Overview of Lab Projects - Molecular Microbiology Laboratory
- Real-Time PCR of Clostridium difficile and Toxin Genes from Enteric Pathogens
- DNA Pyrosequencing-based Identification of Bacterial and Fungal Pathogens
- Liquid Bead Array-Based Diagnostics of Pediatric Sepsis
- Rep-PCR-Based DNA Profiling of Bacterial Pathogens (Molecular Epidemiology)
- Immuno-PCR Strategies for Ultrasensitive Protein Quantitation
- The Infectious Diseases DNA Chip Project
Real-Time PCR of Clostridium difficile and Toxin Genes from Enteric Pathogens
Real-time PCR assays are being developed for direct toxin gene detection in human fecal specimens. Direct DNA detection and possible sequencing of toxin genes will enable the laboratory to markedly improve diagnostic testing for antimicrobial-associated diarrhea, colitis, and pseudomembranous colitis caused by C. difficile. In addition to C. difficile, new assays are being developed for enterotoxigenic Escherichia coli (ETEC) and enterohemorrhagic E. coli (EHEC) that target toxin genes. The overall strategy is to improve molecular strategies for direct toxin gene (DNA) detection in human intestinal tissue and fecal specimens and facilitate molecular diagnostics of infectious gastroenteritis and colitis.
Gram Stain of Clostridium difficile
DNA Pyrosequencing-based Identification of Bacterial and Fungal Pathogens
DNA pyrosequencing methods have been developed for bacterial and fungal pathogen identification. Pyrosequencing is used routinely for pathogen identification when biochemical methods are insufficient to provide definitive genus- or species-level identification. DNA pyrosequencing assays are being extended to explore the applications of different genes and DNA targets for improved species-level identification of different pathogens.
DNA Pyrogram
Liquid Bead Array-Based Diagnostics of Pediatric Sepsis
Liquid bead arrays are in development for new multiplex approaches that will facilitate the diagnosis of bacteremia and sepsis in children. Highly parallel strategies using arrays in solution will enable the molecular microbiology laboratory to develop tests targeting multiple bacterial and fungal pathogens in peripheral blood specimens. New DNA primers and probes are being developed in order to combine nucleic acid amplification of positive blood cultures with liquid bead array hybridization.
Rep-PCR-Based DNA Profiling of Bacterial Pathogens (Molecular Epidemiology)
Repetitive DNA element-based PCR (rep-PCR) strategies have been implemented for molecular epidemiology. The molecular microbiology laboratory works closely with the Infection Control department at Texas Children's Hospital and also functions as a reference laboratory for DNA typing. Chromosomal DNA profiles are stored and archived digitally so that bacterial isolates from disease clusters or isolated infections can be compared with an extensive DNA profile database of bacterial and yeast pathogens. PCR-based DNA typing approaches are being refined and expanded to improve infectious disease surveillance in the hospital and local community.
DNA Typing Profiles
Immuno-PCR Strategies for Ultrasensitive Protein Quantitation
Real-time immuno-PCR methods are being developed for ultrasensitive protein detection and quantitation in animal model and human specimens. Cytokines are primary targets for new immuno-PCR strategies that will enable monitoring of inflammation status in patients when quantitative immunoassays are insufficiently sensitive. Other protein targets for immuno-PCR include antibodies and antigens for rapid, ultrasensitive diagnosis of pediatric infections.
Proposed real-time immuno-PCR strategy Target DNA
will be directly conjugated to secondary polyclonal
antibodies that recognize the protein of interest.
The Infectious Diseases DNA Chip Project
Our primary goal for this project is to design comprehensive "DNA Chips" or array-based technologies that will enable us to rapidly and accurately diagnose a variety of bacterial or viral infections in children. Initially we are focusing on solid phase microarrays (chips) or liquid bead arrays in order to create disease- or system-specific panels. For example, a sepsis panel is already in progress and other infectious disease-oriented arrays are being planned. These projects mark the beginning of a shift to highly parallel, array-based diagnostics for pediatric infections.
Efforts to develop new infectious disease-oriented DNA chips are partly supported by the Grant-A-Starr Foundation. If you want more information about this foundation or would like to make a contribution, please see the link below.
Probiotic Lactobacillus