SEMINAR 1

AUGUST 14, 2021 PCR

COVID 19 RT-PCR and Other Viral Molecular
Diagnostics
 Physician should pull out case investigation form
 Endorsed to Molecular lab together with CIF
form
 Swab test (others: saliva, nasopharyngeal or
oropharyngeal swab) by medtech
 Ensure proper specimen transport
 Processing of PCR by medtech
 Raw PCR results forwarded to the pathologist
for interpretation
 Patient’s individual PCR result (encoded by
laboratory encoders)
 Reporting PCR results to DOH
Basic Principle of Nucleic Acids and Amplification
 Cell inside it is the nucleus that contains DNA.
 DNA contains genetic information
 Transcribed to RNA
 Translated to proteins by cytoplasmic enzyme
 Some viruses have reverse transcriptase that
allows reverse transcription of DNA from RNA
 DNA is a molecule in the form of double-
stranded helix
 Each strand of DNA is composed of 4 types of
nitrogen bases—adenine, cytosine, guanine,
and thymine
 The doubles stranded helix is the most
energetically favorables state of DNA
 Target DNA (from spx)
 Primers
 Taq polymerase (acts in extreme heat)
 Deoxyribonucleotide triphosphates
 Thermocycler that raises and lowers
temperature of the sample
Three main steps in PCR
 Denaturation- unwinding of strands through heat
 Annealing- attachment of primer to the target
segment through a cooler temperature
 Extension- addition of nucleotides to the primer
with a warm temperature
Cont. of PCR
 PCR allows nucleic acid amplification or
production of multiple copies of a target DNA
segment
 With large number of DNA copies, the presence
of a target DNA segment is easier detected
RT-PCR
 If the target is an RNA, a complementary DNA
(cDNA) is first produced via reverse transcription
o COVID has reverse transcriptase
enzyme
 The cDNA is then amplified through PCR
Viral Molecular Diagnostics
 New gold standard for viral testing for most
viruses

1. DNA is double stranded while RNA is single

stranded (where uracil is used than thymine)
2. In RNA, thymine is replaced by uracil
3. The sugar in DNA is

DNA Replication

 DNA duplication uses each strand of the present

DNA to synthesize daughter cells
 Though DNA replication is conceptually simple,
the process is complex and involves a number of
accessory proteins and enzymes
 Helicase- for unzipping of DNA double strand
 Primase- stop the process
 Polymerase- siya magpapahaba
 Primer- make small piece of RNA
 DNA polymerase binds to the primer (5’ to 3’)

Unwinding DNA

 Extreme heat (melting)

 50% of dsDNA is converted to single stranded
DNA is called the melting point
 Melting points depends on amount of adenine-
thymine and cytosine to guanine