Microbial Detection Quiz

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Questions and Answers

What is a probe in the context of nucleic acid hybridization?

A probe is a single-stranded segment of DNA or RNA, specifically designed to hybridize to its complementary sequence in a microorganism.

How are nucleic acid probes labeled?

By a variety of reporter molecules like chemiluminescent, fluorescent, enzymatic, or antigenic molecules (correct)

Exclusively by digoxigenin, a highly specific label

Only by fluorescent dyes to allow for visualization under a microscope

Only conjugated with alkaline phosphatase for signal detection

Probe selection and labeling have no impact on the efficiency of a hybridization assay.

False

Which of the following characteristics describes an ideal nucleic acid probe?

Single-stranded and avoids self-annealing Signup and view all the answers

What is the primary determinant of sensitivity in a nucleic acid hybridization assay?

The abundance of the target sequence in the original sample. Signup and view all the answers

Why is ribosomal RNA (rRNA) a popular target for identifying microorganisms?

rRNA is highly conserved and abundant in all living cells, providing a reliable target for identification Signup and view all the answers

Which of the following is NOT a major format used in nucleic acid probe hybridization assays?

Gas phase Signup and view all the answers

Describe one advantage of in situ hybridization in terms of its application?

In situ hybridization allows for the precise localization of a specific nucleic acid sequence within a tissue or cell sample. Signup and view all the answers

What is a common type of label used in in situ hybridization?

All of the above Signup and view all the answers

What is the purpose of FISH (Fluorescent In Situ Hybridization)?

FISH is a technique that uses fluorescently labeled probes to visualize specific nucleic acid sequences in cells or tissues. Signup and view all the answers

Which of the following is NOT a method used for detecting nucleic acids using nonamplified probes?

Polymerase chain reaction (PCR) Signup and view all the answers

What is the primary advantage of using nonamplified probe-based assays?

All of the above Signup and view all the answers

Explain how the Gen-probe Accuprobe assay works.

This assay releases rRNA from microorganisms and uses a chemiluminescent acridinium ester-labeled DNA probe to bind to a complementary 16S rRNA region, forming DNA:RNA hybrids. Signup and view all the answers

What is the principle behind the Line Probe assay?

The Line Probe assay utilizes biotinylated DNA amplicons that hybridize to specific oligonucleotide probes immobilized on a nitrocellulose membrane. Signup and view all the answers

The Line Probe assay is only used for detecting bacterial pathogens.

False Signup and view all the answers

Study Notes

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).

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Description

Test your knowledge on probe-based assays for microbial detection and identification. This quiz covers the principles of probe design, labeling techniques, and the importance of sequence specificity in detecting microorganisms. Perfect for students and professionals in microbiology and molecular biology.