TaqMan实时荧光定量RT-PCR检测甲型肝炎减毒活疫苗病毒含量的研究

目的：建立一种快速定量检测甲型肝炎减毒活疫苗病毒含量的实时荧光定量RT-PCR方法。方法对Gen-Bank中登陆的甲型肝炎减毒活疫苗株（ L-A-1）和其他甲型肝炎病毒基因组全序列比较分析,根据其高度保守的5′端非编码区设计针对甲型肝炎减毒活疫苗株特异性引物与探针,对荧光定量RT-PCR反应条件进行优化,检测该方法的特异性和灵敏性,并对甲型肝炎减毒活疫苗病毒含量进行定量检测。结果该方法对甲型肝炎减毒活疫苗株高度特异,扩增片段为207 bp,不与其他肠道病毒发生非特异性反应。在104 CCID50/管～10-1 CCID50/管之间有良好的扩增曲线,检测的灵敏度可达0．1CCID50～0．01CCID50,比普通RT-PCR高100倍。结论该方法具有快速、灵敏、特异、重复性好等优点,可应用于甲型肝炎减毒活疫苗生产过程中病毒含量滴度测定及指导疫苗成品的配制。     

气相色谱法检测水痘减毒活疫苗原液中二甲基亚砜残留量

目的 建立测定水痘减毒活疫苗原液中二甲基亚砜(dimethyl sulfoxide, DMSO)残留量的气相色谱法,并进行方法学的验证和初步应用。方法 采用气相色谱法检测,对色谱柱、柱温、分流比以及供试品预处理方法进行了优化,并对优化后的方法进行系统适用性和专属性、线性和范围、准确度和精密度、定量限和检测限、耐用性的方法学验证。结果 色谱柱使用DB-624(30 m×0.53 mm, 3.00μm)毛细管柱,以氮气为载气,进样口温度设定为250℃,FID检测器温度设定为270℃,柱温为120℃,分流比为5∶1。供试品在检测前采用乙腈沉淀蛋白的预处理方式(体积比1∶9～1∶19)以去除其中的干扰杂质。方法学验证结果显示,系统适用性和专属性良好,DMSO色谱峰与杂质分离良好,在50～1 000μg/mL范围内线性关系良好(R²=0.999 9)。检测限为20μg/mL,定量限为50μg/mL;准确度结果均在90%～110%之间,精密度的RSD均<10%。水痘减毒活疫苗原液中的DMSO残留量均未检出。结论 建立的气相色谱法能稳定、准确、快速地检测水痘减毒活疫苗原液...     

ELISA法检测乙型脑炎减毒活疫苗中卡那霉素和庆大霉素的残留量

用ELISA试剂盒检测乙脑减毒活疫苗中卡那霉素和庆大霉素的残留量。以间接竞争ELISA法检测线性范围内加入高、中、低3种浓度的卡那霉素和庆大霉素,测定其在疫苗稳定剂、疫苗稳定剂10倍稀释溶液、试剂盒稀释液中的回收率,以及在10倍稀释乙脑减毒活疫苗中的回收率。高、中、低浓度的卡那霉素在疫苗稳定剂、疫苗稳定剂10倍稀释溶液、试剂盒稀释液中的回收率为101.40%～124.80%之间;原倍疫苗稳定剂对庆大霉素的测定有明显干扰,在原倍疫苗稳定剂中低浓度和中浓度的庆大霉素的回收率高达2280%和575%,但其它测定条件下回收率在90%～125%之间。在10倍稀释的乙脑疫苗中加入一定量的卡那霉素和庆大霉素,回收率分别为124%和103.25%。用10倍稀释法测定1人份规格的乙脑减毒活疫苗17批、5人份规格的乙脑减毒活疫苗19批。乙脑减毒活疫苗10倍稀释后,可用ELISA试剂盒检测卡那霉素和庆大霉素残留量。     

麻疹减毒活疫苗检定用Vero细胞代次对病毒滴度稳定性试验的影响

目的了解麻疹减毒活疫苗检定用Vero细胞代次的稳定性范围。方法用DMEM培养液将Vero细胞137代连续传至173代,检测其细胞活力,并观察Vero细胞的形态,接种麻疹减毒活疫苗参考品进行病毒滴定及热稳定性试验。结果 170代以下Vero细胞形态良好,细胞活力在85%以上,麻疹减毒活疫苗病毒滴度热稳定性试验的结果:137~169代Vero细胞活力及原麻疹减毒活疫苗病毒滴度热稳定性试验无统计学差异(P>0.05),170代以上代次的Vero细胞活力及原麻疹减毒活疫苗热稳定性试验有明显统计学差异(P<0.01)。结论 169代以下Vero细胞为检定用麻疹减毒活疫苗最佳细胞代次。     

麻腮风水痘联合减毒活疫苗病毒滴定方法研究

目的建立并验证MMRV联合减毒活疫苗病毒滴定方法。方法滴定MMRV疫苗中的每种病毒时,首先用特异性抗血清有选择地中和其他病毒成分,再根据每种抗血清与相应病毒的完全中和能力,确定各抗病毒血清的使用浓度。分别用CCID50法(麻、腮、风病毒)和蚀斑法(水痘病毒)检测MMRV疫苗中各病毒的滴度。结果使用该方法对MMRV联合减毒活疫苗进行滴定的实测值与理论值无显著差异,经验证其准确性和可重复性均显示良好。结论建立的MMRV联合减毒活疫苗病毒滴定方法可行。     

流感减毒活疫苗效力试验检测新方法的建立

目的建立基于NP蛋白检测的流感减毒活疫苗病毒滴度检测方法,并进行初步应用。方法对病毒接种方式、流感病毒感染MDCK细胞培养时间、一抗和酶标二抗的稀释度、封闭液等ELISA反应条件进行筛选优化。采用建立的方法检测3批流感减毒活疫苗原液和1批疫苗成品,并与鸡胚培养法检测结果进行比较。结果选择直接接种法作为接种方式,病毒感染细胞后培养48 h进行NP蛋白表达的检测; ELISA检测条件一抗稀释度为1∶4 000,酶标二抗稀释度为1∶4 000,2%牛血清白蛋白封闭2 h,检测结果 P/N值最大。用建立的方法检测流感减毒活疫苗原液及疫苗成品效力,与鸡胚培养法检测结果差异无统计学意义(P0.05),具有较好的一致性。结论建立了基于NP蛋白检测的流感减毒活疫苗效力试验方法,可用于生产过程中流感减毒活疫苗原液和成品的检定。     

离心、超滤技术在甲型肝炎减毒活疫苗生产工艺中的应用

采用离心和超滤技术进行甲型肝炎减毒活疫苗生产 ,达到提高病毒的收获率。将含有甲型肝炎病毒的细胞收获液离心 ,上清进行超滤浓缩 ,回收病毒 ,病毒回收效果好。实验证明 ,该方法适合规模化生产。     

红外吸收法检测西林瓶包装冻干制品中的氧含量

目的应用HSA-P型激光检漏仪建立西林瓶包装冻干制品中氧气含量的测定方法。方法考察冻干制品中氧含量测定的操作条件:标准样瓶对仪器的校准;氮气吹扫前后样品中氧含量值的比较;不同纯度氮气吹扫标准样瓶后的氧含量值的变化。深化红外吸收法在测定西林瓶包装冻干疫苗中氧含量的应用。结果将该方法用于检测A群C群脑膜炎球菌多糖疫苗400批、麻疹减毒活疫苗86批、乙型脑炎减毒活疫苗46批,氧含量分别为1.64%±1.99%、1.36%±1.64%和0.99%±1.58%。结论该法测定冻干疫苗中的氧含量具有灵敏度高、速度快和易操作的优点,为冻干制品真空度的测定提供了定量检测方法。     

乙型脑炎减毒活疫苗37℃孵放一周后不同时间的滴度测定

为了了解乙型脑炎减毒活疫苗(简称JEV)的热稳定性,将乙型脑炎减毒活疫苗在37℃孵放一周后2～8℃保存于不同时间进行病毒滴度检测。结果显示,疫苗在37℃孵放一周后2～8℃保存2个月内进行测定,滴度无明显下降趋势。在乙型脑炎减毒活疫苗的热稳定性检测中,37℃孵放一周后2～8℃保存,可在2个月内进行检测,对其热稳定性检测无明显影响。     

BHK_(21)细胞对蚀斑法检测乙型脑炎减毒活疫苗结果的影响分析

目的分析BHK_(21)细胞对蚀斑法检测乙型脑炎减毒活疫苗系统的影响,降低检测系统误差。方法比较BHK_(21)细胞以不同频次传代培养时的细胞生长状态,以及以不同接种浓度进行疫苗病毒滴度检测时,对检测系统稳定性的影响。结果 BHK_(21)细胞培养4 d传代,其形态良好、边缘光滑、胞质透光性好、细胞分散均匀、细胞活率达到95%以上;BHK_(21)以105细胞/m L浓度接种时,乙型脑炎减毒活疫苗及其病毒滴度参考品的变异系数最小,分别为0.66%和0.64%。结论 BHK_(21)细胞培养4 d传代、以105个/m L浓度接种时用蚀斑法检测病毒滴度系统最稳定,可供乙型脑炎减毒活疫苗滴度检测参考。     

一株甲烷利用菌的分离及其在甲烷气体测定中的应用

从山西太原晋阳湖水样中分离得到一株能以甲烷为唯一碳源生长的菌株ME16.气相色谱分析表明ME16菌株能利用甲烷.ME16菌株的16S rDNA 序列与铜绿假单胞菌(Pseudomonasaeruginosa,ATCC 10145,AF094713)相似性为99%.该菌株最适培养条件为30℃、2%接种量、25%甲烷含量和培养基pH为6.0.用电化学法研究了ME16固定化细胞体系中不同含量甲烷对溶氧的响应时间以及溶氧变化与甲烷含量的关系.结果表明,加入固定化细胞后.溶氧变化在100s内达到平衡,溶氧消耗量与通入甲烷气体含量在0～16%呈线性关系,相关系数为0.9954.对样品气体8次测量,RSD为3.34%,表明该反应体系重现性良好,为该菌株进一步研究甲烷传感器奠定基础.     

A chemical ionization selected ion monitoring assay for methylphenidate and ritalinic acid

A methane chemical ionization quantitative assay for methylphenidate and its major metabolite, ritalinic acid, is described. Methylphenidate and the internal standard, ethylphenidate, were extracted from plasma samples and derivatized to prevent thermal decomposition in the gas chromatography. Ritalinic acid was esterified with diazomethane and extracted as methylphenidate. The intensity of the protonated molecular ion of the derivatized drug and internal standard was measured by selected ion monitoring. Calibration curves were prepared from drug standards dissolved in drug-free plasma, and the lower limit of the curves extended to 0.5 ng methylphenidate per ml plasma. The method was used to generate plasma decay curves for pediatric patients undergoing methylphenidate therapy.     

甲烷利用菌培养条件的优化及其初步应用

利用统计学实验设计（RSM）对能够利用甲烷的假单胞菌菌株ME16主要培养条件进行了优化。以液体无机盐和甲烷气体作为培养基分别进行了温度、接种量、甲烷含量和培养pH对细菌生长影响的研究,并在此基础上,利用响应面法分析优化了ME16菌株的主要培养条件,得到最佳培养条件为：温度29.4℃,接种量1.8%,甲烷含量25%。采用优化培养条件进行培养,细菌生物量增大0.8倍,达到稳定期的培养时间缩短了50h。该菌株初步应用于甲烷气体的脱除,脱除率达65.7 %,表明该菌株能良好的脱除空气中甲烷。     

Comparison of standard methods and gas chromatography method in determination of formaldehyde emission from MDF bonded with formaldehyde-based resins

Formaldehyde emissions from MDF bonded with urea-formaldehyde resin (UF), melamine-formaldehyde resin (MF) and the co-polycondensation resin of urea-melamine-formaldehyde (UMF) and melamine-formaldehyde, measured by the Japanese standard method of determining formaldehyde emission with a desiccator (JIS A 5908) and the DIN EN 120 (European Committee For Standardization, 1991) method using the perforator value, were used as the typical standard methods. While the UF resin showed a desiccator value of 7.05 ppm and a perforator value of 12.1 mg/100 g panel, the MF resin exhibited a desiccator value of 0.6 ppm and a perforator value of 2.88 mg/100 g panel. According to the Japanese industrial standard and the European standard, the formaldehyde emission level of the MDF panels made with UF resin in this study was E(2) grade. The formaldehyde emission level was dramatically reduced by the addition of MF resin. This is because the addition of formaldehyde to melamine occurs more easily and completely than its addition to urea, even though the condensation reaction of melamine with formaldehyde is similar to that between urea and formaldehyde. These two methods, the desiccator method and the perforator method, produced proportionally equivalent results. Gas chromatography, a more sensitive and advanced method, was also used. The samples used for gas chromatography were gathered during the experiment involving the perforator method. The formaldehyde emission levels obtained from gas chromatography were similar to those obtained from the perforator method. The formaldehyde contents measured by gas chromatography were directly proportional to the perforator values.     

Methane content in bottom sediments and water mass of the Black Sea

The methane content in bottom sediments and water column of the Black Sea was determined using various methods of desorption and analysis of gases and various methods of calculating their concentrations. The head-space method with the use of salting out and calculation by internal standard proved to be the most accurate procedure for the analysis of methane concentration in bottom sediments. The methane content in bottom sediments increased with the depth of sediment sampling. In the upper 50-70 cm of shelf sediments, two minimums of methane concentration were revealed; in deep-sea sediments, only one minimum was recorded (in the 20-50 cm horizons). In the water column, methane concentrations slowly grew from the surface to a depth of 150-200 m and abruptly increased to a depth of 700-1200 m, remaining virtually constant in underlying layers. In certain deep-sea regions, peaks of methane content in the 1000-1200 m horizons of the water column were revealed, which were most probably due to local influx of abyssal waters enriched with this gas.     

大柳塔长焰煤中灰分和无机矿物对生物产气的影响

【目的】以不同密度等级大柳塔长焰煤作为产气底物,前期驯化培养厌氧菌群进行生物模拟产气实验,研究不同密度等级煤中的灰分和无机矿物对生物产气的影响。【方法】利用小浮沉将大柳塔长焰煤分成不同密度等级的煤样,采用工业分析、XRD、XRF分析小浮沉处理得到煤样的理化性质,利用这些煤样进行生物产气模拟实验,以甲烷产量作为评价指标,分析不同密度等级煤样中灰分对产气的影响。最后,通过添加几种标准矿物方式比较了煤中无机矿物对生物产气的可能影响。【结果】不同密度等级煤样中灰分对产气量存在一般显著影响(P=0.035),且灰分与甲烷含量呈负相关关系,其灰分中的无机矿物如高岭土、菱铁矿、氧化亚铁镁等的积累对产气有抑制作用。不同矿物配比产气实验证实低含量的粘土矿物促进甲烷的生成,高含量的粘土矿物抑制产气。【结论】不同密度等级煤中的灰分对生物产气存在一般显著的影响,高灰分煤的产气量低,而低灰分煤的产气量高。     

Development of highly sensitive sensor system for methane utilizing cataluminescence

A gaseous sensor system was developed for the detection of methane based on its cataluminescence emission. Cataluminescence characteristics and optimal conditions were studied in detail under optimized experimental conditions. Results showed that the methane cataluminescence sensor system could cover a linear detection range from 10 to 5800 ppm (R = 0.9963, n = 7) and the detection limit was about 7 ppm (S/N = 3), which was below the standard permitted concentration. Moreover, a linear discriminant analysis method was used to test the recognizable performance of the methane sensor. It was found that methane, ethane, propane and pentane could be distinguished clearly. Its methane sensing properties, including improved sensitivity, selectivity, stability and recognition demonstrated the TiO₂/SnO₂ materials to be promising candidates for constructing a cataluminescence‐based gas sensor that could be used for detecting explosive gas contaminants. Copyright © 2015 John Wiley & Sons, Ltd.     

Methane Content in the Bottom Sediments and Water Column of the Black Sea

The methane content in the bottom sediments and water column of the Black Sea was determined using various methods of desorption and analysis of gases and various methods of calculating their concentrations. The head-space method with the use of salting out and calculation by an internal standard proved to be the most accurate procedure for the analysis of methane concentration in bottom sediments. The methane content in bottom sediments increased downward along the sediment thickness. In the upper 50–70 cm of shelf sediments, two minimums of methane concentration were revealed; in deep-sea sediments, only one minimum was recorded (in the 20–50 cm horizons). In the water column, methane concentrations slowly grew from the surface to a depth of 150–200 m and abruptly increased to a depth of 700–1200 m, remaining virtually constant in underlying layers. In certain deep-sea regions, peaks of methane content in the 1000–1200 m horizons of the water column were revealed, which were most probably due to local influx of abyssal waters enriched with this gas.     

Determination of imidazobenzodiazepine-3-car☐amide, a new anxiolytic agent, in human plasma by gas chromatography—negative chemical-ionization mass spectrometry

A method is described for measuring imidazobenzodiazepine-3-car☐yamide, a new anxiolytic agent, in human plasma. A tetradeuterated analogue of the analyte is used as the internal standard. The drug and its internal standard are (1) extracted from plasma at pH 9 with benzene containing 20% 1, 2-dichloroethane, (2) derivatized with pentafluoropropionic anhydride in the presence of triethylamine and (3) the nitrile derivative of the analyte and internal standard are analyzed by gas chromatography (GC)—negative chemical-ionization mass spectrometry (CIMS) using methane as both GC carrier gas and CI reagent gas. The mass spectrometer is set to monitor the intense (M-HCl)⁻- ions of imidazobenzodiazepine-3-nitrile and its tetradeuterated analogue atm/z 316 andm/z 320, respectively. Quantitation of an experimental plasma sample is based on the comparison of them/z 316 tom/z 320 ion ratio in each sample to that obtained from the analyses of control plasma spiked with various amounts of the drug and a fixed amount of internal standard. The limit of quantitation of the method is approximately 100 pg ml⁻¹ of plasma and the precision (relative standard deviation) at a plasma concentration of 1 ng ml⁻¹ is 4%.