Determination of SARS-coronavirus by a microfluidic chip system

We have developed a new experimental system based on a microfluidic chip to determine severe acute respiratory syndrome coronavirus (SARS-Cov). The system includes a laser-induced fluorescence microfluidic chip analyzer, a glass microchip for both polymerase chain reaction (PCR) and capillary electrophoresis, a chip thermal cycler based on dual Peltier thermoelectric elements, a reverse transcription-polymerase chain reaction (RT-PCR) SARS diagnostic kit, and a DNA electrophoretic sizing kit. The system allows efficient cDNA amplification of SARS-CoV followed by electrophoretic sizing and detection on the same chip. To enhance the reliability of RT-PCR on SARS-CoV detection, duplex PCR was developed on the microchip. The assay was carried out on a home-made microfluidic chip system. The positive and the negative control were cDNA fragments of SARS-CoV and parainfluenza virus, respectively. The test results showed that 17 positive samples were obtained among 18 samples of nasopharyngeal swabs from clinically diagnosed SARS patients. However, 12 positive results from the same 18 samples were obtained by the conventional RT-PCR with agarose gel electrophoresis detection. The SARS virus species can be analyzed with high positive rate and rapidity on the microfluidic chip system.

Frankia基因组DNA提取及其PCR带型

本文采用溶菌酶法、CTAB法、微波（MW）法及CTAB＋MW法等4种方法提取Frankia菌CpIl基因组DNA。结果表明四种方式均可满足试验要求，其中以CTAB法及微波法最为有效。在比较提取方法与PCR带型间的关系中，发现提取上的差异不影响PCR带型的变化。

Development of functionalized terbium fluorescent nanoparticles for antibody labeling and time-resolved fluoroimmunoassay application

Silica-based functionalized terbium fluorescent nanoparticles were prepared, characterized and developed as a fluorescence probe for antibody labeling and time-resolved fluoroimmunoassay. The nanoparticles were prepared in a water-in-oil (W/O) microemulsion containing a strongly fluorescent Tb3+ chelate. N,N.N-1,N-1-12,6-bis(3'-aminomethyl-1'-pyrazolyl)phenylpyridine] tetrakis(acetate)-Tb3+ (BPTA-Tb3+), Triton X-100, octanol, and cyclohexane by controlling copolymerization of tetraethyl orthosilicate (TEOS) and 3-[2-(2- aminoethylamino)-ethylamino]propyl-trimethoxysilane (AEPS) with ammonia water. The characterizations by transmission electron microscopy and fluorometric quantum methods show that the nanoparticles are spherical and uniform in size, 45 +/- 3 nm in diameter, strongly fluorescent with fluorescence yield of 10% and a long fluorescence lifetime of 2.0 ms. The amino groups directly introduced to the nanoparticle's surface by using AEPS in the preparation made the surface modification and bioconjugation of the nanoparticles easier. The nanoparticle-labeled anti-human alpha-fetoprotein antibody was prepared and used for time-resolved fluoroimmunoassay of (x-fetoprotein (AFP) in human serum samples. The assay response is linear from 0.10 ng ml(-1) to about 100 ng ml(-1) with the detection limit of 0.10 ng ml(-1). The coefficient variations (CVs) of the method are less than 9.0%. and the recoveries are in the range of 84-98% for human serum sample measurements. (C) 2004 Elsevier B.V. All rights reserved.

长白山赤杨丛枝菌根真菌多样性的半巢式LP-PCR-SSCP检测分析

利用半巢式LP PCR SSCP技术 ,对中国吉林长白山不同海拔生境下 3种赤杨共生丛枝菌根真菌的多样性进行检测分析 .结果表明 ,该地区赤杨属东北赤杨、西伯利亚赤杨及色赤杨共生丛枝菌根真菌在科乃至种的水平上并未随宿主的变化表现出丰富的多样性 ;3个树种在自身属的水平上与共生的球囊霉科 (Glomaceae)至少 1种AMF ,即G .intraradix ,在种的水平上表现出不相关于宿主海拔高度的某种相互选择性 .

长白山四种赤杨丛枝菌根真菌侵染多样性的巢式PCR-RFLP分析

采集中国吉林长白山不同海拔的4种赤杨根须样本,利用巢式PCR-RFLP方法检测丛枝菌根真菌(AMF)对样品的侵染情况,PCR结果经限制性内切酶分析.结果表明,赤杨根内AMF存在丰富的基因多样性.AMF的侵染有从宿主混乱性向宿主专一性发展的趋势.东北赤杨AMF的宿主专一性水平最强,球囊霉属已成为东北赤杨的优势侵染类群;其余3种赤杨的AMF则出现宿主混乱现象.宿主因素比海拔因素对AMF侵染有更重要的影响.

Development of a precolumn derivatization method for the determination of free amines in wastewater by high-performance liquid chromatography via fluorescent detection with 9-(2-hydroxyethyl)acridone

A simple, sensitive, and mild method for the determination of amino compounds based on a condensation reaction with fluorescence detection has been developed. 9-(2-Hydroxyethyl)acridone reacts with coupling agent N,N-carbonyldiimidazole at ambient temperature to form activated amide intermediate 9-(2-acridone)oxyethylcarbonylimidazole (AOCD). The amide intermediate (AOCD) preferably reacts with amino compounds under mild reactions in the presence of 4-(dimethylamino)pyridine (base catalyst) in acetonitrile to give the corresponding sensitively fluorescent derivatives with an excitation maximum lambda(ex) 404 mn and an emission maximum at lambda(em) 440 nm. The labeled derivatives exhibit high stability under reversed-phase conditions. The fluorescence intensities of derivatives in various solvents or at different temperatures were investigated. The method, in conjunction with a gradient elution, offers a baseline resolution of the common amine derivatives on a reversed-phase C-18 column. The LC separation for the derivatized amines shows good reproducibility with acetonitrile-water including 2.5% DMF as mobile phase. The relative standard deviations (n = 6) for each amine derivative are <4.5%. The detection limits (at a signal-to-noise ratio of 3) per injection were 0.16-12.8 ng/mL. Further research for the field of application, based on the AOCD amide intermediate as derivatization reagent, for the determination of free amines in real water samples is achieved.

Highly efficient separation of dsDNA fragments on glass chips by using an ultralow viscosity sieving matrix

A novel protocol has been established to separate dsDNA fragments with high efficiency on glass chips by using an ultralow viscosity sieving matrix with added glucose. Low-molecular-weight hydroxypropylmethylcellulose (HPMC), with a viscosity nearly equivalent to that of water, was used to electrophoretically separate fluorescent inter-calator-labeled double-stranded DNA (dsDNA) fragments on microfluidic glass chips. In comparison with conventional sieving protocols, low-molecular-weight HPMC as sieving matrix could result in reduced running cost and analysis time, in addition to a comparable separation efficiency of dsDNA fragments. In this paper, the addition of glucose was investigated to enhance the separation of DNA in the lowest viscosity polymer evaluated. The effect of staining dye and field strength were also evaluated. At an applied electric field strength of 200 V/cm, satisfactory resolution of the PBR322/HaeIII DNA marker could be achieved within 4 min by using 2% HPMC-5 with 6% glucose added. Coelectrophoresing PCR product along with phiX174/HaeIII DNA sizing marker was also demonstrated by using the ultralow viscosity HPMC-5 solution on a glass chip.