4 Pre-analytical Factors

• Nucleases is an inhibitor that is present everywhere, and it degrades Nucleic Acids
• Heparin is an inhibitor that is an anticoagulant and it inhibits Reverse Transcriptase and DNA Polymerases
• Disodium EDTA is an inhibitor of enzymes
• Hemoglobin and other variants are DNA Polymerase inhibitor that prevents amplification of Nucleic Acids
• Acidic Polysaccharides is an inhibitor that is commonly found in sputum; it inhibits DNA Polymerase
• Nitrite, Hemoglobin, HCG are inhibitors found in Urine, that also inhibits DNA Polymerase
• Hemoglobin is a chromogenic protein
• Urine is not used when it comes to the isolation of genetic material from our bodies and by the microorganisms, because of the different inhibitors present
• A) BAL

  B) Tracheal aspirate

  
• A) Sputum

  
• A) Nasopharyngeal swab

  
• A) Oropharyngeal swab

  
• A) Nasopharyngeal wash/aspirate

  
• Bronchioalveolar lavage is most accurate specimen (if respiratory)
• Tracheal aspirate is also accurate but has lower sensitivity
• Viral transport medium is used specific for viruses from swabs
• Universal Transport Medium can be used for bacterial & fungal
• 2 Solution components in VTM/UTM:

  • Antibacterial → Gentamicin Sulfate Solution
  • Anti-fungal → Amphotericin B
• The function of VTM is to maintain integrity of Nucleic Acid

  • Prevents bacteria and fungi growth
  • Can be made in the lab or purchased
  • Contain protein stabilizer, antibiotics, and buffer solution
• VTM is pink colored because of the pH indicator (phenol red), which signifies if there is contamination
• If pH indicator turns from red to yellow: It produces acid

  • Yellow color is due to bacteria and fungi
• Calcium alginate swabs or wooden tip swabs is avoided because they may contain substances that inactivate some viruses and inhibit PCR testing
• Synthetic fiber swabs with plastic shafts is used only in molecular biology
• Amies transport medium is a general transport medium for all microorganisms
• Sterile saline is a buffer with guanidine, which is used for isolation of nucleic acids
• There are two type of interference of RNAse:

  • Endogenous RNase → from Human tissues and cells
  • Exogenous RNase → from environment (Bacteria and Fungi)
• Restriction Endonucleases are nucleases produce by bacteria
• Remedies for RNAse interferences:

  • Correct storage to avoid RNA degradation
  • Long term storage → ultra-low freezer (-70°C to 80°C)
  • Keep RNA Aliquot on ice with lid closed when performing on bench tops
  • Use Cold Racks to slow down RNase action
• For sample inactivation, chemical is always included in the buffers

  • Guanidine HCl: for DNA Isolation
  • Guanidine Isothiocyanate: for RNA Isolation
• Heat-dry baths is used for heating the specimen, which will inactivate microorganisms

  • 56°C for 30 minutes
  • 65°C for 10 minutes
• As you increase the temperature you are also increasing the risk of degrading nucleic acids, so you need to lower the time incubation of the specimen from the heat

  
• Inactivation or lysis is the first step in extraction procedure to inactivate/destroy organism while preserving its nucleic acids
• Purification is the second step in extraction procedure to removing contaminants

  (e.g., other nucleic acids from other spp., proteins, reagents)
• Washing is the third step in extraction procedure

  • washing buffer: has ↑ salts concentration and ↓ alcohol concentration
  • rinsing buffer: ↑ alcohol concentration and ↓ salts concentration
• Drying is the fourth step in extraction procedure; it is the process of evaporation of alcohol that was present in the rinsing and washing buffer
• Elution is the last step in extraction procedure; it is done by adding nuclease free water/PCR water, which undergo “harvesting” process
• Carrier RNA is added/applied to enhance recovery, preventing irreversible binding of the small amount of target DNA
• Protease K is an enzyme used to degrade proteins; it can even degrade the strongest protein, which is the Keratin
• Lysate buffer has a ratio of 1:1 (equal amount of lysis buffer and the sample)
• Phenol & Chloroform or Isoamyl Alcohol has a ratio of 25:24:1 and is used in organic purifcation

  • Reagent: Sample 1:1
  • Three layers formed:
  • 1st: Nucleic Acids (aqueous phase)
  • 2nd: Interface
  • 3rd: Precipitated proteins;