Week 1

• RT-PCR quantification involves qPCR of dilutions of references samples of known concentrations.
• The Ct value of an analysed sample on the standard curve gives the original sample DNA concentration.
• A standard curve is plotted with known DNA concentrations against Ct values.
• Examples of typing include outbreak investigation and defining subtypes within species.
• Whole genome sequencing of bacteria is a technique used in molecular diagnostic and typing.
• PCR relies on the culture of the organism or bacterial growth.
• Colony morphology is a physical and chemical property of the cell wall that is used in bacterial identification.
• Physiology and biochemical/serological markers are used as physiological and biochemical markers in bacterial identification.
• Antibiotic sensitivity test, also known as antibiogram, is a technique used in bacterial identification.
• Nucleic acids markers such as DNA and RNA regions are used in PFGE and 16sRNA.
• Cultural methods in microbiology diagnostic labs have a minimal turnaround time of 24 hours, usually 48 hours to 72 hours.
• Cultural methods in microbiology diagnostic labs are established techniques, qualified experienced professionals, and work well.
• Low cost techniques are often used in molecular diagnostic tests.
• Molecular diagnostic tests are important for accurate and timely diagnosis, better patient outcome, reducing spread of antibiotic resistance, and preventing the spread of a contagious disease.
• Main molecular methods used in clinical microbiology are Polymerase chain reaction (PCR), MALDI-TOF mass spectrometry, and Emergent use of next generation sequencing (NGS) methods and whole genome sequence (WGS) analysis.
• PCR involves the 5’>3’ orientation of primers, forward and reverse primer, and exponential amplification.
• PCR applications in microbiology include determining if an organism is present in a clinical sample or culture, determining if a gene of interest is present in a clinical sample or culture, identifying what organisms are present in a clinical sample or culture, and requiring sequence analysis.
• Probes in Real-time PCR are degraded by 5’ exonuclease activity of DNA Polymerase.
• Real-time quantitative PCR uses standards of known amounts (dilutions) for quantification of patient samples.
• In Real-time PCR, fluorescence is measured during elongation, with the number of cycles at which the fluorescence exceeds the threshold being called the cycle threshold, Ct.
• In Real-time quantitative PCR, amplification plot is generated using serial dilutions, with Ct values and dilution standard dilutions of total DNA from 1mg to 1pg.
• Real-time PCR uses Sybr Green intercalating fluorescent dye that binds double stranded DNA, with fluorescence of dye highly amplified when bound to dsDNA.
• Specific probes (FRET principle) used in Real-time PCR are TaqMan/hydrolysis probes.
• Non specific DNA binding dye used in Real-time PCR is SYBR green.
• In Real-time PCR, denatured target, binding of primer to target, and synthesis of second strand allow dye to bind - fluorescence proportional to amount of ds DNA.
• PCR is used in the Clinical Microbiology lab for culture confirmation, non-culturable agents, fastidious, slow-growing agents, Mycobacterium tuberculosis, Legionella pneumophilia, and agents present in low numbers.
• Detection and quantification in Real-time PCR is done via fluorescence (fluorescent dye or probes), with the fluorescence level being proportional to the amount of PCR product.
• Probes in Real-time PCR are non-fluorescent.
• Quenchers are molecules that quench the fluorescence of dyes in their proximity.
• Real-time or Quantitative real-time PCR (RT-PCR or qPCR) allows detection of DNA and quantitation of reaction product at every cycle.
• Real-time PCR uses fluorescent dyes or probes which binds amplified DNA and/or target DNA.
• Real-time PCR uses Taqman principle, with probes specific for internal sequence of target DNA, optimal length 18 to 22 bases, labelled 5’ and 3’ by Quencher fluorophore and Reporter fluorophore.
• The cycle threshold, Ct, value in Real-time PCR is a relative measure of target concentration.
• PCR is also used in Forensic samples, Molecular epidemiology to identify point sources for hospital and community-based outbreaks, and to predict virulence.
• Diagnostic tests

  • To treat or prevent a disease we have to know the cause

  • To know the cause we have to accurately diagnose it
• Identifying bacteria via culture on agar plates

  • Physical features and phenotypic tests
  • Biochemical tests
  • Antibiotic susceptibility
  • MALDI-TOF mass spectroscopy
  • PCR
  • Genome sequencing

  Identification of bacteria directly from a sample

  • PCR, PCR/Mass spectrometry
  • FISH
  • Microarrays
  • Genome sequencing
• Sensitivity

  • Ability to detect all true positive samples for a test

  Specificity

  • Accurate enough to only detect true positive samples for a test
• Trial of a new PCR test through preliminary tests with well-characterised samples

  Steps to check

  1. Primers work on a chosen target
  2. PCR conditions correct
  3. Extraction of DNA from bacteria works
  4. Extraction of DNA from bacteria in relevant material/tissue works
  5. Extract is free from inhibitors
  6. Detection of amplified product works
  7. No amplification when target is absent
  8. Test detects known gene/species variants
  9. Test differentiates target gene/species from related
• Trial of a new PCR test via further tests with real clinical samples

  • Use known positive and negative samples

  • Include samples positive for other pathogens

  • Always control for PCR performance (positive control)

  • Always control for contaminants (negative control)

  • Control for inhibitors (internal positive control
• PCR principles and Real Time PCR are reviewed in the context of bacterial typing.