用Ames试验和小鼠原代细胞彗星试验检测A市生活饮用水遗传毒性

目的　用Ames试验和小鼠原代肝细胞彗星试验检测A市生活饮用水遗传毒性并比较两种方法的敏感性。方法　采用Ames试验和彗星试验分别对A市南郊水厂的水源水、出厂水、自来水的有机浓集物的诱变性进行检测。结果　出厂水和自来水的Ames试验于试样浓度为 3 0L/皿时才表现为阳性结果 ,而肝细胞的彗星试验在水样量为0 1L时就出现明显的阳性结果 (与阴性对照比P <0 0 1) ,后者的浓度比前者低 3 0倍。水源水的Ames试验于试样浓度为 6 0L/皿时仍表现为阴性结果 ,而原代肝细胞的彗星试验在水样量为 0 5L时就出现明确的阳性结果。结论　Ames试验只能检测出氯化消毒后饮用水的致突变阳性 ,而彗星试验能同时检测出水源水和氯化消毒后饮用水的致突变性 ,后者的敏感性远远高于前者。     

ATP生物荧光检测法快速检测水细菌污染的评价

目的:探讨ATP生物荧光检测法用于水质污染检测的可行性,为水质监测的快速检测提供科学依据。方法:同时对珠江水样和大肠杆菌悬溶液进行ATP生物荧光检测和国家标准法(琼脂平板计数法),并将结果进行比较。结果:ATP生物荧光检测法得出的RLU(relative light unit,相对光单位)数值越高,其对应的活菌总数越多,ATP生物荧光检测法与国家标准法具有相关性。结论:ATP生物荧光检测法可应用于现场水质监测快速检测和污染程度评估。     

酶联免疫吸附试验监测梅毒的应用评价

目的：评价酶联免疫吸附试验（ELISA）对梅毒病人诊断的敏感性和特异性。方法：使用目前常用的梅毒密螺旋体感染诊断酶联免疫吸附试验（ELISA）对3718例2006-7-1至2007-7-1门诊及住院手术输血病人进行检测。若结果阳性再做梅毒简介血凝试验（TPHA）。结果：ELISA共检阳性37例,其中30例经临床诊断为阳性。结论：该方法灵敏度高,操作方便。     

外周微量血单细胞凝胶电泳技术的正交试验

目的研究外周微量血单细胞凝脉电泳技术的最佳实验条件.方法采用四因素三水平的正交表L9(34)对电泳电压、电泳时间、解旋时间、铺胶层数四因素作正交试验.结果解旋时间为最主要影响因素,其次分别为铺胶层数、电泳时间和电泳电压;并且各因素下的第一水平为最佳水平.结论外周微量血单细胞凝胶电泳技术的最佳实验条件为:解旋20min、铺胶3层、电泳20?min、电泳电压20V.     

Standardization of whole blood assay for determination of pyrogenic activity in organic dust samples

To characterize bioaerosol exposure at workplaces standardized methods are necessary. Activity of endotoxin, one component of organic dust, can be quantified with the Limulus-Amoebocyte Lysat test (LAL test). Further information with respect to pyrogenic activity of the organic dust can be achieved by measuring cytokine release of human blood after stimulation with the dust or its extract (whole blood assay).The aim of our study was the standardization of the whole blood assay (WBA) while using cryo-preserved human blood (Qualis Laboratorium) and to compare the outcome of the different cytokines determined by incubation of the blood cells with extracts from dust samples collected at various workplaces.Cytokine release (IL-1β, IL-6, IL-8, TNF-α, MCP-1) was measured by ELISA after stimulation of fresh blood from ten donors as well as cryo-preserved human blood. In both cases blood was stimulated with E. coli endotoxin as well as with 30 dust filter extracts from various workplaces. All dust filter extracts were investigated in the WBA using cryo-preserved blood as well as with LAL test.E. coli endotoxin stimulated the release of IL-1β, IL-6, IL-8, TNF-α and MCP-1 in a dose-dependent manner in fresh as well as cryo-preserved human whole blood.200 pg/ml E. coli endotoxin induced maximal cytokine release in cryo-preserved blood (mean value for IL-1β 2509±418 pg/ml; n=13 experiments) whereas fresh blood of single donors reached a maximum release when stimulated with 50 ng/ml endotoxin (mean value of ten donors 1125±553 pg/ml IL-1β).Using cryo-preserved blood the coefficient of variation (CV) regarding the interassay variability was below 21% for all cytokines measured.Regarding 26 dust sample extracts correlation coefficient r² for LAL test and WBA was between 0.90 and 0.93 (Pearson) for IL-1β, IL-6, IL-8 and TNF-α whereas correlation for MCP-1 was lower (r²=0.59).Two dust sample extracts which showed similar reactivity patterns in LAL test as well as in WBA with respect to IL-1β, IL-6, IL-8 and TNF-α could be differentiated by measuring MCP-1.In conclusion, cryo-preserved blood pools are suitable to standardize WBA.Combination of different outcome variables like IL-1β and MCP-1 improve the characterization from the inflammatory potency of workplace related dust samples.     

Assessment of tryptophol genotoxicity in four cell lines in vitro: a pilot study with alkaline comet assay

Tryptophol is an aromatic alcohol and secondary metabolite of the opportunistic fungus Candida albicans. Although its toxicity profile at cell level has been poorly investigated, recent data point to cytotoxic, cytostatic, and genotoxic effects in lymphocytes and the induction of apoptosis in leukaemic blood monocytes. In this pilot study we evaluated the genotoxicity of tryptophol in vitro on four permanent cell lines of animal and human origin: ovary cells, alveolar epithelium, liver cells, and blood monocytes using the alkaline comet assay. We selected cells that might be principal targets of tryptophol and other low-molecular geno(toxins) secreted by Candida albicans during host invasion. Our results suggest that tryptophol applied in vitro at 2 mmol L(-1) for 24 h damages DNA in HepG2, A549 and THP-1 cells, obviously due to bioactivation and/or decomposition of the parent compound, which results in the formation of more genotoxic compound(s) and production of reactive species that additionally damage DNA. On the other hand, notably lower levels of primary DNA damage were recorded in CHO cells, which lack metabolic activity. Future studies with tryptophol should look further into mechanisms involved in its toxic action and should focus on other cell types prone to infection with Candida spp. such as vaginal epithelial cells or keratinocytes of human origin.     

胶体金免疫层法在鼠疫监测中应用效果评价

目的 了解胶体金免疫层析法在鼠疫监测中的作用及其优劣.同时观察56℃30 min灭活与升汞盐水作用1 min对鼠疫菌F1抗体检测的影响.方法 对2006年从广西隆林县、西林县鼠疫监测中收集的鼠脏器、鼠血清以及既往鼠疫抗体阳性患者血清,利用胶体金免疫层析技术进行鼠疫菌F1抗原(脏器)与F1抗体(血清)检测.结果 鼠肝脏120份、鼠脾脏99份,胶体金法检测鼠疫菌Fl抗原均为阴性;鼠血清235份胶体金法检测鼠疫菌F1抗体为阴性、既往鼠疫阳性病人27例,胶体金检测鼠疫菌Fl抗体灭活组阳性2例,阳性率为7.41%(2/27),升汞组均为阴性.结论 升汞对鼠疫菌F1抗体的检测有抑制作用;胶体金免疫层析法具有特异性强、敏感性高、操作简便、快速等优点,在鼠疫监测中具有重要意义.     

Development of an enzyme-linked immunosorbent assay for determination of pretilachlor in water and soil

An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) has been developed for detection of pretilachlor in water and soil. An immunogen was prepared from haptens of pretilachlor conjugated to bovine serum albumin(BSA). The specific polyclonal antibodies were obtained by immunizing New Zealand white rabbits. The influence of parameters including concentrations of methanol, ionic strength and pH values were optimized to improve the sensitivity of the assay. The optimized ELISA was shown to have a high sensitivity and specificity for pretilachlor. Under optimal conditions, the ELISA has demonstrated an 50% inhibitory concentration (IC₅₀) value of 0.0359 mg/L with a limit of detection (LOD, IC₁₀) of 6.9 ng/L. The cross-reactivities to some analogs of pretilachlor (acetochlor, butachlor, metazachlor and metalaxyl) were below 1.5%. The average recoveries of pretilachlor from distilled water, tap water, paddy water and soil were in the range of 77.0-115.2% between 0.005 and 5.0 mg/L. The results of ELISA for spiked samples were confirmed by GC-ECD with a high correlation coefficient of 0.9950(n=11). Thus, the ELISA proven to be a sensitive, specific, inexpensive and quantitative tool for detection of pretilachlor from four kinds of spiked samples.     

鼠疫监测中胶体金免疫层析法的应用效果评价

目的 了解胶体金免疫层析法在鼠疫监测中的作用及其影响因素.方法 对2006年自广西隆林县、西林县鼠疫监测中收集的鼠脏器、鼠血清以及既往鼠疫阳性病人血清,利用胶体金免疫层析技术进行鼠疫菌F1抗原(脏器)与F1抗体(血清)检测.结果 鼠肝脏120份、鼠脾脏99份,胶体金法检测鼠疫菌F1抗原均为阴性;鼠血清235份胶体金法检测鼠疫菌F1抗体为阴性、既往鼠疫阳性病人27例胶体金检测鼠疫菌F1抗体灭活组阳性2例,阳性率为7.41%(2/27),升汞组均为阴性.结论 升汞对鼠疫菌F1抗体的检测有抑制作用;胶体金免疫层析法具有特异性强、敏感性高、操作简便、快速等优点,在鼠疫监测中具有重要的流行病学意义.     

Discovering and differentiating new and emerging clonal populations of Chlamydia trachomatis with a novel shotgun cell culture harvest assay

Chlamydia trachomatis is the leading cause of preventable blindness and bacterial sexually transmitted diseases worldwide. Plaque assays have been used to clonally segregate laboratory-adapted C. trachomatis strains from mixed infections, but no assays have been reported to segregate clones from recent clinical samples. We developed a novel shotgun cell culture harvest assay for this purpose because we found that recent clinical samples do not form plaques. Clones were strain-typed by using outer membrane protein A and 16S rRNA sequences. Surprisingly, ocular trachoma reference strain A/SA-1 contained clones of Chlamydophila abortus. C. abortus primarily infects ruminants and pigs and has never been identified in populations where trachoma is endemic. Three clonal variants of reference strain Ba/Apache-2 were also identified. Our findings reflect the importance of clonal isolation in identifying constituents of mixed infections containing new or emerging strains and of viable clones for research to more fully understand the dynamics of in vivo strain-mixing, evolution, and disease pathogenesis.     

ATP生物荧光检测法快速检测手污染细菌总数的评价

目的:探探索ATP生物荧光检测法用于手部细菌检查的可行性,为口岸食品从业人员手部卫生现场快速检测提供科学依据。方法:采用ATP生物荧光检测法与国标法(琼脂平板计数法)比较,检测某单位30名员工双手经洗手后、自然污染、纸币污染后细菌污染情况。结果:随着污染程度加重,ATP生物荧光检测仪上检测出的RLU(relative light units,相对荧光强度)值增大,琼脂平板计数法检出对应的手部细菌菌数也越大,对检出的RLU值的对数值与菌落总数的对数值作统计学线性相关分析发现,r=0.995,P<0.001,具有统计学意义,两者呈正相关。结论:ATP生物荧光检测法可应用于食品卫生中手部卫生现场快速检查。     

Evaluation of estrogenic activities of pesticides using an in vitro reporter gene assay

The estrogenic activities of 32 pesticides in agricultural products were evaluated using the E-CALUX assay system developed by Xenobiotic Detection Systems Inc (North Carolina, USA). This system utilizes human ovarian carcinoma cells (BG1) stably transfected with an estrogen-responsive luciferase reporter gene plasmid. It was found that tolclofos-methyl, prothiofos, diazinon, Thiabenclazole (TBZ) and pyriproxyfen had estrogenic activity. Several pesticides are often present in agricultural products. Therefore the estrogenicity of the mixtures of two kinds of pesticides was evaluated. The activity of diazinon/tolclofos-methyl, pyriproxyfen/prothiofos and TBZ/o-phenylphenol (OPP) was increased up to 1.2-5.3 fold. On the other hand, chlorfluazuron, imazalil and chlorfenapyr had anti-estrogenic activity. Further, to evaluate the change in the estrogenic activity of pesticide metabolites, an experimental system was established using a rat S9 mixture. Metabolites of permethrin and OPP had no estrogenic activity, but they had weak activity after the metabolism. On the other hand, the metabolites of TBZ exhibited less estrogenic activity than the original compounds.     

血样低温放置时间对黏多糖贮积症酶学检测的影响

目的探讨4 ℃条件下血样放置时间对黏多糖贮积症(MPS)酶学检测的影响。 方法选择2020年1月5日至9日,采集自5例健康志愿者(男性为2例,女性为3例,年龄为24~30岁)的血样各6管为研究对象。将其依次命名为血样1、2、3、4、5(研究组)。将采集的5×6管血样,置于4 ℃条件保存,于不同放置时间点(0、24、48、72、96、120 h)对其进行MPS酶学检测。同时收集本院医学遗传科20例健康受试者进行外周血细胞染色体检查后的剩余血样,将其混合与研究组同批次检测作为MPS酶活性正常值(对照组)。①观察研究组血样放置不同时间点,血浆颜色变化,并进行血浆游离血红蛋白(f-Hb)检测,评估血样溶血情况。②进行白细胞分离,并检测放置不同时间点白细胞中α-硫酸艾杜糖醛酸酶(IDUA)、α-N-乙酰葡萄糖胺酶(NAGLU)和β-葡萄糖醛酸酶(GUSB)活性水平,比较各种酶活性水平在不同检测时间点的差异。所有受试者均签署临床研究知情同意书,本研究遵循的程序符合四川大学华西第二医院伦理委员会制定的伦理学批准,并得到该委员会批准(审批文号：20180094)。 结果①血样在放置72 h时血浆颜色开始逐渐变红,放置时间越长,变红的程度越深;血浆f-Hb水平提示放置72 h呈增高趋势,并且随着放置时间延长,溶血程度也越来越严重。②血样1~5放置24、48、72、96、120 h时,IDUA、NAGLU活性水平分别与自身未放置血样比较,差异均无统计学意义(P>0.05)。③血样1~5在放置24、48、72、96 h的GUSB活性水平分别与未放置血样比较,差异亦均无统计学意义(P>0.05);但是,放置120 h时GUSB活性水平明显低于未放置血样,2组比较,差异有统计学意义(t＝4.601、P＝0.002)。 结论综合溶血情况和酶活性水平等多种因素,建议进行MPS酶学检测的外周血血样于采集后72 h(3 d)内送达检测实验室,72~120 h(3~5 d)送达实验室的血样,需取决于血样自身质量和待检测溶酶体酶类型,于120 h(5 d)时送达的检测失败可能性极大。     

Application of cell sorting for enhancing the performance of the cytokinesis-block micronucleus assay

Among the numerous methods available to assess genotoxicity, the cytokinesis-block micronucleus (CBMN) assay is very popular due its relative simplicity and power to detect both clastogenic and aneugenic compounds. A problem with the CBMN assay is that all DNA damaging agents also inhibit the ability of cells to progress through mitosis, leading to a low number of binucleated cells (BNCs). One method to resolve this issue is to ensure a sufficient proportion of BNCs in the samples. In the current study, the applicability of a cell sorting system capable of isolating cell fractions containing abundant BNCs was investigated. Furthermore, to investigate the relationship between the cell division delay due to radiation exposure and the generation of BNCs and micronuclei (MN), we assessed a series of lag times between radiation exposure and addition of cytochalasin-B (Cyt-B). Cells from the human chronic myelogenous leukemia cell line K562 were exposed to X-rays (2 Gy and 4 Gy), and Cyt-B was subsequently added at 0, 6 and 12 h following irradiation. After treatment with Cyt-B for 24 h, the percentage of BNCs, the MN frequency and the cell cycle distribution were analyzed. In addition, cells displaying the DNA contents corresponding to BNCs were isolated and analyzed. The results indicate that applying the cell sorter to the CBMN assay increased the percentage of BNCs compared with the standard method. Thus, this technique is a promising way of enhancing the capacity of the CBMN assay.     

CT检查在石棉肺诊断中的价值

目的　研究石棉肺的肺实质和胸膜病变的ＣＴ 检查所见。方法　对１９ 例石棉肺患者进行胸部Ｘ 线和ＣＴ 检查, 比较Ｘ 线和ＣＴ 检查对石棉肺患者肺实质改变和胸膜改变的表现特点和检出结果。结果　Ｘ 线胸片有胸膜改变的５例, 其中１ 例为胸膜斑。ＣＴ 检查发现有胸膜改变的１６ 例, 其中６ 例为胸膜斑; ６ 例有胸膜斑的病例中, 有３ 例为Ｘ 线胸片上难以发现的脊椎旁胸膜斑。肺实质改变, 在Ｘ 线胸片上, 在３ 个病例各发现１ 个大阴影; 而ＣＴ 检查, 在４ 个病例中, 共发现５ 个大阴影, 其中１ 例伴有胸膜下曲线状肺纤维化阴影。肺内小阴影的显示, ＣＴ 检查所见不如Ｘ 线所见的密集。结论　联合应用胸部Ｘ 线和ＣＴ 检查结果, 可为石棉肺的诊断和分级提供较有价值的信息。     

Quantitative assay for the transforming DNA activity in cell culture products for human use

DNA isolates from a number of established cell lines have been tested for their transforming capacity in the NIH3T3 transformation assay. It has been found that this assay can be used to quantify the amount of transforming DNA at a submicrogram to milligram level. This assay system has been applied to validate the reduction of transforming DNA in various purification steps of cell culture products. The NIH3T3 assay system has been shown useful for in-process control and release of cell culture derived vaccines or therapeutics.     

水体中苯并芘检测试剂盒研制及效果评价

目的 研制间接竞争酶联免疫吸附法(ELISA)检测苯并芘试剂盒。方法 采用棋盘滴定法确定抗原抗体的最佳用量;采用间接ELISA分析法确定苯并芘检测的最适条件,包括pH值、盐离子浓度及甲醇浓度。结果 抗原最佳包被浓度为10 μg/mL,单克隆抗体稀释倍数为 1:5000;在最优pH值为7.4、盐离子浓度为0.01 mol/L及甲醇浓度为10%的条件下,检测方法在5~50 ng/mL范围内线性相关,线性方程为y=-28.105 Ln(x)+123.66,R²=0.9937,IC₅₀为13.74 ng/mL,苯并芘的最低检出限为3.3 ng/mL;在水样中的加标回收率为94.8%~112.3%;变异系数为4.38%~9.72%。结论 该方法适用于水体中苯并芘的检测,可用于大批量样品的快速筛查。     

四种梅毒血清学检测方法临床应用评价

目的:了解快速血浆反应素试验(RPR)、梅毒螺旋体明胶凝集试验(TPPA)、胶体金免疫层析法和酶联免疫吸附试验(ELISA)的临床应用和价值。方法:采用RPR、TPPA、胶体金法和ELISA同时对4241例监测对象的标本进行检测,以TPPA作为参考标准,分别统计RPR、胶体金法和ELISA的敏感性、特异性、阳性预测值和阴性预测值。结果:RPR、胶体金法、ELISA诊断梅毒的敏感性分别为66.80%、97.27%、98.18%,特异性分别为99.72%、99.43%、99.55%;胶体金法、ELISA与TPPA检测阳性率差异无统计学意义(P>0.05),RPR与TPPA法检测结果差异有统计学意义(P<0.05)。结论:TP-ELISA法具有较高的敏感性和特异性法,可用于临床筛查和梅毒感染的确证试验。     

稳健统计-迭代法在环境样品中四种阴离子测定不确定度评估中的应用

目的探讨稳健统计-迭代法在环境样品中四种阴离子测定不确定度评估中的应用。方法对水中氟化物、氯化物、硝酸盐、硫酸盐四种阴离子的质控样品的检测数据进行不确定度评估。结果使用稳健统计-迭代法评估的不确定度和相对标准偏差都与质控样品的结果基本一致。结论该方法适用于环境样品中四种阴离子测定不确定度的评估。