Ionic solution and nanoparticle assisted MALDI-MS as bacterial biosensors for rapid analysis of yogurt

Bacterial analysis from food samples is a highly challenging task because food samples contain intensive interferences from proteins and carbohydrates. Three different conditions of yogurt were analyzed: (1) the fresh yogurt immediately after purchasing, (2) the yogurt after expiry date stored in the refrigerator and (3) the yogurt left outside, without refrigeration. The shelf lives of both these yogurt was compared in terms of the decrease in bacterial signals. AB which initially contained 10(9) cells/mL drastically reduced to 10(7) cells/mL. However, Lin (Feng-Yin) yogurt which initially (fresh) had 10(8) cells/mL, even after two weeks beyond the expiry period showed no marked drop in bacterial count. Conventional MALDI-MS analysis showed limited sensitivity for analysis of yogurt bacteria amidst the complex milk proteins present in yogurt. A cost effective ionic solution, CrO(4)(2-) solution was used to enable the successful detection of bacterial signals (40-fold increased in sensitivity) selectively without the interference of the milk proteins. 0.035 mg of Ag nanoparticles (NPs) were also found to improve the detection of bacteria 2-6 times in yogurt samples. The current approach can be further applied as a rapid, sensitive and effective platform for bacterial analysis from food.     

Fabrication of titanium based MALDI bacterial chips for rapid, sensitive and direct analysis of pathogenic bacteria

For the first time, we report the fabrication of a titanium bacterial chip for MALDI-MS produced from a simple, cost effective and rapid heat treatment process. This bacterial chip can be reused many times and is highly versatile. These bacterial chips serve dual roles: (1) They can be applied as MALDI-MS target plates for direct and highly sensitive bacterial analysis. (2) They can be used as bacterial sensors for direct analysis of the captured bacteria using MALDI-MS. The sensitivity of these chips when used as bacterial sensors is <10(3)cfu/mL. The lowest detectable concentration for direct MALDI-MS analysis was found to be 10(4)cfu/mL. The results were further justified by using standard plate counting method combined with Tukey-Kramer statistical analysis and fluorescence imaging followed by image processing for fluorescence quantification using ImageJ software to substantiate the MALDI-MS results.     

Rapid and direct detection of Invivo kinetics of pathogenic bacterial infection from mouse blood and urine

This study demonstrates the first use of matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) to trace the Invivo infection kinetics of the well known deadly pathogen Staphylococcus aureus in Swiss albino mice. The growth curve of the bacteria from the point of injection (200μL of bacterial suspension (10(8)cfu/mL)) into the mouse blood till mortality (death) was periodically analyzed using the plate counting method and MALDI-MS. Bacterial counts of 10(3)cfu/mL were observed in the log phase of the growth curve in the blood and 10(2)cfu/mL were observed in the urine samples. Death occurred in the log phase of the growth curve, where the bacterial counts showed steady increase. In other cases, the bacteria counts started decreasing after 48h and by 96h the bacteria got totally eliminated from the mouse and these mice survived. Direct MALDI-MS was not feasible for tracking the bacteria in the infected blood. However, ionic liquid 1-Butyl-3-methylimidazolium tetrafluoroborate was successful in enabling bacterial detection amidst the strong blood peaks. But, in the case of the urine analysis, it was observed that direct MALDI-MS was adequate to enable detection. The results obtained prove the efficacy of MALDI-MS for analyzing pathogenic bacteria in clinical samples. This article is part of a Special Issue entitled: Proteomics: The clinical link.     

免疫捕获PCR法快速检测金黄色葡萄球菌

旨在建立一种快速检测金黄色葡萄球菌(Staphylococcus aureus,SA)免疫捕获PCR技术,并探讨其灵敏度和特异性。SA特异性抗体包被PCR管以富集待测样品中目标菌,之后在同一PCR管里直接进行免疫捕获PCR,并和直接PCR比较。免疫捕获PCR法可特异性检测2株SA菌株,而无法检测到其它8种常见食源性致病菌,说明该方法对SA具有良好的特异性;该方法对纯菌液而言,检测灵敏度可达到2.35×102CFU/mL,是直接PCR的100倍;对5种食品模拟带菌检测发现,无需增菌培养,其灵敏度可达到2.35×103-2.35×104CFU/mL,是直接PCR的10-100倍。免疫捕获PCR法集免疫学与分子生物学检测技术于一体,具有高特异性、高灵敏度、检测快速、易于操作、成本低廉等诸多优点,是一种适合基层实验室使用的检测技术。     

The development of a matrix-assisted laser desorption/ionization mass spectrometry-based method for the protein fingerprinting and identification of Aeromonas species using whole cells

This report describes the development of a method to detect the waterborne pathogen Aeromonas using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The genus Aeromonas is one of several medically significant genera that have gained prominence due to their evolving taxonomy and controversial role in human diseases. In this study, MALDI-MS was applied to the characterization of seventeen species of Aeromonas. These seventeen species were represented by thirty-two strains, which included type, reference and clinical isolates. Intact cells from each strain were used to generate a reproducible library of protein mass spectral fingerprints or m/z signatures. Under the test conditions used, peak lists of the mass ions observed in each species revealed that three mass ions were conserved among all the seventeen species tested. These common mass ions having an average m/z of 6301, 12,160 or 12,254, and 13,450, can be potentially used as genus-specific biomarkers to identify Aeromonas in unknown samples. A dendrogram generated using the m/z signatures of all the strains tested indicated that the mass spectral data contained sufficient information to distinguish between genera, species, and strains. There are several advantages of using MALDI-MS based protein mass spectral fingerprinting of whole cells for the identification of microorganisms as well as for their differentiation at the sub-species level: (1) the capability to detect proteins, (2) high throughput, and (3) relatively simple sample preparation techniques. The accuracy and speed with which data can be obtained makes MALDI-MS a powerful tool especially suited for environmental monitoring and detection of biological hazards.     

Preparation of highly luminescent mannose-gold nanodots for detection and inhibition of growth of Escherichia coli

In this paper, we describe a novel, simple, and convenient method for preparing water-soluble biofunctional gold nanodots (Au NDs) for the sensitive and selective detection of Escherichia coli (E. coli) and the inhibition of its growth. We obtained luminescent mannose-capped Au NDs (Man-Au NDs) from as-prepared 2.9-nm Au nanoparticles (Au NPs) and 29,29'-dithio bis(3',6',9',12',15',18'-hexaoxa-nonacosyl α-D-mannopyranoside) (Man-RSSR-Man). To obtain improved quantum yield (>20%), luminescent Man-Au NDs (1.8 nm) were prepared from Au NPs (0.47 μM) and Man-RSSR-Man (2.5 mM) in the presence of sodium borohydride (NaBH(4); 1.0 mM). The highly luminescent properties of Man-Au NDs prepared by the NaBH(4)-assisted method were characterized by UV-vis absorption, photoluminescence, and X-ray photoelectron spectroscopies. The results supported the high-density coverage of the NDs surface by Man-RS ligands. Multivalent interactions between Man-Au NDs and FimH proteins located on the bacterial pili of E. coli resulted in the formation of aggregated cell clusters. After concentrating this agglutinative E. coli from a large-volume cell solution (5 mL), Man-Au NDs were displaced by mannose (100 mM) and stabilized by Man-RSSR-Man (5 mM). Monitoring the luminescence of Man-Au NDs allowed the detection of E. coli at levels as low as 150 CFU/mL. Man-Au NDs were also found to be efficient antibacterial agents, selectively inhibiting the growth of E. coli through Man-Au ND-induced agglutination. Our small-diameter Man-Au NDs, which provided an ultra high ligand density (local concentration) of mannose units for multivalent interactions with E. coli, have great potential for use as an antibacterial agent in other applications.     

Oxygen tolerance of an implantable polymer/enzyme composite glutamate biosensor displaying polycation-enhanced substrate sensitivity

Biosensors were fabricated at neutral pH by sequentially depositing the polycation polyethyleneimine (PEI), the stereoselective enzyme L-glutamate oxidase (GluOx) and the permselective barrier poly-ortho-phenylenediamine (PPD) onto 125-microm diameter Pt wire electrodes (Pt/PEI/GluOx/PPD). These devices were calibrated amperometrically at 0.7 V versus SCE to determine the Michaelis-Menten parameters for enzyme substrate, l-glutamate (Glu) and co-substrate, dioxygen. The presence of PEI produced a 10-fold enhancement in the detection limit for Glu (approximately 20 nM) compared with the corresponding PEI-free configurations (Pt/GluOx/PPD), without undermining their fast response time (approximately 2 s). Most remarkable was the finding that, although some designs of PEI-containing biosensors showed a 10-fold increase in linear region sensitivity to Glu, their oxygen dependence remained low.     

Ultra-fine Pt nanoparticles supported on ionic liquid polymer-functionalized ordered mesoporous carbons for nonenzymatic hydrogen peroxide detection

Poly(ionic liquid) (PIL) coated ordered mesoporous carbons (OMCs) were prepared by in situ polymerization of 3-ethyl-1-vinylimidazolium tetrafluoroborate ([VEIM]BF(4)) monomer on OMCs matrix. PIL on the surface of OMCs can provide sufficient binding sites to anchor the precursors of metal ion. PIL/OMCs were employed as support material for the deposition and formation of ultra-fine Pt nanoparticles, via the self-assembly between the negative Pt precursor and positively charged functional groups of PIL-functionalized OMCs. The combination of the unique properties of each component endows Pt/PIL/OMCs as a good electrode material. Compared with the Pt/OMCs nanocomposite, the Pt/PIL/OMCs modified electrode displays high electrocatalytic activity towards hydrogen peroxide (H(2)O(2)) and gives linear range from 1.0 × 10(-7) to 3.2 × 10(-3) M (R=0.999). The Pt/PIL/OMCs responds very rapidly to the changes in the level of H(2)O(2), producing steady-state signals within 4-5s. A high sensitivity of 24.43 μA mM(-1) and low detection limit of 0.08 μM was obtained at Pt/PIL/OMCs modified electrode towards the reduction of H(2)O(2). The improved activity makes Pt/PIL/OMCs nanocomposite promising for being developed as an attractive robust and new electrode material for electrochemical sensors and biosensors design.     

Nanoflow liquid chromatography coupled to matrix-assisted laser desorption/ionization mass spectrometry: sample preparation, data analysis, and application to the analysis of complex peptide mixtures

We report the development of a robust interface for off-line coupling of nano liquid chromatography (LC) to matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) and its application to the analysis of proteolytic digests of proteins, both isolated and in mixtures. The interface makes use of prestructured MALDI sample supports to concentrate the effluent to a small sample plate area and localize the MALDI sample to a predefined array, thereby enriching the analyte molecules and facilitating automated MALDI-MS analysis. Parameters that influence the preparation of MALDI samples from the LC effluent were evaluated with regard to detection sensitivity, spectra quality, and reproducibility of the method. A procedure for data processing is described. The presented nano LC MALDI-MS system allowed the detection of several peptides from a tryptic digest of bovine serum albumin, at analyzed amounts corresponding to one femtomole of the digested protein. For the identification of native proteins isolated from mouse brain by two-dimensional gel electrophoresis, nano LC MALDI-MS increased the number of detected peptides, thereby allowing identification of proteins that could not be identified by direct MALDI-MS analysis. The ability to identify proteins in complex mixtures was evaluated for the analysis of Escherichia coli 50S ribosomal subunit. Out of the 33 expected proteins, 30 were identified by MALDI tandem time of flight fragment ion fingerprinting.     

Facile fabrication of magnetic gold electrode for magnetic beads-based electrochemical immunoassay: application to the diagnosis of Japanese encephalitis virus

A novel magnetic beads-based electrochemical immunoassay strategy has been developed for the detection of Japanese encephalitis virus (JEV). The magnetic gold electrode was fabricated to manipulate magnetic beads for the direct sensing applications. Gold-coated magnetic beads were employed as the platforms for the immobilization and immunoreaction process, and horseradish peroxidase was chosen as an enzymatic tracer. The proteins (e.g., antibodies or immunocomplexes) attached on the surface of magnetic beads were found to induce a significant decline in their electric conductivity. Multiwalled carbon nanotubes were introduced to improve sensitivity of the assay. The envelope (E) protein, a major immunogenic protein of JEV, was utilized to optimize the assay parameters. Under the optimal conditions, the linear response range of E protein was 0.84 to 11,200 ng/mL with a detection limit of 0.56 ng/mL. When applied for detection of JEV, the proposed method generated a linear response range between 2×10(3) and 5×10(5) PFU/mL. The detection limit for JEV was 2.0×10(3) PFU/mL, which was 2 orders of magnitude lower than that of immunochromatographic strip and similar to that obtained from RT-PCR. This method was also successfully applied to detect JEV in clinical specimens.     

Electrochemiluminescent biosensing of carbohydrate-functionalized CdS nanocomposites for in situ label-free analysis of cell surface carbohydrate

A facile electrochemiluminescent (ECL) strategy for in situ label-free monitoring of carbohydrate expression on living cells was designed by integrating the specific recognition of lectin to carbohydrate with a carbohydrate-functionalized CdS nanocomposite. The mercaptopropionic acid-capped CdS quantum dots were firstly immobilized on carbon nanotubes modified electrode and then functionalized with carbohydrate using mannan as a model on the surface. The carbohydrate-functionalized CdS nanocomposite showed high ECL sensitivity and good stability, and could be used for competitive recognition to concanavalin A with the target cells in solution, which led to a change of ECL intensity due to the resistance of concanavalin A. The change depended on both the cell number and the expression level of cell surface carbohydrate. A wide linear response to cells ranging from 2×10(3) to 1×10(7) cells mL(-1) with a detection limit of 1.2×10(3) cells mL(-1) was obtained. The proposed biosensor could be used to in situ evaluate cell surface glycan, and the average number of mannose moieties on single living BGC cell was detected to be 8.7×10(7). This sensitive strategy was further used for facile monitoring of dynamic carbohydrate expression on living cells in response to drugs. The proposed method could be further expanded to high-throughput detection with the addition of more specific glycan-lectin pairs to the repertoire.     

A DNA sequence-specific electrochemical biosensor based on alginic acid-coated cobalt magnetic beads for the detection of E. coli

A new type of DNA sequence-specific electrochemical biosensor based on magnetic beads for the detection of Escherichia coli is reported in the present work. Alginic acid-coated cobalt magnetic beads, capped with 5'-(NH(2)) oligonucleotide and employed not only for magnetic separation but also as the solid adsorbent, were used as DNA probes to hybridize with the target E. coli DNA sequence. This assay was specific for E. coli detection depending on the uid A gene, which encodes for the enzyme β-d-glucuronidase produced by E. coli strains. When daunomycin (DNR) was used as DNA hybridization indicator, the target sequences of E. coli hybridized with the probes resulted in the decrease of DNR reduction peak current, which was proportional to the E. coli concentration. The optimization of the hybridization detection was carried out and the specificity of the probes was also demonstrated. This DNA biosensor can be employed to detect a complementary target sequence for 3.0×10(-10) mol/L and denatured PCR products for 0.5 ng/μL. The linear range of the developed biosensor for the detection of E. coli cells was from 1.0×10(2) to 2.0×10(3) cells/mL with a detection limit of 50 cells/mL. After a brief enrichment process, a concentration of 10 cells/mL E. coli in real water samples was detected by the electrochemical biosensor.     

Platinum-induced mutations to 8-azaguanine resistance in Chinese hamster ovary cells.

6 platinum (Pt) compounds were compared in suspension cultured Chinese hamster ovary (CHO-S) cells with respect to their inhibition of growth, their reduction of cloning efficiency, and their induction of mutants resistant to 200 microM (30 micrograms/ml) 8-azaguanine (8-AG) and 3 mM ouabain (OUA), respectively. The toxicity of these compounds can be ranked by the medium concentrations which decrease suspension growth/or cloning efficiency by 50%: cis-Pt(NH3)2-Cl2 (0.9/1.5 microM) greater than Pt(SO4)2 + methylcobalamin (MeB-12) methylation product (20/10 microM) greater than K2PtCl4 (32/50 microM) = K2PtCl6 (34/50 microM) = MePtCl2-3 (60/50 microM) greater than Pt(SO4)2 (66/105 microM). Following 20 h exposures to concentrations which resulted in relative survivals of 80-2%, none of the foregoing compounds increased consistently the frequency of OUA(R) mutants above the spontaneous frequency (6.0 x 10(-6)). Parallel treatments with 800 microM (100 micrograms/ml) ethyl methanesulfonate (EMS) increased the OUA(R) mutant frequency 10--12-fold. Using 8-AG for mutant selection, dose-dependent increases of 5--7-fold above the spontaneous frequency (3--8 x 10(-5) were obtained with cis-Pt(NH3)2Cl2, Pt(S04)2, and the product from Pt(SO4)2 + MeB-12. Identical 20 h exposures to varying amounts of K2PtCl4, K2PtCl6, and MePtCl2-3 did not induce 8-AG(R) mutants. Optimal detection of Pt-induced 8-AG(R) mutants required 7 post-treatments, expression doublings in suspension culture. Under our selection conditions 8/8 spontaneous and 24/24 Pt-induced 8-AG(R) variants contained reduced hypoxanthine-guanine phosphoribosyl transferase (HGPRT) specific activities (means ranging from 3 to 11% of the parental CHO-S cells). When compared from linear plots of the 8-Ag(r) frequency against the initial medium concentration, cis-Pt(NH3)2Cl2 is 134 times and Pt(SO4)2 si 3.5 times more mutagenic than EMS. However, on a cell-survival basis EMS is 8--10-fold more mutagenic than these two Pt-compounds. 6-Thioguanine (10 microM) can be substituted for 8-AG to assay mutant induction by cis-Pt(NH3)2Cl2 and Pt(SO4)2 in CHO-S cells. The sensitivity of the CHO-S HGPRT locus for detecting mutagenesis by Pt complexes can be increased several fold by continuous subculture in the presence of these agents for 10--25 population doublings. By this procedure K2PtCl6 is seen to be weakly mutagenic and 20 microM Pt(SO4)2 produces 8-AG(R) mutants at frequencies requiring 7--8-fold higher concentrations when a fixed 20 h exposure is used.     

Highly reproducible immunoassay of cancer markers on a gold-patterned microarray chip using surface-enhanced Raman scattering imaging

This paper reports a highly reproducible immunoassay of cancer markers using surface-enhanced Raman scattering (SERS) imaging. SERS is a highly sensitive detection method but it is limited in its ability to achieve reproducible signal enhancement because of the difficulty with precisely controlling the uniform distribution of hot junctions. Consequently, inconsistent enhancement prevents the wide exploitation of SERS detection as a bio-detection tool for quantitative analysis. To resolve this problem, we explored the use of a SERS imaging-based immunoassay. For this purpose, Raman reporter-labeled hollow gold nanospheres (HGNs), were manufactured and antibodies were immobilized onto their surfaces for targeting specific antigens. After the formation of sandwich immunocomplexes using these functional HGNs on the surfaces of gold patterned wells, the SERS mapping images were measured. For target protein markers, 12×9 pixels were imaged using a Raman mapping technique in the 0-10(-4) g/mL concentration range, and the SERS signals for 66 pixels were averaged. Here, the SERS imaging-based assay shows much better correlations between concentration and intensity than does the conventional point-based assay. The limits of detection were determined to be 0.1 pg/mL and 1.0 pg/mL for angiogenin (ANG) and alpha-fetoprotein (AFP), respectively. This detection sensitivity is increased by three or four orders of magnitude over that of conventional ELISA method. The detectable dynamic range for SERS imaging (10(-4)-10(-12) g/mL) is also much wider than that for ELISA (10(-6)-10(-9) g/mL).     

Improved chemical analysis of cellulose ethers using dialkylamine derivatization and mass spectrometry

Oligosaccharides of hydroxypropylmethyl cellulose, hydroxypropyl cellulose, and methyl cellulose were investigated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The cellulose ether oligosaccharides were produced either by enzymatic depolymerization utilizing the purified family 5 endoglucanase from Bacillus agaradhaerens or by partial acidic depolymerization. To lower the limit of detection in MALDI-MS three dilakylamines, dimethyl-, diethyl-, and dipropylamine were studied as reagents for reductive amination of the oligosaccharides. All three amines contributed to a significant increase in sensitivity in MALDI-MS, especially for oligosaccharides with a degree of polymerization (DP) < 3. These reagents were also attractive due to their high volatility, which facilitated the purification of the reaction mixtures. It was established that low-mass discrimination in MALDI-MS in the DP range 1-7 was substantially reduced with dialkylamine derivatization. Hence, dialkylamine derivatization of cellulose ether oligosaccharides obtained by endoglucanase depolymerization increased the number of detected analyte components. Dimethylamine was concluded to be the preferred reagent of those evaluated.     

农杆菌介导转化莱茵衣藻体系的优化

[目的]为建立根癌农杆菌介导的莱茵衣藻快速简便高效的遗传转化体系,本研究以模式生物莱茵衣藻为受体材料,从转化方法和转化子快速鉴定两个方面进行了优化.[方法]比较了固体培养基共培养转化方法和液体培养基共培养转化方法对根癌农杆菌LBA 4404介导的莱茵衣藻CC425转化效率的影响;研究并比较了(1)首先经过TE裂解再进行...     

3种菊花病毒/类病毒实时荧光定量RT-PCR检测体系的构建与准确性评价

菊花容易受到病毒感染而造成品质下降,目前国内对菊花病毒的检测主要根据外观表现或者定性PCR检测,无法准确判定病毒载量。为构建一种可同时用于检测菊花B病毒(Chrysanthemum virus B,CVB)、番茄不孕病毒(Tomato aspermy virus,TAV)和菊花褪绿斑驳类病毒(Chrysanthemum chloritic mottle viroid,CChMVd)的实时荧光定量RT-PCR检测方法,本研究分别以保守区域作为靶标设计相应的引物探针,通过优化扩增体系中CVB、TAV、CChMVd 3种病毒/类病毒探针浓度、引物浓度、Mg²⁺浓度、dNTPs浓度,摸索扩增程序中反转录时间、退火温度和扩增循环数,构建了一种可同时用于CVB、TAV、CChMVd的3重实时荧光定量RT-PCR检测体系,优化后的扩扩增体系中CVB、TAV和CChMVd的探针浓度分别为100 nmol/L、120 nmol/L和80 nmol/L,引物浓度分别为200 nmol/L、240 nmol/L和160 nmol/L,Mg²⁺浓度为3.0 mmol/L;dNTPs浓度200μmol/L;最适反转录时间为25 min,退火温度为60℃,循环数为40。敏感性实验结果表明,该反应体系对3种病毒/类病毒的敏感性为1.0×¹⁰3拷贝/mL,敏感性好;定量线性范围为1.0×¹⁰3拷贝/mL~1.0×¹⁰10拷贝/mL,线性范围宽;特异性好,对菊花矮化类病毒、烟草花叶病毒和黄瓜花叶病毒核酸检测结果为阴性;对1.0×¹⁰4拷贝/mL的低浓度参考品平行检测10次,定量结果lg值偏差(CV%)为4.81%,重复性好。在南京农业大学"中国菊花种质资源保存中心"基地随机选择菊花20株进行本研究试剂检测,检出6例CVB病毒株和4例TAV病毒株,其病毒载量为2.5×¹⁰4拷贝/mL~5.5×¹⁰7拷贝/mL,随机选择1株CVB病毒株定量PCR,产物进行TA克隆后经测序与NCBI Blast比对,其与MH678704.1的同源性为100%。因此,本研究建立了一种能同时检测CVB、TAV、CChMVd 3种菊花常见病毒/类病毒的灵敏、快速、可定量的检测方法。     

Processing of autologous bone marrow cells by apheresis technology for cell-based cardiovascular regeneration

Background aims. Bone marrow (BM)-derived mononuclear cell (MNC) preparations are increasingly used in experimental studies exploring the potential effect of progenitor cell-derived therapies in cardiocirculatory diseases. We analyzed the cellular BM composition, side-effects and other process-related variables of BM harvest and BM-MNC preparation in 80 patients with cardiovascular disease. Methods. BM (median 828 mL, range 223-1038 mL) was collected from the iliac crest. After BM harvest the MNC fraction was enriched by semi-automatic apheresis to reduce the total volume of the transplant. Autologous red blood cells (RBC) were salvaged from the initial BM harvest and autotransfused to the patients. Results. There were no serious side-effects related to BM collection, particularly no serious bleeding complications. Twenty- five of 80 (31%) patients developed mild pain. BM harvest resulted in the collection of a median of 2.8 × 10(9) MNC, containing a median of 66.5 × 10(6) CD34/45 cells, 39.5 × 10(6) CD133/45 cells and 50.3 × 10(6) CD34/CD133 cells. Apheresis technology-based MNC enrichment of harvested BM resulted in a progenitor cell recovery of 69-75.3% of total cells. Additional salvage of RBC from the initial BM harvest resulted in the recovery of a median of 175.0 mL autologous RBC mass. Transfusion of salvaged RBC was well tolerated and resulted in a significant increase in hemoglobin levels. Conclusions. Collection of BM of up to 1 L in combination with in vitro processing using a semi-automated apheresis device is a safe and feasible approach to increasing the number of progenitor cells necessary for cellular therapies, particularly when combined with RBC salvage.     

翘嘴鳜病原嗜水气单胞菌分子特征及LAMP检测方法的建立

嗜水气单胞菌(Aeromonas hydrophila)是一种危害鳜鱼养殖生产的重要病原细菌, 为进一步明确该病原菌的分子特征及建立快速检测技术, 实验对引起翘嘴鳜(Siniperca chuatsi)暴发性死亡的病原嗜水气单胞菌进行了致病性、菌株毒力特征研究, 同时以嗜水气单胞菌气溶素基因aerA为分子靶标设计引物, 利用环介导等温扩增技术(Loop-mediated isothermal amplification, LAMP)建立了病原嗜水气单胞菌的快速检测方法。结果表明, 本次引起翘嘴鳜暴发性死亡的病原嗜水气单胞菌半致死浓度为1.6×106 CFU/mL, 携带aerA等14种毒力基因, 此14种毒力基因可用于其致病性分析及分子检测。以气溶素基因aerA设计引物进行的环介导恒温扩增, 结果显示可扩增出阶梯状条带, 加入SYBR Green I染色后呈现绿色的阳性反应, 而对照组均未出现任何扩增条带且反应体系呈现橙色, 表明LAMP检测方法对于嗜水气单胞菌检测具有很好的特异性; 灵敏度检测的最低检测限为4.6×101 CFU/mL; 10种经人工感染的淡水养殖鱼虾组织匀浆增菌液, 提取DNA后进行LAMP方法检测, 结果均可获得阳性扩增结果, 而对照未染菌组呈阴性, 表明该方法具有较好的应用性, 可应用于嗜水气单胞菌引起的水生动物疾病的检测。     

Simple and sensitive bacterial quantification by a flow-based kinetic exclusion fluorescence immunoassay

A flow-based immunoassay system utilizing secondary-antibody coated microbeads and Cy5-secondary antibody for signal production was successfully developed to quantitate target bacteria with a kinetic exclusion assay (KinExA 3000 Instrument). It directly measured the concentration of unliganded antibody separated from the equilibrated mixture of antibody and bacteria through a 0.2 microm polyethersulfone membrane, enabling it to quantify the concentration of bacteria. The novel method demonstrated the qualities of rapidness, sensitivity, high accuracy and reproducibility, and ease to perform. Detection of Pseudomonas aeruginosa and Staphylococcus aureus was accomplished with low detection limits of 4.10 x 10(6) and 5.20 x l0(4)cells/mL, respectively, with an assay time of less than 15 min. The working ranges for quantification were 4.10 x l0(6) to 1.64 x l0(10)cells/mL for P. aeruginosa, and 5.20 x l0(4) to 1.04 x l0(9)cells/mL for S. aureus. It yielded an assay with at least 10-fold greater sensitivity than ELISA and could correctly assess the concentration of predominant bacterium spiked in the mixture of P. aeruginosa and S. aureus. With this reliable platform, the average amount of antibody bound by one cell in the maximum capability could be further provided: (1.6-2.5) x l0(5) antibodies for one P. aeruginosa cell and (2.2-2.7) x l0(8) antibodies for one S. aureus cell. The KinExA system is flexible to determine different kinds of bacteria conveniently by using anti-mouse IgG as the same immobilizing agent. However, a higher specificity of the antibodies to the target bacteria will be required for the use of this system with higher detection sensitivity.