Probes Based Microbial Identificatn PDF

Nonamplified Probe-Based Microbial Detection and Identification  Probe-Based Assay Design A probe is a single-stranded segment of DNA or RNA specifically designed to hybridize to its complementary sequence in the microorganism to be identified.  The nucleic acid probe i...

Nonamplified Probe-Based Microbial Detection and Identification  Probe-Based Assay Design A probe is a single-stranded segment of DNA or RNA specifically designed to hybridize to its complementary sequence in the microorganism to be identified.  The nucleic acid probe is labeled Probe- by a variety of reporter molecules that can be chemiluminescent, Based Assay fluorescent, enzymatic, or antigenic in order to detect the Design double-stranded hybrids. Probe-Based Assay Design There are a variety of probes and targets that are carefully selected when designing diagnostic assays.  Probe Selection Probe selection and labeling have direct impact on hybridization assay efficiency. The ideal probe is single stranded, lacks secondary structure, and does not self-anneal. to generate a probe-target base-paired double-stranded hybrid. contain greater than 50% guanine and cytosine (or GC content) It typically is either a short DNA or RNA oligonucleotide that is usually 15–25 bases long.  Probe Selection Broad range of reporter molecules alkaline phosphatase Antibodies chemiluminescence fluorescence dyes The most used probe labels to detect DNA or RNA within tissue or cells are enzymatically linked antibody-based reporter molecules like digoxigenin. Probe Selection Digoxigenin (DIG) Target Selection Sensitivity primarily determined by the abundance of the target in the original sample ribosomal RNA (rRNA) has been selected as a favorite target sequence as many as 104–5 copies of 5S, 16S, and 23S rRNA per cell Ribosomes are highly conserved and essential organelles responsible for protein synthesis and are therefore present in all living cells in high quantity Target Selection The nucleotide sequence of rRNA is well conserved within a species, yet quite variable among different microbial species rRNA an ideal target for species identification for medically important organisms The 16S and 23S rRNA molecules contain variable sequence motifs that reflect their phylogenetic origins The 16S rRNA gene is approximately 1600 base pairs long and includes nine hypervariable regions of varying conservation (V1-V9) variability allows the design of species-specific probes for organism identification Microbiome analysis 16S rRNA sequencing targets  Nucleic acid probe hybridization assays commonly used in diagnostics occur in three major formats: In solution or liquid phase both probe and target rapidly interact in a homogeneous Nucleic Acid solution Hybridization Solid phase Formats occur on a solid surface (nylon membranes) to which the nucleic acid probe is bound In situ hybridization.  In situ hybridization Type of solid-phase format that fixes intact cells or tissues onto glass slides for detection of target nucleic acid. The controlled enzymatic digestion of cellular membranes and other proteins allows the probes to gain access to the target sequences. The labels for the nucleic acid probes, which can be biotin or digoxigenin, incorporate a signal compound, such as a colorimetric or a fluorescent compound.  In situ hybridization is a technique that allows for precise localization of a specific segment of nucleic acid within a histologic section. The underlying basis of ISH is that nucleic acids, if preserved adequately within a histologic specimen, can be detected through the application of a complementary strand of nucleic acid to which a reporter molecule is attached.  In situ hybridization Digoxigenin-labeled ISH probes are detected enzymatically with anti-digoxigenin antibodies conjugated with alkaline phosphatase or horseradish peroxidase. Biotin is another popular label that can be detected with enzyme conjugates of avidin, streptavidin, or anti- biotin antibodies. These enzymes convert soluble substrates into insoluble precipitates that appear as dark, localized cellular or subcellular stains. Clinical Gen-Probe Nucleic Acid Detection Methods Application of Nonamplified Hybrid Capture Assay Probe-Based Assays Affirm DNA Assay Line Probe Assay Peptide-Nucleic Acid FISH Assays Gen-Probe Nucleic Acid Detection Methods Gen-Probe AccuProbe assays (Hologic, Inc., San Diego, CA) among the first companies to utilize nonamplified nucleic acid hybridization for routine identification of microorganisms In this assay, rRNA molecules are released from the organisms by sonication. A single-stranded chemiluminescent acridinium ester-labeled DNA probe binds to a complementary 16S rRNA region of the target organism to form DNA:RNA hybrids  Hybrid Capture Assay A relatively fast, liquid-based hybridization assay to detect specific viruses and bacteria. The basic steps of the assay involve the combination of specific RNA probes to target DNA, generating RNA/DNA hybrids. The RNA/ DNA hybrids are detected with multiple signaling antibodies conjugated to alkaline phosphatase, resulting in chemiluminescence, which is measured in a luminometer. N. gonorrhoeae, and human papillomavirus (HPV).  Affirm DNA Assay Detects complementary targeted DNA sequences of three major agents that can cause vaginitis/vaginosis: Candida, Gardnerella, and Trichomonas.  Line Probe Assay A solid-phase assay based on the principle of reverse hybridization technology Biotinylated DNA amplicons hybridize to specific oligonucleotide probes that are immobilized as parallel lines on nitrocellulose membrane-based strips. Each line represents a specific probe. After hybridization and stringent wash steps, specific hybrids can be detected by colorimetric reactions. mycobacteria, hepatitis B virus, hepatitis C virus, and HPV genotyping.