Detection of thiopurine s-methyltransferase mutations by template directed determinator in incorporation with fluorescence polarization (TDI-FP)

Objective: To develop a new method for the detection of TPMT gene mutations and determine the frequencies of four TPMT alleles, TPMT *1, *3A , *3B and *3C in a healthy Chinese population. Methods: A TDI-FP assay system was set up in out lab. To evaluate this system, 220 healthy individuals were analyzed for the polymorphic sites at positions 460(G→A)and 719(A→G)of the TPMP gene using our new TDI–FP method. Results: Three TPMP*3C(G 460→G 719) heterozygotes were identified, TPMP *3A and TPMP *3B were not found. All mutations were confirmed by conventional DNA sequencing analysis. Conclusion: TDI-FP method has proven to be very efficient as a rapid and accurate approach for TPMP genotyping. TPMP *3C was the only polymorphism identified in this clinical samples we have registered.

Determining a Diagnostic Cut-Off on Fluorescence Polarization Assay(FPA)for Bovine Brucellosis in Carchi,Ecuador

Serology is the foundation of any brucellosis control and eradication program worldwide, thus it is important to define accuracy diagnostics assays and cut-off of those assays, due to variations from country to country and even among specific areas in the country. The variation of cutoff values depended on: prevalence of disease, vaccination status, animal management, and control and eradication programs. Therefore, a cut-off for the diagnosis of bovine brucellosis through fluorescence polarization assay (FPA) in Carchi—Ecuador was determined. The survey has been carried out in Carchi province of Ecuador, who is considered a province of high prevalence of brucellosis and the vaccination status is unknown due to the lack of registers. Sera samples (n = 200) were obtained from individual cows from randomly selected herds. Blood sera were tested through Fluoresce Polarization Assay (FPA) and competitive enzyme-linked inmunosorbent assay (cELISA) as confirmatory test, and then receiver operating characteristic (ROC) analysis was done. The sensitivity and specificity values of FPA were 88.7% and 92.50% respectively using a cut-off of 89.90 mP. Moreover, the area under the curve showed that 92.2% is the probability accuracy of the test. The advantage of the FPA is that it is a test with good characteristics of sensitivity and specificity as well as a simple and quick test.

The Development of A Fluorescence Polarization Immunoassay for Aflatoxin Detection

A fluorescence polarization immunoassay （FPIA） was developed for the analysis ofaflatoxins （AFs） using an anti-aflatoxin B1 （AFB1） monoclonal antibody and a novel fluorescein-labeled AFB1 tracer. The FPIA showed an IC50 value of 23.33 ng/mL with a limit of detection of 13.12 ng/mL for AFB1. The cross-reactivities of AFB1, AFB2, AFG1, AFG2, AFM1, and AFM2 with the antibody were 100%, 65.7%, 143%, 23.5%, 111.4%, and 2%, respectively. The group-specificity of anti-AFB1mAb indicated that the FPIA could potentially be used in a screening method for the detection of total AFs, albeit not AFG2 and AFM2. The total time required for analyzing 96 samples in one microplate was less than 5 rain. This study demonstrates the potential usefulness of the FPIA as a rapid and simple technique for monitoring AFs.

Simultaneous Determination of Fluorescein, Rhodamine 6G and Rhodamine B in Turbid Solution by Polarization Variable-Angle Synchronous Fluorescence Spectrometry

Polarization variable-angle synchronous fluorescence spectrometry was proposed to determine samples in turbid solution. A mixture of fluorescein, rhodamine 6G and rhodamine B was used to evaluate the technique. The background caused by scattering light was decreased remarkably. The limits of detection were 0.6 ng/ml for fluorescein, 2.3 ng/ml for rhodamine 6G and 4.1 ng/ml for rhodamine B, respectively.

Chlorophyll Fluorescence Extraction from Water-Leaving Radiance of Algae-Containing Water Through Polarization

When measuring reflectance spectra, it is very important to accurately extract chlorophyll fluorescence from elastic-scattering light in water-leaving radiance. The elastic scattering of light by water particles produces partially polarized light. In contrast, chlorophyll fluorescence in planktonic algae yields completely unpolarized light. These properties can be used to separate fluorescent signals from the water-leaving radiance and thus to determine chlorophyll concentration. The algal species Aureococcus anophagefferens was used to conduct a laboratory polarization experiment. For the tests, we used a field spectroradiometer and a polarizer; measurements were collected using two different observation modes. The chlorophyll fluorescence curve extracted through polarization shows an excellent match with the results obtained using the fluorospectro photometer for both measurement modes, suggesting that polarization-based chlorophyll fluorescence extraction may be feasible. The extracted fluorescence is more reliable at incident zenith angles ranging from 30? to 60?. For algae-containing water, the results improve with increasing chlorophyll concentration. This method could help improve chlorophyll concentration measurement and the remote-sensing detection of resulting harmful algae blooms.

Super-resolution fluorescence polarization microscopy

Fluorescence polarization is related to the dipole orientation of chromophores,making fuores-cence polarization microscopy possible to_reveal structures and functions of tagged cellularorganelles and biological macromolecules.Several recent super resolution techniques have beenapplied to fluorescence polarization microscopy,achieving dipole measurement at nanoscale.In this review,we summarize both difraction limited and super resolution fluorescence polari-zation microscopy techniques,as well as their applications in biological imaging.

A rapid and sensitive method to detect mycobacterium tuberculosis DNA by fluorescence polarization

Objective: To develop a new high sensitivity, rapid and simple mycobacterium tuberculosis DNA detection method using fluorescence polarization technology. Methods: In our asymmetric PCR protocol, 100 times mole of TB-R primer was added than in the usual symmetric PCR to get enough single strands PCR product. The probe TB-5′-TAMRA and PCR products were mixed in a tube and incubate for 5-15 min at 46 ℃.The polarization (mp) was measured using victor2 Multilabel Plate Reader. Results: Asymmetric and symmetric PCR products were analyzed by the FP method. Asymmetric PCR products are detected more sensitively than symmetric ones. The polarization values of probe associated with asymmetric products were much higher than with symmetric products. Conclusion: This fluorescence polarization assay in conjunction with asymmetric PCR is a powerful and widely applicable method for the rapid and sensitive detection of micro-organisms in clinical laboratories.

Rapid detection of PPAR_γ gene Pro12Ala polymorphism with fluorescence polarization in Chinese population

Objective: Peroxisome proliferator-activated receptor γ (PPARγ) plays a critical role in adipocyte differentiation and the development of type 2 diabetes mellitus (T2DM). Numerous studies across several populations have indicated that Pro12Ala polymorphism of PPARγ is associated with decreased insulin resistance and decreased risk of T2DM. The aims of this study are to develop a simple and sensitive detection of Pro12Ala polymorphism and examined the distribution of this polymorphism in Chinese population. Methods: The PPAR-γ gene fragment containing Pro12Ala variant of 101 T2DM patients and 104 controls were amplified by PCR amplification and the extension reaction was performed using primer that adjacent to the single nucleotide polymorphic site in presence of two different dye-labeled terminators. The primer's specially extending reactions make the increase of their fluorescence polarization (FP) that mean special genotype. The variant frequencies of the two groups were compared. Results: We detected the Pro12Ala variant successfully by TDI-FP method and we found no significant association between this polymorphism and T2DM in case-control study. Conclusion: The TDI-FP technology is a new specific and sensitive method that is suitable for automatic detection of large number of clinical samples. Pro12Ala mutation in PPAR-γ2 gene does not play a significant role in T2DM risk in Chinese population.

Fluorescence emission difference microscopy based on polarization modulation

In this paper,we propose a new fluorescence emission difference microscopy(FED)technique based on polarization modulation.An electro-optical modulator(EOM)is used to switch the excitation beam between the horizontal and vertical polarization states at a high frequency,which leads to solid-and donut-shaped beams after spatial light modulation.Experiment on the fluorescent nanoparticles demonstrates that the proposed method can achieve~λ=4 spatial resolution.Using the proposed system,the dynamic imaging of subcellular structures in living cells over time is achieved.

Antibody Production for a Rapid Fluorescence Polarization Immunoassay of Estrone

Estrone has been identified as a potential endocrine-disrupting chemical （EDC）[1]. Estrone is usually quantified by gas chromatography-mass spectrometry （GC-MS）, GC-MS/MS, high performance liquid chromatography （HPLC）, HPLC- MS, and HPLC-MS/MS, etc.[2-3]. Meanwhile, several immunoassays based on radioimmunoassay, enzyme linked immunosorbent assay （ELISA） or chemiluminescence immunoassay （CLIA） for determination of estrone in real samples have been developed[2＇4]. Although these methods are sensitive, they need multistage separation and are thus time-consuming and laborious. A very promising way for the simplification of immunoassays for routine applications is a shift from heterogeneous methods （with separation） to homogeneous assays （without separation）[5]. Fluorescence polarization immunoassay （FPIA） is one of the homogeneous techniques that meets the requirements of a simple, reliable, fast, and cost-effective analysis[6]. Therefore, the present study is focused on the development of FPIA in order to analyze estrone based on antibody production.

两种咔唑基-吡啶-N-氧化物内盐荧光极性探针研究

合成了4-(9H-咔唑-9-基)吡啶1-氧化物(CPNO)和4-(4-(9H-咔唑-9-基)苯基)吡啶1-氧化物(CPPNO)两种咔唑基-吡啶-N-氧化物内盐,测定了它们在不同溶剂中的紫外-可见吸收和荧光光谱,均表现出对溶剂极性较好的敏感性。计算表明,两个化合物都具有较大的激发态偶极矩,是化合物溶剂极性敏感性的原因。研究为开发新型的荧光极性探针提供了一种新思路。

比较HPLC和FPIA法测定人血清中卡马西平的血药浓度

目的比较治疗药物监测中高效液相色谱法(HPLC)和荧光偏振免疫法(FPIA)测定血清中卡马西平(CBZ)的浓度.方法收集上海华山医院和北京天坛医院服用CBZ的癫痫患者608例的稳态谷浓度血样,分别用HPLC法和FPIA法进行测定,用回归法和图解法考察2种测定方法的相关程度以及环氧化卡马西平(CBZE)的交叉反应率.结果CBZE能干扰CBZ的测定,CBZ测定值FPIA较HPLC法高4.58%(95%置信区间:-5.7%～17.6%).CBZE的交叉反应率随CBZE浓度增加而减小.结论在CBZ治疗药物监测中,对不同测定方法测定差异,应予以关注并作相应调整.

RAM-HPLC法和FPIA法测定人血中甲氨蝶呤浓度的比较

建立一种直接进样浸透限制介质高效液相色谱 (RAM- HPL C)法测定白血病人中甲氨蝶呤 (MTX)的血药浓度 ,并与荧光偏振免疫 (FPIA)测定结果相比较 .RAM- HPL C法具有样品用量小 ,专一性强 ,几乎 10 0 %回收率和重现性好的特点 .线性回归表明 RAM- HPL C和 FPIA两法测定结果之间有良好的相关性 ,MTX输注期的相关系数 (r )为 0 .95 5 ,消除期相关系数 (r )为 0 .932 .经 t检验 ,两法测定结果在消除期相差显著 (p <0 .0 5 ) ,说明 FPIA法较高地估计了 MTX的浓度 .RAM- HPLC可用于治疗和药物监测 .

基于FP法的EPOR配体高通量筛选模型

目的 寻找疗效好、副作用小的EPO功能类似物。方法 用FITC标记EPO，从FVA细胞中提取含EPO受体的细胞膜，建立了用FP法筛选EPOR配体的模型，并对实验条件进行了优化和可能影响模型的因素（DMSO）进行了分析，此外用Z’因子对此模型进行了评估。结果 样品中含0％～5％DMSO不会对实验结果产生影响，Z’因子的值为0．78．结论 建立的筛选模型稳定性好和准确率高。

多重、非对称PCR-FP技术同步检测沙眼衣原体和解脲支原体

目的:应用多重PCR-荧光偏振(FP)技术建立一种可靠、简单经济、快速的同步检测沙眼衣原体、解脲支原体的新方法,并推向临床应用.方法:以与沙眼衣原体dnaB样蛋白、解脲支原体UreC各血清型保守区序列互补的2对特异而无交叉反应的引物在同一反应体系中行多重、非对称聚合酶链反应扩增246例临床样本,应用沙眼衣原体、解脲支原体特异探针混合物与上述非对称扩增PCR产物孵育杂交,荧光偏振检测技术检测,并与常规检测方法测定结果比较.结果:应用多重、非对称PCR-FP技术同步检测泌尿生殖系感染临床标本246例,其中沙眼衣原体感染阳性56例,占22.7%;解脲支原体感染阳性62例,占25.2%;沙眼衣原体、解脲支原体混合感染阳性28例,占11.2%.与常规方法检测结果比较,两者对感染阳性的检测符合率为100%.结论:初步建立了具有良好的特异性、仅需一次扩增即可方便简单地检测沙眼衣原体、解脲支原体的同步检测新方法,对于相关感染的筛查及预防、预后判断具有重要价值.

FPIA法和HPLC-MS/MS法测定甲氨蝶呤血药浓度的相关性研究

目的评价荧光偏振免疫分析法（FPIA）和液质联用法（HPLC-MS/MS）测定人血浆中甲氨蝶呤（MTX）浓度的相关性。方法分别用FPIA法和HPLC-MS/MS法测定人血浆中MTX的浓度,并比较两者的相关性。结果当MTX浓度在0.05-1μmol·L^-1时,Wilcoxon检验结果表明FPIA法和HPLC-MS/MS法的测定结果差异有统计学意义,回归方程为：CFPIA=1.0923·CHPLC-MS/MS＋0.001 084,r=0.975。当MTX浓度在1-100μmol·L^-1时,配对t检验结果表明FPIA法和HPLC-MS/MS法的测定结果差异无统计学意义,回归方程为：CFPIA=0.9811·CHPLC-MS/MS-0.4405,r=0.939。结论 FPIA法和HPLC-MS/MS法是否存在显著性差异取决于MTX浓度,MTX浓度可通过公式换算后供临床医师参考。

TDI-FP技术检测耐乙胺丁醇结核分支杆菌embB306点突变

目的 应用TDI FP技术分析结核分支杆菌embB 30 6点突变与乙胺丁醇耐药性的关系 ,并建立一种准确、快速的诊断结核分支杆菌乙胺丁醇耐药的新方法。方法 对临床 82例耐多药结核病患者所感染的结核分支杆菌菌株进行常规培养和药敏实验 ;提取结核分支杆菌DNA ,并PCR扩增 2 0 5bp的embB基因片段 ,消化降解掉PCR产物中残余的dNTPs和引物 ,进行模板指导的荧光标记终止碱基掺入反应 ;应用Victor2测定反应产物的荧光偏振值 ,分析所有样品的embB基因 30 6位点的基因型 ;对分析结果进行测序验证。结果 5例乙胺丁醇高度耐药患者中有 3例所感染的结核分支杆菌发生embB 30 6的ATG→GTG突变 ;4 7例乙胺丁醇低度耐药患者中有 15例为embB 30 6突变和未突变的混合感染 ;30例乙胺丁醇敏感患者的结核分支杆菌未发现embB 30 6突变。测序结果与实验相符合。结论 embB 30 6突变与结核分支杆菌对乙胺丁醇产生耐药性密切有关 ;应用TDI FP技术可以准确、快速简便检测embB 30 6突变 ,在结核分支杆菌耐乙胺丁醇的临床诊断中具有潜在的应用前景。

非对称PCR-FP技术同步检测HSV-1/-2、CMV和EBV

目的应用非对称PCR-荧光偏振（FP）技术建立一种同步检测全血中4种主要疱疹病毒（单纯疱疹病毒1／2型，巨细胞病毒、EB病毒）的新方法。方法以人疱疹病毒属通用引物行非对称PCR，扩增从179例样本中抽提的DNA。PCR扩增产物与特异性单纯疱疹病毒1／2型（HSV-1／-2）、巨细胞病毒（CMV）和EB病毒（EBV）寡核苷酸探针混合物温育杂交，荧光偏振检测技术检测杂交液荧光偏振值，据荧光偏振值判断病毒感染类型，并以DNA序列测定结果为参照。结果与DNA序列测定的阳性检测符合率为100％，但DNA序列测定检测均未检出多重混合感染。非对称PCR-FP方法对HSV-1／-2检测的灵敏度达到1．0×10^3拷贝／ml．对EBV和CMV检测的灵敏度达到2．0×10^3拷贝／ml。结论本法对于4种主要疱疹病毒感染的筛查及预防、预后判断具重要价值。

EMIT法和FPIA法监测全血环孢素A浓度相关性比较

目的比较酶放大免疫法(EMIT)与荧光偏振免疫法(FPIA)监测全血中环孢素A(Cs A)浓度的测定结果,并进行相关性分析。方法收集126例/次临床使用环孢素A的患者稳态浓度血样,分别用EMIT法及FPIA法测定,并考察两种方法测定结果的相关程度。结果 2种不同方法测定Cs A血药浓度平均值(±s)分别为EMIT法:(205.77±117.65)ng/m L;FPIA法:(186.49±112.16)ng/m L,以SPSS19.0对经对数转换后的两组浓度数据进行配对样本T检验,其P=0.00,即两种测定方法有显著性差异。将患者分为肾移植组(63例/次),肝移植组(17例/次),干细胞移植组(31例/次),肾病综合征组(15例/次)。以SPSS19.0对四组浓度数据分别进行组内的配对样本T检验,得到肾移植组与肝移植组P<0.05,差异具统计学意义。干细胞移植组与肾病综合征组P>0.05,差异无统计学意义。结论对CsA进行治疗药物监测时应考虑不同分析方法的影响。