Biomedical subjects

Si-Yuan Wen

Publications and source records attributed to Si-Yuan Wen.

6 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↗

PubMed2006

[DNA microarrays and their application in detecting and identifying intestinal pathogens].

DNA microarrays offer many advantages of high throughout, automation, rapid detection, and so on. Therefore, this technology had been used in many fields such as molecular epidemiology of bacteria, microbial gene identification, disease mechanism, gene mutation, gene expression identification, DNA sequencing and medicine screening etc. The assays for identifying pathogens using DNA microarrays reported aboard recently are introduced. The application of DNA microarrays in detecting and identifying intestinal pathogens mainly includes three aspects: the identification of toxin and characteristic genes of pathogens, the identification of bacterial DNA or RNA directly, the simultaneous detection of a large number of intestinal pathogens with the target - gene of ribosomal RNA. Because of its high efficiency, DNA microarrays is superior to other biological method. Obviously DNA microarrays technology may be useful in identifying intestinal pathogens and have a wide prospect.

Animals↗

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

[Study on PBPs genes of penicillin-resistant Streptococcus pneumonia in Zhejiang province].

To elucidate alternations in gene/amino acid sequence of penicillin-binding proteins (PBPs) 1A, 2B, 2X from clinical isolates of penicillin-resistant Streptococcus pneumonia (PRSP) in Zhejiang Province, 26 strains of Streptococcus pneumonia were collected from November 2001 to January 2004. The antibiotics susceptibility of these strains was detected. PCR amplification and direct sequencing of PBP1A, 2B, 2X genes were performed. The sequence variations of PBP genes of the PRSPs in this region were studied by sequence BLAST analysis. It was shown that the main alternations of PBP1A were the four consecutive amino acid substitutions (Thr574Ala, Ser575Thr, Gln576Gly, Phe577Tyr) following the conservative motif KTG and the amino acid substitution Thr371Ser in the conservative motif STMK. The main alternation of PBP2B was Thr451Ala following the conservative motif SSN, and the main alternation of PBP2X was Thr338Ala in conservative motif STMK. The above mutation sites and drug resistant level were consistent to the data reported previously. Neither new gene mutation specific to these strains nor certain amino acid substitutions related to penicillin resistance reported was identified in the genes.

Amino Acid Sequence↗

PubMed2004

Preparation and property analysis of a hepatocyte targeting pH-sensitive liposome.

AIM: To develop a hepatocyte targeting pH-sensitive liposome for drug delivery based on active targeting technology mediated by asialoglycoprotein receptors. METHODS: Four types of targeting molecules with galactose residue were synthesized and mixed with pH-sensitive lipids DC-chol/DOPE to prepare liposome with integrated property of hepatocyte specificity and pH sensitivity. Liposome 18-gal was selected with the best transfection activity through cellular uptake experiment. Property analysis was made through experiments of competitive inhibition of receptors, red blood cell hemolysis, in vitro cytotoxicity test by MTS assay and mediation of inhibitory effects of antisense phosphorothioate ODN on gene expression, etc. RESULTS: Liposome 18-gal had the desired properties of hepatocyte specificity, pH sensitivity, low cytotoxicity, and high transfection efficiency. CONCLUSION: Liposome 18-gal can be further developed as a potential hepatocyte- targeting delivery system.

Cell Line, Tumor↗

PubMed2003

Rapid detection of the known SNPs of CYP2C9 using oligonucleotide microarray.

AIM: Cytochrome P450 2C9 (CYP2C9) is a polymorphic enzyme responsible for the metabolism of a large number of clinically important drugs. Individuals with mutant enzymes may risk serious side effects under routine therapy with certain drugs metabolized by CYP2C9. In order to facilitate the detection of the known SNPs of CYP2C9, an allele-specific oligonucleotide (ASO) based microarray was made. METHODS: An oligonucleotide microarray was made to facilitate the SNP (single nucleotide polymorphism) screening and was applied for the detection of CYP2C9 polymorphism in 62 high blood pressure (HBP) patients who received Irbesartan for treatment. Part of the genotyping results was confirmed by direct sequencing. And the relation between CYP2C9 polymorphism and therapeutic outcome of Irbesartan was statistically analyzed. RESULTS: Heterozygous alleles of CYP2C9*1/*3 were found in 7 out of 62 subjects. No mutant alleles of CYP2C9*2, *4 and *5 and no homozygous mutant alleles were detected. The 7 heterozygous CYP2C9*1/*3 and 13 random wild type DNA samples were subjected to direct sequencing with purified PCR products and same genotyping results were obtained with the 20 DNA samples. There was no significant difference in the odds of effectiveness of Irbesartan between the wild type (normal) group and CYP2C9*1/*3 (mutant) group (P>0.05). CONCLUSION: The oligonucleotide microarray made in this study is a reliable assay for detecting the CYP2C9 known alleles and the heterozygous CYP2C9*1/*3 has no significant effects on the therapeutic outcome of Irbesartan.

Alleles↗