毛细管内均相辣根过氧化物酶（HRP）与核酸杂交偶联HRP催化化学发光比较研究

目的：了解毛细管内核酸偶联和游离辣根过氧化物酶（HRP）催化化学发光差异。方法：不同浓度HRP和经核酸杂交固定于毛细管内壁HRP催化化学发光反应。结果：①游离HRP催化化学发光线性检测范围窄（2.7×10-5-1.3×10-6mg/ml,R2〉0.96）,下限为1.0×10-7mg/ml;②2.0×1014-2.0×106copies/ml的5μl单链DNA杂交后,1.0-10min时DNA浓度M对数（lgM）与化学发光I值线性相关（R2〉0.99）,且大于阴性I值平均数＋3倍标准偏差（s.d）;③5.0×1011-5.0×106copies/ml的5μl PCR产物杂交后,10min内PCR产物对数lgM与I值线性相关（R2〉0.97）,且大于阴性I值平均数＋3倍标准偏差（s.d）。4.0-7.0min内lgM与I值的R2〉0.99,3次平行检测标准偏差〈5.0%。结论：毛细管内核酸杂交的HRP催化化学发光检测线性范围宽、灵敏度高、底物用量少,有望用于临床核酸分子杂交检测。     

毛细管内均相辣根过氧化物酶(HRP)与核酸杂交偶联HRP 催化化学 发光比较研究

目的:了解毛细管内核酸偶联和游离辣根过氧化物酶(HRP)催化化学发光差异。方法:不同浓度HRP和经核酸杂交固定于毛细管内壁HRP催化化学发光反应。结果:①游离HRP催化化学发光线性检测范围窄(2.7×10-5-1.3×10-6mg/ml,R2>0.96),下限为1.0×10-7mg/ml;②2.0×1014-2.0×106copies/ml的5μl单链DNA杂交后,1.0-10min时DNA浓度M对数(lgM)与化学发光I值线性相关(R2>0.99),且大于阴性I值平均数+3倍标准偏差(s.d);③5.0×1011-5.0×106copies/ml的5μl PCR产物杂交后,10min内PCR产物对数lgM与I值线性相关(R2>0.97),且大于阴性I值平均数+3倍标准偏差(s.d)。4.0-7.0min内lgM与I值的R2>0.99,3次平行检测标准偏差<5.0%。结论:毛细管内核酸杂交的HRP催化化学发光检测线性范围宽、灵敏度高、底物用量少,有望用于临床核酸分子杂交检测。     

基于SYBR Green I的双链DNA定量方法

摘要 基于SYBR Green I荧光染料与双链DNA（dsDNA）结合产生荧光的原理,建立一种高精度、高通量的双链DNA 定量方法。将梯度稀释后的基因组DNA及已知浓度的?DNA与等体积的SYBR Green I（4×）充分混合后,利用荧光定量PCR仪采集荧光信号,以ROX（1×）作为校正染料进行定量分析;同时利用紫外分光光度计对样品进行平行测定,比较该方法与紫外分光光度法的检测限与准确度。紫外分光光度法的检测限为2 ng/?l,而SYBR Green I荧光定量法的检测限可达到0.015 ng/?l,并且在0.015~2 ng/?l范围内,SYBR Green I荧光强度与?DNA浓度呈线性关系（R2=0.9999）,比紫外分光光度法灵敏100倍以上,并可准确定量低纯度的DNA样品。此方法具有重复性好、高通量的特点,仅需少量的生物样本即可满足定量要求,为分子生物学研究及临床检验等多个领域提供了一种可靠的dsDNA定量方法。     

毛细管气相色谱法测定甲磺酸伊马替尼中残留有机溶剂

目的：建立可同时测定甲磺酸伊马替尼原料药中甲醇、乙醇和N''N - 二甲基甲酰胺（DMF）等3 种残留有机溶剂的毛细管气相色谱法。方法：采用WondaCAP WAX 毛细管柱为色谱柱，程序升温，N2 为载气，载气流速为4.0 mL ? min-1，氢火焰离子化检测器，检测器温度为250 ℃，进样口温度为200 ℃，进样量为1.0 μL。以正丁醇为内标，通过内标法计算样品中3 种有机溶剂残留量。结果：甲醇、乙醇和DMF 在所建立的色谱条件下均能完全分离，分别在2.94~44.11、4.91~73.68 和0.084~12.60 mg ? L-1 的质量浓度范围内线性关系良好（r =0.9940~0.9985），进样精密度（RSD）均小于5.0%（n =6），平均回收率为104.8%~105.8%（RSD<5.0%，n =9），定量限分别为0.20、0.33 和0.08 ng（信噪比为10 ∶ 1），检测限分别为0.05、0.11 和0.02 ng（信噪比为3 ∶ 1）。样品中未检出甲醇和DMF，乙醇含量低于有关限度规定。结论：该方法简便、准确、灵敏、重复性好，可用于甲磺酸伊马替尼原料药中残留有机溶剂的检测。     

微芯片电泳-紫外检测系统分析蛋白质

微芯片电泳是基于微机电加工技术(MEMS)工艺,在芯片上的微管道中完成电泳检测过程的新型技术.依据紫外吸光度分析法,对蛋白质样品进行电泳分离与紫外检测.实验采用自控接口模板对进样及分离电压进行了系统的程序化控制,从而准确地控制整个电泳、检测流程,提高了微芯片电泳的分离效率和检测灵敏度.实验结果表明,夹流进样的方法可以有效分离混合蛋白,可用于蛋白质样品的分离检测.     

胶束电动毛细管色谱法测定红丝线提取物中紫蓝素的含量

建立了胶束电动毛细管电泳色谱法(MECC)测定红丝线提取物中紫蓝素的含量的方法。毛细管柱内径75μm,长50.2cm;运行电压25kV,检测波长585nm,温度25℃;缓冲液为25mmol/L,β-CD10mmol/L,硼酸盐-20%乙腈(pH值8.0);进样方式:压力进样,进样时间5s。结果表明紫蓝素在10～100μmol/L浓度范围内具有良好的线性关系(r=0.9995),回收率为95.3%～103.2%。该方法快速、简便、并且较为准确,适用于测定红丝线提取物中紫蓝素的含量。     

高效毛细管电泳法直接测定红豆杉悬浮培养细胞中游离芳香族氨基酸

建立了红豆杉悬浮培养细胞中游离芳香族氨基酸的高效毛细管电泳的分离直接紫外吸收测定方法。在优化的实验条件下 ,以间苯二酚为内标 ,未涂层融硅毛细管 (75 μmi.d .,总长 37cm ,有效分离长度 30cm)为分离通道 ,40mmol/L磷酸缓冲液 (pH10 .46 )为电泳介质 ,3sec(0 .5 psi)压力进样 ,15kV恒压电泳 ,2 0 0nm检测 ,在 9min内三种氨基酸得到分离测定。色氨酸、苯丙氨酸和酪氨酸的线性范围分别为 2 .5 0～ 2 5 0 μmol/L(r =0 .996 2 ,RSD =2 .5 2 % )、1.2 5～ 2 5 0 μmol/L(r =0 .996 6 ,RSD =2 .12 % )和 2 .5 0～ 2 5 0 μmol/L(r=0 .9940 ,RSD =2 .93% ) ,苯丙氨酸回收率为 96 .8%± 1.37% ,酪氨酸回收率为 99.2 %± 3.0 4%。     

基于纳米金核酸探针的新型试纸条对灿烂弧菌快速检测

灿烂弧菌Vibrio splendidus作为一种水产条件致病菌,可以感染多种水产养殖动物,给水产养殖业带来巨大的经济损失。文中将核酸外切酶Ⅲ酶切信号放大策略和纳米金标记DNA探针核酸试纸条相结合,建立了一种新型高效的灿烂弧菌检测方法,检测结果可凭肉眼直接判定,并突破了常规免疫试纸条单克隆抗体制备困难的障碍。经过实验条件优化,该核酸试纸条对灿烂弧菌人工合成寡核苷酸DNA片段的检测限是5 ng/mL,对灿烂弧菌基因组DNA实际样本的检测限是10 ng/mL,较PCR法灵敏度高,并对灿烂弧菌具有检测特异性。研究结果实现了核酸试纸条的快捷制备及对灿烂弧菌的高效检测,为水产病害的防治开辟了新的途径。     

PCR—ELISA检测HBV DNA的研究

目的：建立一种敏感、特异的乙型肝炎病毒（HBV）DNA检测方法。方法：应用PCR扩增技术和核酸杂交技术结合酶促显色技术（即PCR－ELISA技术）来检测血清中的HBV DNA。结果：应用PCR－ELISA技术能够检出许多PCR琼脂张胶电泳所检测不到的HBV DNA,大大地提高了检出率,而且,特异性强。结论：PCR－ELISA方法灵敏度高,行异性强,检测结果数据化,不受主观因素的影响。     

阿特拉津高灵敏酶联免疫吸附检测法的建立

目的：建立高灵敏度的阿特拉津酶联免疫吸附检测法。方法：将间接竞争ELISA进行条件优化以提高检测灵敏度,包括包被抗原与一抗的最佳工作浓度筛选、选择一抗的最佳稀释度对包被抗原进行细化筛选、不同有机溶剂对竞争结合反应的影响、酶标二抗稀释度筛选等。用建立的酶联免疫检测法检测实际样品,再与高效液相色谱法（HPLC）检测进行比较。结果：利用优化后条件建立了阿特拉津间接竞争ELISA检测曲线,标准曲线的相关系数R²=0.9958,相关性较好。另由此标准曲线可得LOD （最低检出限）为1.972 ng/ml。用于检测实际样品,回收率在80%-120%之间。当添加样品浓度为（0~6） ng/ml时,该法的检测灵敏度高于HPLC。结论：新建立的阿特拉津ELISA特异性好、精密度高,可代替大型仪器用于阿特拉津实际样品检测。     

On-column preconcentration of glutathione and glutathione disulfide using pH-mediated base stacking for the analysis of microdialysis samples by capillary electrophoresis

Capillary electrophoresis (CE) has become a useful analytical tool for the analysis of microdialysis samples. However, CE with UV detection (CE-UV) does not provide detection limits sufficient to quantify glutathione (GSH) and glutathione disulfide (GSSG) in biological samples such as liver microdialysates, because of the small optical path length in the capillary. To overcome this limitation, an on-column preconcentration technique, pH-mediated base stacking, was used in this study to improve the sensitivity of CE-UV. This stacking technique allowed large volumes of high ionic strength sample injection without deterioration of the separation efficiency and resolution. A 26-fold increase in sensitivity was achieved for both GSH and GSSG using the pH-mediated base stacking, relative to normal injection without stacking. The limit of detection for GSH and GSSG was found to be 0.75 microM (S/N=6) and 0.25 microM (S/N=6), respectively. The developed method was used to analyze GSH and GSSG in liver microdialysates of anesthetized Sprague Dawley male rats. The basal concentrations of GSH and GSSG in the liver microdialysates of male rats were found to be 4.73+/-2.08 microM (n=7) and 5.52+/-3.66 microM (n=7), respectively.     

Determination of heterocyclic amines by capillary electrophoresis with UV-DAD detection using on-line preconcentration

This paper proposed a method with capillary zone electrophoresis (CZE) for analysis of eight heterocyclic amines in a commercial meat matrix. The influence of composition, pH and concentration of buffer, and applied voltage were investigated. A 5 mmol/L formic acid-ammonium formate solution at pH 2.20 was chosen as the running electrolyte. Also several solid-phase extraction actions were performed for the clean-up of the samples. With 3s hydrodynamic injection, detection limits ranging from 0.554 to 1.783 microg/g was obtained. To improve sensitivity, field-amplified sample injection (FASI) was used with the conditions of 3 s hydrodynamic injection of a water plug and 25 s electrokinetic injection of the sample. And methanol-water (1:1 in volume) was applied as the sample solvent. Under above conditions, detection limits ranged from 1.329 to 19.39 ng/g, which were at least 50 times lower than those with hydrodynamic injection.     

三种提取RNA的方法对汉滩病毒检测敏感性影响的研究

分别采用标准法、ＧＰ法和ＭＢＰ法抽提汉滩病毒ＲＮＡ,结合套式ＰＣＲ比较这三种方法的敏感性。结果表明,所用引物对这两株病毒具有相同的特异性。这三种提取ＲＮＡ的敏感性差异不大,敏感性从高到低依次为：ＧＰ、ＭＢＰ和标准法,检测汉滩病毒的效价分别为０．０１、０．０１和０．１ＴＣＩＤ５０／２００μｌ;提取ＲＮＡ的时间长短分别为：标准法为２．５ｈ,ＧＰ为１ｈ,ＭＢＰ为０．５ｈ,可见ＭＢＰ用时最少。使用ＧＰ和ＭＢＰ提取ＲＮＡ时,可以在室温下进行,不需要低温离心。所以,ＭＢＰ最适合于实验室和临床病原体的快速检测,特别是对采集的环境样品的快速检测     

Determination of metronidazole in vaginal tissue by high-performance liquid chromatography using solid-phase extraction

A sensitive high-performance liquid chromatographic (HPLC) method for the determination of metronidazole in vaginal tissue is reported. The method uses a Zorbax SB phenyl column with a 0.01 M aqueous monobasic potassium phosphate buffer (pH 4.0)-absolute methanol (85:15, v/v) as mobile phase at a flow-rate of 1.0 ml/min and detection at 313 nm. Tinidazole was used as the internal standard. The method employed homogenization of tissue followed by solid-phase extraction. The quantitation was achieved within 30 min with sensitivity in the ng/g range. Metronidazole was linear in the 100–2000 ng/g range. The accuracy and precision were in the 1–4% range for the drug and the limit of detection was approximately 100 ng/g based on a signal-to-noise ratio of 3 and a 100-μl injection.     

Determination of amoxicillin in plasma samples by capillary electrophoresis

We have developed a capillary zone electrophoresis (CZE) method for determining amoxicillin in animal plasma samples. Sample clean-up involved solid-phase extraction onto Sep-Pak C₁₈ cartridges followed by elution with water–methanol (85:15). This paper describes two different techniques to increase the sensitivity of the CZE method: field-amplified sample injection (FASI) and electrokinetic injection. We have enhanced the detection limit to 280 μg l⁻¹ by the FASI technique.     

Recovery of DNA of Giardia intestinalis cysts from surface water concentrates measured with PCR and real time PCR

The most important restriction for the detection in water samples is the low concentration of Giardia intestinalis cysts, additional difficulty is the presence of PCR inhibitors. We have carried out trials in order to assess the sensitivity of semi-nested PCR and TaqMan real time PCR on the basis of DNA extracted from G. intestinalis cysts coming from spiked environmental and distilled water samples, filtrated with the use of Filta-Max^® equipment (1623 Method). Removal of inhibitors was carried out with addition of BSA in different concentrations. During the filtration and concentration of water samples, losses of cysts have been recorded. Moreover, addition of BSA to the PCR and real time PCR mix increases the sensitivity of reaction. The optimal concentration of BSA for semi-nested PCR was 15 and 20 ng/μl, whereas for real time PCR 5 ng/μl.     

Sensitive gas-liquid chromatographic method for the assay of the neuroleptic drug cis(Z)-flupentixol in human serum or plasma

A gas-liquid chromatographic (GLC) assay suitable for the analysis of the cis(Z)-stereoisomer of the antipsychotic drug flupentixol in human serum or plasma was developed. The minimal quantifiable concentration was 0.5 ng/ml and the day-to-day coefficient of variation was 11.2% at 1 ng/ml and 8.7% at 10 ng/ml. Following addition of perphenazine as the internal standard (I.S.) and aqueous NaOH, samples (2 ml) are extracted with n-hexane-isoamyl alcohol (98.5:1.5, v/v) (solvent), back-extracted to 0.1 M HCl and after one washing-step and addition of aqueous NaOH again extracted into 100 μl solvent. After evaporation to dryness, the extract is reconstituted in 20 μl solvent and evaporated to approximative 10 μl. A 4-μl aliquot is injected cool on-column onto the GLC system. A gas chromatograph HP 5890 with on-column injection port, nitrogen-phosphorus detector (NPD), a HP-1 25 m × 0.32 mm I.D., 0.5 μm capillary and hydrogen (3 ml/min, automated pressure control) as the carrier gas was applied. The negative influence of light on the assay was measured and discussed. The suitability of this method for clinical pharmacokinetic studies and therapeutic drug monitoring (TDM) was determined by the analysis of serum samples of 12 schizophrenic patients.     

Modified method of nalbuphine determination in plasma: validation and application to pharmacokinetics of the rectal route

A solid-phase extraction (SPE) procedure was developed for the quantification of nalbuphine in a small volume (500 μl) of human plasma with subsequent assay by high-performance liquid chromatography (HPLC) and electrochemical detection using 6-monoacetylmorphine as internal standard. Plasma was extracted using Bond Elute certified extraction columns (LCR: 10 ml, 130 mg) after conditioning with methanol and 0.2 M Tris buffer (pH 8). Elution was performed with a CH₂Cl₂-isopropanol-NH₄OH (79:20:, v/v). The organic phase was evaporated to dryness and resuspended in HPLC mobile phase containing 2% isopropanol. Linearity was assessed over the 5–100 ng/ml concentration range and a straight line passing through the origin was obtained. Experiments with spiked plasma samples resulted in recoveries of 95±5.4% and 98±6.2% for nalbuphine and 6-monoacetylmorphine, respectively. The optimal pH conditions for the SPE were found at pH 8. The intra-day coefficients of variation (C.V.) for 5, 40, and 100 ng/ml were 5.3, 3.0 and 2.3% (n=8) and the inter-day C.V.s were 7.7, 3.2 and 3.5% (n=10), respectively. The detection limit for 500 μl plasma sample was 0.02 ng/ml and the limit of quantification 0.1 ng/ml (C.V.=12.4%). The ease of the proposed method of analysis, as well as its high accuracy and sensitivity allow its application to pharmacokinetic studies. A preliminary kinetic profile of nalbuphine after rectal administration in a pediatric patient is presented.     

Detection of phytoplasma by loop-mediated isothermal amplification of DNA (LAMP)

Napier stunt phytoplasma (16SrXI and 16SrIII) in eastern Africa is a serious threat to the expansion of Napier grass (Pennisetum purpureum) farming in the region, where it is widely cultivated as fodder in zero grazing livestock systems. The grass has high potential for bio-fuel production, and has been adopted by farmers as a countermeasure to cereal stem borer Lepidoptera, since it attracts and traps the insect. Diagnosis of stunt phytoplasma have been largely by nested polymerase chain reaction (nPCR) targeting the 16S rRNA gene. However, the method is laborious, costly and technically demanding. This investigation has developed a simpler but effective phytoplasma diagnostic tool, called; loop-mediated isothermal amplification of DNA (LAMP). The assay was tested on 8 symptomatic and 8 asymptomatic plants, while its detection limit was compared to nested PCR using samples serially diluted from 3 ng/μl to 0.38 pg/μl. Molecular typing of LAMP products was determined by BsrI restriction digestion and Southern blot analysis. The assay sensitivity, positive and negative predictive values were estimated, while the specificity was tested on 11 phytoplasma groups. LAMP was specific to 5 phytoplasma groups: 16SrVI, X, XI and XVI. BsrI restriction digestion produced two predicted fragments, and there was specific binding of probe DNA to the LAMP amplicons in Southern blot analysis. The assay sensitivity was 100%, while the positive and negative predictive values were 63 and 100% respectively. LAMP was 20-fold more sensitive than nested PCR. This study validates LAMP for routine diagnosis of Napier stunt and other closely related phytoplasmas.     

Determination of amdinocillin in plasma and urine by high-performance liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of amdinocillin (formerly mecillinam) in human plasma and urine. The assay is performed by direct injection of a plasma protein-free supernatant or a dilution of urine. A 10-μm μBondapak phenyl column with an eluting solvent of water—methanol—1 M phosphate buffer, pH 7 (70:30:0.5) was used, with UV detection of the effluent at 220 nm. Azidocillin potassium salt [potassium-6-(d-(-)-α-azidophenyacetamido)-penicillanate] was used as the internal standard and quantitation was based on peak height ratio of amdinocillin to that of the internal standard. The assay has a recovery of 74.4 ± 6.3% (S.D.) in the concentration ranges of 0.1–20 μg per 0.2 ml of plasma with a limit of detection equivalent to 0.5 μg/ml plasma. The urine assay was validated over a concentration range of 0.025–5 mg/ml of urine, and has a limit of detection of 0.025 mg/ml (25 μg/ml) using a 0.1-ml urine specimen per assay.The assay was applied to the determination of plasma and urine concentrations of amdinocillin following intravenous administration of a 10 mg/kg dose of amdinocillin to two human subjects. The HPLC and microbiological assays were shown to correlate well for these samples.