Microbial Detection Quiz

Nonamplified Probe-Based Microbial Detection and Identification

• Probe-based assays identify microorganisms by targeting specific DNA or RNA sequences.
• A probe is a single-stranded DNA or RNA segment designed to hybridize with a complementary sequence in the microorganism.
• Probes are labeled with reporter molecules (e.g., chemiluminescent, fluorescent, enzymatic) for detection of hybridized double-stranded DNA or RNA.
• Probe selection and labeling directly influence hybridization assay efficiency.
• Ideal probes lack secondary structures, are single-stranded, and do not self-anneal.
• Probes typically consist of short DNA or RNA oligonucleotides (15-25 bases long).
• They often contain a high guanine-cytosine (GC) content.
• Common probe labels include alkaline phosphatase, antibodies (chemiluminescence), fluorescence, and digoxigenin (enzymatically linked).

Probe-Based Assay Design

• The nucleic acid probe is tagged with reporter molecules to detect a double-stranded hybrid.
• The reporter molecules can be chemiluminescent, fluorescent, enzymatic or antigenic.

Probe Selection

• Probe selection and labeling directly affect hybridization assay efficiency.
• An ideal probe is single-stranded, avoids secondary structures and self-annealing.
• Probe content often contains more than 50% guanine-cytosine (GC).
• Common reporter molecules include alkaline phosphatase, antibodies (chemiluminescence), dyes, and digoxigenin (linked to antibodies).

Target Selection

• Sensitivity relies on target abundance in the original sample.
• Ribosomal RNA (rRNA) is a common target.
• 16S and 23S rRNA molecules often have variable regions that reflect phylogenetic origins, which are useful for identification.
• The 16S rRNA genes (used to identify species) are typically ~1600 base pairs long and include 9 variable regions.
• rRNA sequence variations allow species-specific probes.

Microbiome analysis

• 16S rRNA genes are used in analysing microbiomes.
• 16S rRNA has variable regions.
• Specific primers are used to target these regions.

16S rRNA sequencing targets

• Specific regions of the 16S rRNA gene are targeted for sequencing, e.g., V1-V3, V3-V4, V3-V5, V6-V9, and V1-V9 (full-length).
• Fragments are sized to suit different sequencing platforms (Roche 454, MiSeq, HiSeq).

Nucleic Acid Hybridization Formats

• Nucleic acid probe hybridization assays commonly occur in three formats: solution/liquid phase, solid phase, and in situ hybridization.
• In solution, nucleic acid probes and targets rapidly interact in a homogeneous solution.
• In solid phase, hybridization occurs on a solid surface such as nylon membranes where nucleic acid probes are attached.

In situ hybridization

• This is a solid-phase format where intact cells/tissues are fixed on glass slides.
• Controlled enzymatic digestion of cellular membranes/proteins allows probes to access target sequences.
• Probe labels (e.g., biotin, digoxigenin) incorporate signal compounds (e.g., colorimetric, fluorescent).

FISH (Fluorescent In Situ Hybridization)

• FISH uses fluorescent probes to detect specific DNA or RNA sequences in fixed cells or tissues.
• FISH probes are often labeled with digoxigenin or biotin.

FISH for Bacterial Pathogen Identification

• Procedure for identifying bacterial pathogens using FISH:
  1. Collect tissue sample.
  2. Synthesise complementary oligonucleotide for suspected pathogen.
  3. Treat to permeabilize cells for fluorescent oligonucleotide binding.
  4. Add fluorescently tagged oligonucleotides.
  5. The fluorescently tagged oligonucleotides bind to the pathogenic DNA.
  6. View under a microscope to detect fluorescence in pathogenic cells.

Clinical Application of Nonamplified Probe-Based Assays

• Gen-Probe AccuProbe assays use nonamplified probes for routine microbial identification.
• Hybrid Capture Assays use RNA or DNA probes to detect specific viruses and bacteria.
• Affirm DNA Assays detect the DNA sequences of three major vaginitis/vaginosis agents (Candida, Gardnerella, Trichomonas).
• Line Probe Assays use biotinylated DNA amplicons for specific identification.

Gen-Probe Nucleic Acid Detection Methods

• rRNA molecules from organisms are released by sonication.
• A single-stranded, chemiluminescent probe binds to a complementary 16S rRNA region of the target organism to form DNA-RNA hybrids.

Hybrid Capture Assay

• Relatively fast, liquid-based hybridization assay for viral and bacterial detection.
• RNA probes bind to target DNA forming RNA/DNA hybrids.
• RNA-DNA hybrids are detected with multiple antibodies linked to alkaline phosphatase, which produces chemiluminescence.

Affirm DNA Assay

• Detects complementary targeted DNA sequences of three major vaginal agents (Candida, Gardnerella, Trichomonas).

Line Probe Assay

• Solid-phase assay using reverse hybridization technology.
• Biotinylated DNA amplicons bind to specific probes on nitrocellulose strips to identify specific microorganisms (e.g., mycobacteria, hepatitis viruses, HPV).