphery and posterior pole at the early stage and no leakage at the late stage. ICGA detected more lesions than FFA. All of the patients were recovered without any visual complications within 6-8 weeks. Conclusions MEWDS patients have multiple fundus gray-white dots, and hyper-fluorescence and the abnormal retinal vessels by FFA, and multiple weak hypo-fluorescent spots throughout the posterior pole and the mid-periphery areas clearly on ICGA. The ICGA showed more lesions than the ophthalmoscope and FFA examination.     

Fluorimetric analysis of mebendazole and flubendazole with hydrogen peroxide

Analytically useful fluorescence was obtained from mebendazole and flubendazole after reaction with hydrogen peroxide in alkaline solution. The fluorescence produced (lambda(ex) = 300 nm; lambda(em) = 360 nm) is stable and concentrations of 0.1 mug ml(-1) can be determined. The method has been applied to the analysis of some commercially available anthelmintic preparations.     

乙肝患者肝组织中HBVcccDNA与血清中HBVDNA、HBeAg的关系

目的 探讨乙肝患者肝组织中HBVcccDNA与血清中HBVDNA、HBeAg的关系.方法 采用实时荧光定量聚合酶链反应检测乙肝患者肝组织中HBVcccDNA及血清HBVDNA,同时采用ELISA检测其血清中免疫指标HBeAg.结果 30例HBsAg、HBeAg、抗HBc阳性的乙肝患者血清中HBVDNA阳性28例阳性率93.33%,肝组织中HBVcccDNA阳性19例阳性率63.33%,两者比较差异有显著性P＜0.05.30例HBsAg、抗HBe、抗HBc阳性的乙肝患者血清中HBVDNA阳性8例阳性率26.67%,肝组织中HBVcccDNA阳性5例阳性率16.67%,两者比较差异无显著性P＞0.05.10例脂肪肝患者血清HBVDNA阴性,肝组织中未检出HBVcccDNA.结论 (1)血清HBVDNA或HBeAg阳性并不能完全确定肝内一定存在乙肝病毒正在复制.(2)HBeAg阴性抗HBe阳性不能完全确定肝内一定没有乙肝病毒复制.(3)要确定肝内病毒足否存在复制必须通过肝组织HB-VcccDNA检测来证实.     

用荧光偏振免疫法和微生物法比较研究庆大霉素在人体内的药代动力学

本文用新建立的荧光偏振免疫法(FPIA)和微生物法(MA)平行测定90例临床庆大霉素血清浓度,线性回归分析发现两种方法有较高的相关性(r=0.98)。但用MA法测得庆大霉素的平均浓度明显高于FPIA的平均浓度。5例病人庆大霉素药动学研究表明,两种方法所得血浓数据经计算机拟合符合单室模型,动力学参数Vd,k,T_(1/2)和CL及由此推算的给药剂量均无显著差异(p>0.05)。     

A cell membrane correlate of tardive dyskinesia in patients treated with phenothiazines

Phenothiazine administration to psychiatric patients is associated with an increase in the structural order of platelet membranes as determined by steady-state fluorescence polarization measurements with 1,6-diphenyl-1,3,5-hexatriene (DPH), a fluorescent probe that localizes preferentially in the hydrocarbon region of cell membranes (Zubenko and Cohen 1984, 1985a, b). In this study, platelet membranes prepared from a group of psychiatric patients who developed tardive dyskinesia following chronic treatment with phenothiazines exhibited a significant elevation in DPH fluorescence polarization when compared to similar preparations from an otherwise matched group of patients who had no symptoms or history of tardive dyskinesia. The distribution of polarization values obtained for the tardive dyskinesia group displayed minimal overlap with that of an unmedicated, psychiatrically-healthy control group matched for age and gender. The fluorescence polarization of DPH-labelled platelet membranes was not significantly correlated with phenothiazine daily dose or serum cholesterol concentration in the phenothiazine-treated patient groups, or with dyskinesia severity (AIMS rating) in the tardive dyskinesia group. Patient gender and the presence of an affective disorder did not significantly correlate with DPH fluorescence polarization. The potential physiological and clinical significance of these findings is discussed.     

Acute lymphoblastic leukaemia associated antigen. IV Expression on non-leukaemic ‘lymphoid’ cells

The cellular specificity of the common ALL (cALL) membrane (gp100) antigen and the p28, 33 (Ia-like) antigen has been further explored. Non-leukaemic cells with a variable and usually weak expression of the cALL antigen can be demonstrated in foetal haemopoietic tissue, in bone marrow of normal children, in the marrow of children with a variety of non-leukaemic haematologic and non-haematologic disorders. In normal children the cALL antigen positive cells appear to be restricted to the bone marrow. These cells are regularly detected in the marrow of many leukaemic (ALL, AML) and non-leukaemic children with post-chemotherapy associated lymphocytosis, in marrow transplant recipients, and in neonatal ‘leukaemoid’ reactions. When the number of cALL⁺ cells is monitored in children following cessation of maintenance chemotherapy for ALL, some prognostic correlation can be observed; in general, however, the numbers of ‘lymphoid’ cells bearing these leukaemia associated antigens have no clear predictive value with respect to relapse.Analysis by simultaneous markers coupled with flow microfluorimetry and sorting indicates that the majority of cALL antigen⁺ cells (> 90%) are positive for the p28, 33 (Ia-like) antigen but do not express markers of mature lymphocytes (T antigen, E sheep, E mouse rosettes, surface immunoglobulin), binding sites for complement and IgG or a myeloid membrane antigen. More than 90% of pre-B cells (i.e. cyt IgM⁺, SmIg^?) are p28, 33 positive but only a small proportion (5–15%) are cALL antigen positive. These and additional data support the view that the cALL and p28, 33 (Ia) antigens are normal ‘early’ differentiation antigens of the lymphoid lineage.