Biomedical subjects

Su-Hong Chen

Publications and source records attributed to Su-Hong Chen.

7 recordsLinked to original sources

PubMed2006

Detection and identification of intestinal pathogens in clinical specimens using DNA microarrays.

The detection and identification of intestinal pathogens is critical for clinical patient diagnosis and antimicrobial therapy. No currently available assays with DNA microarrays can simultaneously detect and identify multiple intestinal pathogens, because there is no appropriate method for choosing target probes. To solve the problem we have experimented for facilitating screening of specific probes and developed a rapid (<3h) and reliable assay for simultaneous detection of intestinal pathogens using two universal PCR primers to amplify two variable regions of bacterial 16S and 23S ribosomal DNA (rDNA) genes, and then applied to DNA microarrays, hybridization between probes and amplicons occurred. Through this idea for screening of probes the assay was successful in discriminating 15 genera or species of intestinal pathogens. The limit of detection was approximately 10(3)CFU/mL for one species of pathogen and 10(5)CFU/mL for six species pathogens existing simultaneously in stool. When this assay was applied directly to identify 99 clinical specimens, 80(80.8%) were correctly analyzed, including four with mixed pathogens; 8(8.08%) received negative results due to no corresponding probes in this array and 11(11.11%) belonging to our targets were misidentified due to low-level pathogens and other factors. This approach is also convenient to obtain specific and proper probes while establishing assays for the applications in other aspects using DNA microarrays. In addition, the more species may be added to this system easily and endlessly by screening of candidate target probes in order to increase the power of simultaneous detection.

Bacterial Infections↗

PubMed2005

Sequence confirmation of synthetic phosphorothioate oligodeoxynucleotides using Sanger sequencing reactions in combination with mass spectrometry.

A protocol relying on Sanger sequencing reactions in combination with mass spectrometry (MS) for sequence confirmation of antisense phosphorothioate oligodeoxynucleotides is described. In this procedure, synthetic phosphorothioate oligodeoxynucleotides are used as reverse primers for extension of matched templates with enough length (approximately 150-300 bp) for well-established Sanger sequencing. Because the complementary strand of modified primer is used directly for sequencing primer extension, the base order shown in the sequencing result is reversely complementary to phosphorothioate oligodeoxynucleotide. This sequencing method can be applied not only to phosphorothioate oligodeoxynucleotides with different lengths (13-21 mer) and base composition but also to sequences with bases' switch, deletion, or insertion. In addition, modified primers incorporate the 5' end of polymerase chain reaction (PCR) products conveying the characters of phosphorothioate modification. The method requires only common reagents and instruments and so is better suited to routine sequence analysis in quality control of phosphorothioate antisense drugs.

Base Sequence↗

PubMed2005

Screening of specific antigens for SARS clinical diagnosis using a protein microarray.

In this study several SARS-CoV structural proteins and fragments were expressed in E. coli as GST or TRX fusion proteins. They were fabricated on a microarray and tested with sera from SARS patients. Antigenic screening indicated that recombinant GST-N2 fusion protein, the carboxy-terminus 213aa-423aa of N protein, was strongest positive and weakest non-specific compared with others. An indirect antibody ELISA method was developed and clinical positive and negative sera for their antibodies against GST-N2 fusion protein were assayed. 311 out of the 442 sera from clinical SARS inpatients, as well as 229 out of 302 sera from convalescent patients gave positive reactivities; positive rates were 70.4% and 75.8% respectively. Sera from a total of 2726 non-SARS patients and healthy individuals were tested and the false positive rate was only 0.07%. When the sensitivity control sample was diluted 1 : 64, it yielded OD values above the cutoff value. Reported data showed that this was a relatively high degree of sensitivity and specificity for SARS-CoV antibody testing. The data indicate that GST-N2 fusion protein, which was screened by protein microarray, may be a valuable diagnostic antigen for the development of serological assays for SARS. In addition, protein microarray assay presents a higher positive rate and sensitivity (86.1% and 1 : 200) compared with the traditional ELISA screening method, and could provide a rapid, parallel and high-throughput antigen screening platform.

Antibodies, Viral↗

PubMed2005

Fabrication and optimization of the multiplex PCR-based oligonucleotide microarray for detection of Neisseria gonorrhoeae, Chlamydia trachomatis and Ureaplasma urealyticum.

To detect and identify the pathogens responsible for sexually transmitted diseases (STDs) at the early stage of infection and with a high throughput, a new microarray with a bifunctional probe modification was prepared using Neisseria gonorrhoeae, Chlamydia trachomatis and Ureaplasma urealyticum as a model system. During the fabrication of the microarray, an asymmetric fluorescently labeled multiplex PCR was introduced. The fabrication optimization proved that the best hybridization results would be obtained by spotting N. gonorrhoeae probe at a position near the side of the fluorescently labeled reverse primer within its target gene and spotting each probe at a concentration of 50 microM onto the aldehyde-derived glass slides using spotting solution S1 and using hybridization solution H2 for hybridization. The probes designed by our laboratory could specifically discriminate the pathogens of N. gonorrhoeae, C. trachomatis and U. urealyticum in the presence of the internal control on the microarray simultaneously and separately. By incorporating the key features of DNA microarray with those of multiplex PCR, the microarray provides a fast high throughput platform for multiple infections and multiple samples to be detected and identified simultaneously for STD clinics. It also provides a new platform for other diseases and gene mutations to be detected and identified at a high throughput.

Chlamydia trachomatis↗

PubMed2005

[The distribution of calcium in the stigma and style of tobacco during pollen germination and tube growth].

After pollen grains of tobacco landed on stigma they begin to hydrate and form many small vesicles containing some calcium grains in cytoplasm. The calcium stored in pollen wall is released into tectum of stigma to make a calcium-rich environment. When a pollen tube penetrates the tectum and grows between stigma cells, numerous calcium precipitates appear in the tip tube wall. The length of style of tobacco is 4 cm, and the pollen tube need take 44 h to reach the ovary. The style was artificially divided into 4 stages and each 1 cm respectively. There were only a few of calcium precipitates in the transmitting tissue of style from anthesis to 11 h after pollination. A calcium gradient in the transmitting tissue of style was formed at 22 h after pollination: only a few calcium precipitates found in the transmitting tissue of the style under stigma and at stage 1, 2 and 3, and many of them were located in the transmitting tissue of style near ovary (stage 4). When the flowers were emasculated and unpollinated at 1 d after anthesis, no calcium gradient in the transmitting tissue of style could be identified because some precipitates were also accumulated in the transmitting tissue at stage 1. When a flower without pollination was kept for 3 d, some calcium precipitates were formed in the cells of stigma, and the cells of the whole transmitting tissue contained the same quantity of calcium precipitates. To check the ability of pollen to germinate and grow in a low calcium environment, pollen grains were cultured in a medium containing 0-0.1% CaCl(2).2H(2)O. The result of in vitro assay confirmed that tobacco pollen can germinate and the pollen tube can grow in an environment with a very low concentration of calcium, which may be similar to the environment in the stigma. A few calcium precipitates were accumulated in stigma and upper transmitting tissue of tobacco to make a calcium gradient in the style. If the calcium in the style at 1 cm increases it will be increased more at 4 cm, and more in ovules, and more in synergid cells to keep the calcium gradient. When the emasculated flowers were not pollinated for 3 days the calcium in upper transmitting tissue evidently increases. The calcium in style is abundant in all plants, but the distribution of calcium in style is different between different plant species. For this difference, it may differ from types of style, and in the plants with short style the calcium gradient in the style is too small to be detected. But for tobacco with style 4 cm long, the gradient can be identified using antimonate method.

Calcium↗

PubMed2005

[Isolation of egg cells and zygotes from Torenia fournieri].

Torenia fournieri is a special plant with embryo sac partly protruding through the micropyle of ovule, and its egg cell, two synergids and part of the central cell can be clearly observed using light microscope (Plate I-1, 2). The feature of embryo sac of T. fournieri makes it easy to isolate egg cell and zygote, especially for physiologically mature and vigorous egg cell to study its fertilization mechanism. Egg cells of T. fournieri were isolated from embryo sacs 2 d after anthesis using enzymatic digestion or mechanical dissection after mild osmotic shock (Plate I-3, 4). About 5% egg cells (2-3 from 50 ovules) could be mechanically dissected within 2 h. When 0.1% cellulase and 0.1% pectinase were added into the solution, about 18% egg cells (8-10 from 50 ovules) could be isolated within 2 h (Plate I-5). Although egg cells could be isolated by mechanical dissection, they can be used in in vitro fertilization to probe fertilization mechanism without deleterious effect of enzymatic action on the surface of egg cell. But it is easier to isolate egg cells by using enzymatic digestion, and more egg cells can be obtained for the research of molecular biology. Using enzymatic digestion method, the zygote of Torenia fournieri was also isolated from the pollinated ovules (Plate I-7-10). Basing on our successful isolation of mature sperm cells, the isolation of egg cells of T. fournieri will make in vitro fertilization possible for the first time in a dicot plant.

Cell Separation↗

PubMed2002

[Preparation and analysis of oligonucleotide microarray for expression detection of mouse cytokine-associated gene].

A new method for the preparation of oligonucleotide microarray for gene expression detection was found. The key techniques and standards of quality controlling for preparation of oligonucleotide microarray was explored using gene of human 23 kD highly protein and Luciferase and mouse cytokine-associated genes. By the using of a software system MProbe, oligonucleotide probes were designed and BLAST. All the probes have a very high specificity, i.e. except target sequence, the similarity between the probe and non-target sequences is less than 70% and the hairpin structure are not exist in all probes. All the probes have the same length 40. GC contents in all probes are in a narrow scope (from 45% to 55%). All the probes are modified with amino at 5' or 3' terminal. The satisfied images with good sensitivity and very high specificity were obtained by the using of the methods above and also using of positive and negative controls and some internal controls(house keeping gene) to quantitate and balance expression of genes. High specificity, good sensitivity and stability have been verified by three continuous experiments using the oligonucleotide microarray to study gene expression profile of normal mouse breast grand tissue. The oligonucleotide microarray for expression detection prepared using our method have high specificity, good sensitivity and stability et al. It may be a more advanced method for analysis of gene expression profile.

Animals↗