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MOLECULAR BIOLOGY  It can be processed even in small quantity of

Dr. Gilbert Vergara samples
 It is the method of choice for the rapid identification
Definition of viral and non-cultivable infectious agents
 Deals with the study of structure and function of
macromolecules (CHON and nucleic acid) Viral Nucleic Acid

Techniques of Molecular Biology

 Expression cloning
 Polymerase chain reaction (PCR)
 Gel electrophoresis
 Macromolecule blotting and probing techniques
 Southern blotting
 Northern blotting
 Western blotting
 Eastern blotting
 Arrays Routine Specimens
 Allele specific oligonucleotide  CSF
 Antiquated technologies  Respiratory secretions
 Discharge
Polymerase Chain Reaction (PCR)
 Pure Isolate (bacteria)
 Is a technique in molecular biology to amplify a  Urine
single copy or a few copies of a segmented DNA
 Swabs
across several orders of magnitude, generating
thousands to millions of copies of a particular DNA
Acceptable Clinical samples for TB PCR:
sequence
 Sputum
 Basic information
 Gastric washing
 Role in molecular biology in diagnostics
 Stool
 Accuracy in diagnosis
 Urine
 PCR is a technique to amplify a piece of DNA or
 Paraffin block
RNA very rapidly outside of a cell
 CSF
 The method relies entirely on thermal cycling
or repeated heating and cooling to promote  BAL
enzymatic replication  Tissue (no formalin)
 Uses enzyme of bacterium Thermus aquaticus  Wound discharge
(Taq DNA polymerase)
 Some applications of PCR Unacceptable Clinical samples for TB PCR:
 Genetic disorders  Blood
 Paternity testing  Specimen in anaerobic vial
 Forensic medicine  Viral transport medium
 Infectious disease  Swab
 Tissue formalin fluid
To pinpoint the real culprit:
 PCR is 100% accurate in determining the etiologic Important Pathogens:
agent of an infectious disease  Hepatitis viruses
 Dengue

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 Rabies  Annealing: alteration of the current structure of the

 HIV nucleic acid
 Malaria  Elongation: stretches the target nucleic acid t
 Tuberculosis (shorter TAT) basically insert the Taq gene
 Respiratory viruses
 Leprosy bacteria

PCR Drawback:
 Processing of samples
 Aseptic technique
 Kits (expensive)
 Sample storage (very crucial in TB PCR)
 Temperature (kits)
 Special training for Medtech

PCR vs Culture: which is more accurate for TB?

 Culture

Basic Procedures
 100 ul reaction proceudre

Principle and Procedure

 Denaturation: a process in which CHONs and NA
lose the tertiary structure and secondary structures

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Reading the Results (PCR stages) Interpretaion of Results

 Copies (quantitative)
 Positive/Negative
 Detected/Not Detected

PCR and Hepa C Virus

 Viral load monitoring thru copies (HCV)
 Can monitor drug effectivity (HCV)
 Can detect early sero-conversion thru decreased
viral NA or viral load

Which is more accurate in PCR

 DNA
 RNA
 Mitochondria

Semi PCR: GeneXpert

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