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Microbiology

Molecular Methods

This section provides a comprehensive overview of molecular methods used in bacterial identification. Molecular techniques offer high sensitivity, specificity, and speed, complementing and often surpassing traditional methods

Theory: The Science of Identifying Bacteria at the DNA Level

  • What are Molecular Methods?
    • Molecular methods are laboratory techniques that analyze the DNA or RNA of bacteria
    • They detect and identify bacteria based on their unique genetic sequences
    • They offer high sensitivity, specificity, and speed compared to traditional methods
  • Why Use Molecular Methods?
    • High Sensitivity: Detects very small amounts of bacteria, even in the absence of visible growth
    • High Specificity: Identifies bacteria to the species or even strain level
    • Rapid Results: Provides results much faster than culture-based methods
    • Culture-Independent: Can identify bacteria without the need for prior culture
    • Detect Non-Culturable Organisms: Useful for identifying bacteria that are difficult or impossible to culture
    • Strain Typing: Allows for the differentiation of strains of the same species (e.g., for outbreak investigations)
    • Detection of Resistance Genes: Identifies antibiotic resistance genes directly from clinical samples
  • Key Molecular Techniques
    • Polymerase Chain Reaction (PCR)
    • Real-Time PCR (qPCR)
    • Multiplex PCR
    • Sequencing (e.g., 16S rRNA gene sequencing)
    • Whole Genome Sequencing (WGS)
    • Nucleic Acid Hybridization
    • Microarrays
  • General Principles
    1. Sample Preparation: The bacterial sample is processed to extract the nucleic acid (DNA or RNA)
    2. Target Amplification (if applicable): The specific DNA or RNA target is amplified using PCR or a similar method
    3. Detection/Analysis: The amplified product or the presence of the target sequence is detected using various methods (e.g., gel electrophoresis, real-time PCR, sequencing)
    4. Identification: The presence or absence of the target sequence, or the sequence itself, is used to identify the bacterium

Polymerase Chain Reaction (PCR)

  • Principle: Amplifies specific DNA sequences using primers, a DNA polymerase enzyme, and thermal cycling
  • Procedure
    1. DNA Extraction: Extract DNA from the bacterial sample
    2. Primer Design: Design primers that are specific to the target gene
    3. PCR Reaction: Combine DNA, primers, DNA polymerase, dNTPs, and buffer
    4. Thermal Cycling
      • Denaturation: Heat to separate DNA strands
      • Annealing: Cool to allow primers to bind to the target DNA
      • Extension: Heat to allow DNA polymerase to extend the primers
    5. Detection: Analyze the PCR product using gel electrophoresis, real-time PCR, or sequencing
  • Interpretation: Presence of the amplified product indicates the presence of the target sequence, and thus the presence of the bacterium
  • Applications: Detection of specific pathogens, antibiotic resistance genes, and strain typing

Real-Time PCR (qPCR)

  • Principle: PCR with real-time detection of the amplified product using fluorescent probes or dyes
  • Procedure: Similar to PCR, but includes a fluorescent probe or dye in the reaction
    • The fluorescent signal increases as the target DNA is amplified
  • Interpretation: Measures the amount of the target DNA in the sample
    • Quantitative results are obtained
    • Used for quantifying bacterial load, detecting pathogens, and identifying antibiotic resistance genes
  • Advantages: Faster, more sensitive, and quantitative than standard PCR

Multiplex PCR

  • Principle: Amplifies multiple target sequences simultaneously in a single PCR reaction
  • Procedure: Uses multiple primer sets, each specific for a different target gene
  • Interpretation: Identifies multiple pathogens or genes in a single test
  • Applications: Multiplex PCR is highly useful for the detection of several pathogens that cause similar symptoms, such as respiratory infections

Sequencing (e.g., 16S rRNA gene sequencing)

  • Principle: Determines the exact order of nucleotides in a DNA sequence
  • Procedure
    1. DNA Extraction: Extract DNA from the bacterial sample
    2. PCR Amplification: Amplify a specific gene (e.g., 16S rRNA gene)
    3. Sequencing: Determine the nucleotide sequence of the amplified product
    4. Sequence Analysis: Compare the sequence to a database of known sequences
  • Interpretation: Identifies the bacterium based on its unique DNA sequence
  • Applications: Species identification, strain typing, and phylogenetic analysis

Whole Genome Sequencing (WGS)

  • Principle: Determines the complete DNA sequence of an organism’s genome
  • Procedure
    1. DNA Extraction: Extract DNA from the bacterial sample
    2. Library Preparation: Fragment the DNA and prepare a sequencing library
    3. Sequencing: Determine the complete nucleotide sequence of the genome
    4. Genome Assembly and Analysis: Assemble the sequence and analyze the data
  • Interpretation: Provides a highly detailed genetic profile of the organism
  • Applications: Strain typing, outbreak investigations, identification of virulence factors and resistance genes, and phylogenetic analysis

Nucleic Acid Hybridization

  • Principle: Detects the presence of a specific DNA or RNA sequence by hybridization (binding) to a complementary probe
  • Procedure
    1. Prepare the Sample: Extract nucleic acid and denature it
    2. Hybridization: Incubate the sample with a labeled probe
    3. Detection: Detect the probe-target hybrid using a detection method (e.g., fluorescence)
  • Interpretation: Presence of the probe-target hybrid indicates the presence of the target sequence
  • Applications: Used for the detection of specific pathogens and genes

Microarrays

  • Principle: Uses thousands of DNA probes immobilized on a solid surface to detect multiple target sequences simultaneously
  • Procedure
    1. Prepare the Sample: Extract and label the DNA from the sample
    2. Hybridization: Incubate the labeled DNA with the microarray
    3. Detection: Scan the microarray to detect the hybridization signal
  • Interpretation: Identifies multiple pathogens or genes in a single test
  • Applications: Used for the detection of antibiotic resistance genes, virulence factors, and for strain typing

Interpretation: Deciphering the Genetic Code

  • PCR and qPCR
    • Positive Result: Presence of the amplified product (PCR) or a detectable fluorescent signal (qPCR) indicates the presence of the target sequence and the bacterium
    • Negative Result: Absence of the amplified product or fluorescent signal indicates the absence of the target sequence, and the bacterium is either absent or below the detection limit
    • Quantitative Results (qPCR): The cycle threshold (Ct) value is inversely proportional to the amount of target DNA in the sample. Lower Ct values indicate a higher bacterial load
  • Sequencing
    • Sequence Alignment: The sequence is aligned with a database of known sequences
    • Percentage Identity: The percentage identity between the unknown sequence and the closest match in the database is used to determine the identification
    • Species Identification: If the percentage identity is high enough (typically >98%), the organism is identified to the species level
    • Strain Typing: Sequence variations can be used to differentiate strains of the same species
  • WGS
    • Genome Assembly: The sequencing reads are assembled into a complete genome sequence
    • Annotation: The genome is annotated to identify genes, regulatory elements, and other features
    • Strain Typing: The complete genome sequence allows for highly accurate strain typing and the identification of genetic markers
    • Phylogenetic Analysis: Used to determine the evolutionary relationships between strains
  • Nucleic Acid Hybridization and Microarrays
    • Signal Detection: A positive signal (e.g., fluorescence) indicates the presence of the target sequence
    • Signal Intensity: The intensity of the signal can be used to estimate the amount of target DNA
    • Multiplexing: Multiple targets can be detected simultaneously

Application: Putting Knowledge into Practice

  • Quality Control (QC)
    • Control Strains: Use known positive and negative control organisms for each assay
    • Internal Controls: Include internal controls in each reaction to monitor the amplification process
    • Frequency: Perform QC according to the manufacturer’s recommendations and/or laboratory policies
    • Documentation: Record QC results in a logbook or LIS
    • QC Failure: Investigate the cause and repeat the test if QC fails
  • Procedure
    1. Specimen Collection and Processing: Collect and process the sample according to laboratory protocols
    2. Nucleic Acid Extraction: Extract the DNA or RNA from the sample
    3. Reagent Preparation: Prepare reagents according to the manufacturer’s instructions
    4. Reaction Setup: Set up the PCR, qPCR, or other molecular assay
    5. Amplification/Hybridization: Perform the amplification or hybridization step
    6. Detection/Analysis: Detect and analyze the results using the appropriate method
    7. Interpretation: Interpret the results based on the expected outcomes
    8. Documentation: Record the results in the LIS
    9. Correlation: Correlate the results with other test results and clinical information
    10. Reporting: Report the identification to the clinician
  • Examples of Applications
    • Detection of Clostridioides difficile toxin genes
    • Detection of methicillin resistance in Staphylococcus aureus (MRSA)
    • Identification of Mycobacterium tuberculosis
    • Detection of viral infections
    • Strain typing for outbreak investigations
    • Detection of sexually transmitted infections (STIs)
    • Detection of Legionella pneumophila in respiratory samples
  • Troubleshooting
    • False Positives
      • Contamination: Strict adherence to aseptic technique is essential
      • Primer Dimer Formation: Optimize primer design and reaction conditions
      • Carryover Contamination: Use separate areas for pre- and post-amplification steps
    • False Negatives
      • Poor Sample Quality: Ensure proper sample collection and storage
      • Inhibitors: Remove inhibitors during nucleic acid extraction
      • Primer Issues: Optimize primer design and annealing conditions
      • Low Bacterial Load: The bacterial load may be below the detection limit
    • Inconclusive Results
      • Degraded Nucleic Acid: Use fresh samples and proper storage
      • Poor Amplification: Optimize PCR conditions, use fresh reagents, and check primer specificity
      • Database Limitations: The target sequence may not be present in the database

Key Terms

  • Molecular Methods: Laboratory techniques that analyze the DNA or RNA of bacteria
  • DNA (Deoxyribonucleic Acid): The genetic material of living organisms
  • RNA (Ribonucleic Acid): A molecule involved in protein synthesis
  • PCR (Polymerase Chain Reaction): A technique for amplifying specific DNA sequences
  • qPCR (Real-Time PCR): PCR with real-time detection of the amplified product
  • Multiplex PCR: PCR that amplifies multiple target sequences simultaneously
  • Sequencing: Determining the order of nucleotides in a DNA sequence
  • 16S rRNA gene: A gene that encodes for the small subunit ribosomal RNA, used for bacterial identification
  • Whole Genome Sequencing (WGS): Determining the complete DNA sequence of an organism’s genome
  • Nucleic Acid Hybridization: The process of binding a DNA or RNA probe to a complementary target sequence
  • Microarray: A device that contains thousands of DNA probes for detecting multiple target sequences simultaneously
  • Primer: A short DNA sequence that initiates DNA synthesis
  • Amplification: The process of making multiple copies of a DNA sequence
  • Nucleic Acid Extraction: The process of isolating DNA or RNA from a sample
  • Cycle Threshold (Ct) Value: The cycle number at which the fluorescent signal in qPCR crosses a threshold
  • Percentage Identity: The percentage of identical nucleotides between two DNA sequences
  • Strain Typing: Differentiating strains of the same species
  • Phylogenetic Analysis: Determining the evolutionary relationships between organisms
  • Probe: A short DNA or RNA sequence used to detect a target sequence
  • Target Sequence: The specific DNA or RNA sequence being analyzed
  • Lysis: The breakdown of a cell
  • Annotation: The process of identifying and describing the features of a genome
  • Aseptic Technique: Procedures used to prevent contamination
  • Inhibitors: Substances that can interfere with PCR
  • Degraded Nucleic Acid: DNA or RNA that has been broken down