MOLECULAR BIOLOGY

PRETEST
96 Well PCR These are high quality plastics used in thermocyclers for
PLATES PCR applications. With uniform and thin walls enable a
precise thermal transfer. It is free of DNA, DNase,
RNase, PCR inhibitors and is autoclavable.

BIORAD CFX 96 A Thermocycler or PCR machine, used to amplify

segments of DNA via Polymerase Chain Reaction. It
includes two parts: An Annealing Chamber Thermal
rotation and a Real-time device. The device has a
thermal block with holes where tubes holding the PCR
reaction mixtures can be inserted. The cycler then raises
and lowers the temperature of the block in discrete, pre-
programmed steps.
Cold Block Provides a stable platform for the PCR plates, strips, and
reagents. It is made up of solid aluminum. Once placed
on ice, or kept in the freezer overnight, it keeps the
DNA/RNA samples as well as valuable enzymes cold
and prevents degradation.
Microcentrifuge Used to spin down samples or reagents in a
microcentrifuge tube at high speed.

Microcentrifuge Tube Single use tubes made from polypropylene used for
preparing, mixing, centrifuging, transporting and storing
small volume liquid samples and reagents. It must be
Rnase-free and autoclavable.

PCR Cabinet Is a kind of ventilated enclosure that provides product

PCR Plate Centrifuge
protection only. ventilated enclosures are
enclosures/cabinets with defined airflow that provide
protection. Air coming from outside is filtered first before
entering the cabinet but exhaust air is not filtered, thus
providing product protection only. it can only be only
used for sterile work involving non-pathogenic samples
such as preparation of mastermixes.
Used to spin down all the droplets on the walls of the
PCR plate and the condensation of droplets are
concentrated at the bottom of wells in the PCR plate.

SARS-CoV-2 Detection Kit Consist of Enzymes (Taq DNA Polymerase, Reverse

Transcriptase), Primers, Probes, dNTPs, and a Buffer.
The detection kit also consists of commercially prepared
No Template Control and Positive Template Control. It is
used to detect presence SARS-CoV-2 RNA by
 amplifying a target gene, making multiple copies of it and
read by the machine by its fluorescence signal.
Spin Column Used in solid phase extraction method. its filter is made
up of silica resin that selectively bind DNA/RNA.

Vortex Mixer It is used to mix small volume sample and reagent

rapidly. This device has an electric motor with a drive
shaft that is vertically oriented and also attached to a
cupped rubber piece mounted slightly of the center. The
motor drives the rubber piece at the top in a circular
motion, this causes sample liquids to circulate and
undergo turbulent flow, otherwise known as vortex. You
can also regulate the speed by adjusting its knob.

INTRODUCTION
QIAcube HT

Automated nucleic acid Purification

CRX 96 is a powerful machine for real time

polymerase chain reaction detection.

SPECIMEN COLLECTION, HANDLING, AND TRANSPORT

(NASOPHARYNGEAL AND OROPHARYNGEAL SWAB)
OBJECTIVES
• To gain knowledge on the collection, handling, and transport of samples to be tested in RT-PCR /
Molecular Laboratory (Oropharyngeal and nasopharyngeal swab)
• To gain knowledge on completeness of data in a Case Investigation Form (CIF)
• To gain knowledge in the proper labelling, storage, and transport of Virus Transport Media (VTM)
THREE PHASES OF LABORATORY TESTING
Pre-analytical phase collection, handling, and transport
Analytical phase laboratory testing, processing
Post-analytical phase recording, reporting, interpretation
SPECIMEN COLLECTION
is defined as the first and crucial step in laboratory testing
SPECIMEN COLLECTION MATERIALS
Virus Transport Media (VTM)
Swab
Parafilm, Gauze, Triple Packaging
Case Investigation Form (CIF)
Label should have:
• Full name (including middle name)
• Birthday / Birthdate (mm-dd-yy)
• Age/Gender
• Date of collection (mm-dd-yy)
• Time of collection (hh:min)
• Sample collected → OPS / NPS
Points to remember:
• Always check for the VTM’s expiration date before
using
• Visually check for the VTM’s volume (should be at
least 1 mL or depending on the manufacturer’s guide)
• OPS is thicker than NPS
• Sterile
• For single use only
• Calcium alginate swabs or swabs with wooden shaft
are unacceptable as they may contain substances that
inactivate viruses and inhibit PCR testing
• Handling of swab sample follows the “triple
packaging system” (primary, secondary, tertiary
container)
• VMMC: zip lock (primary), bigger zip lock (secondary),
cooler with ice pack from ref (tertiary)
• Parafilm → used to prevent leakage for screw cap of
VTM
• Gauze → used to absorb any form of leakage coming
from the VTM
• Primary container → serves as the container to
prevent sample spills and contamination to other VTMs
• Secondary container → used to prevent spills and
maintain specimen stability during transport
• Tertiary container → used for transporting VTMs to the
molecular laboratory. Must be sterilized before and after
use
• Ice packs → included in tertiary containers
for the stability of temperature required for VTMs
We follow the “NO CIF, NO SWABBING” rule
• Has 3 pages: it is important to check the completeness
of data before proceeding in the swabbing area
FIRST PAGE
Should be written legibly and basic information
should
be complete (as per DOH guidelines for contact
tracing)
• Name of interviewer (requesting physician)
• Complete name of patient (including middle name,
check for proper spelling)
• Birthday, age, gender, civil status occupation (if
available)

SPECIMEN COLLECTION PROCEDURE
NASOPHARYNGEAL SWAB (NPS)
AND OROPHARYNGEAL SWAB
(OPS)
PREPARATION
• Complete address (house number, street, barangay,
municipality, and province)
o In case of unavailable house number or street,
ask for a landmark
• Cellphone number (landline is not allowed, in case of
no available phone number, look for patient’s relative
and ask for number)
• Email address (optional)
Case investigation details are accomplished by the
requesting physician especially if the patient is under
PUI (probable or suspect)
At the bottom of the first page, write:
• Specific ward (either emergency ward, ward 1, 3 ,4
etc.)
• Date (mm-dd-yy) of collection
• Time (hh:min) of collection
• Name of the collector (Juan, RMT)
• Category
o RPV (retired veteran)
o Vmmcp (VMMC employee)
o CP (civilian patient)
o RPVD, VMMCPD (for their corresponding
dependents)
• ID number (available at Hospital Information
System [HIS])
• Transaction and Document number
o Can be accessed through HIS after patient is
charged for payments
SECOND PAGE: clinical information filled up by the
doctor or attending physician
THIRD PAGE: Case definitions (category of the patient)
Complete all the information before proceding to the
swabbing area.
• Anatomy of the pharynx
o Naso, oro, and hypopharynx
o Lined by mucous membrane
o NPS and OPS are ideal sites for specimen
collection for COVID-19 test
Both nostrils in VMMC
• It is important to have a checklist of the materials
needed for sample collection
• Take note of the donning (clean) and doffing (dirty)
area
• Make sure that your PPE is complete before going to
the donning area
 Complete PPE:
Shoe cover
Hair net
Hazmath (full cover)
N95 (respirator/double mask)
Face shield and gloves
PATIENT IDENTIFICATION AND LABEL FIRST
SWABBING PROCEDURE – SEQUENTIAL • Identify the patient by asking for their full name
SWAB SAMPLE PACKAGING including the middle name, birthday, and age
o In some cases, the px cannot talk (NGT, etc.), you
can check for name on their name tags, or ask the
attending NOD for px ID (take note for the name of
the NOD who identified the px)
• Take note for the correction (if there’s any) before
proceeding to swabbing
• If patient’s identity matches the label, put on the label
on your available VTM
• Briefly orient the px about the procedure of the
swabbing
• Collect sample
SEQUENTIAL SWAB SAMPLE PACKAGING
a. Put label on VTM
b. Apple parafilm (not too tight, make sure screw cap is tightly closed)
c. Apply gauze (not little and not too much)
d. Put inside ziplock (primary container)
e. Put label outside the ziplock/primary container (make sure the ziplock is tightly sealed)
f. Put it inside in another ziplock/secondary container along with the other samples
g. For storage:
o VTM (not in use) → refer to manufacturer’s recommended storage temperature
o VTM (with swab) → 2-8°C (short term storage) or freezer temperature for long term
h. Collected samples are then transferred to tertiary container labelled as biohazard before transporting
to molecular laboratory
o Specimen should be transported within 72 hours
IMPORTANT REMINDERS
• Make sure to change secondary gloves per px every after swabbing
o This is to prevent contamination and transfer of virus
• Avoid physical contact of gloved hands to your mask or eyes
• For emergency wards: do not go to clean area while still wearing PPE if contact with PUI is already
done. Doff first before going to clean area
o Always make sure that the request for swab is encoded in the HIS for charging and payment is
required for civilian px before swabbing
• For in px wards: do not go to the nurse’s station wearing PPE if contact with px is done. Doff first before
going to the station.
o Require the Nurse On Duty to charge the px in the HIS before proceeding to swabbing
• Always perform proper handwashing after each contact with possible contaminated surfaces
• Do not remove mask wherever you go around the hospital premises
• It’s best to practice “BUDDY SYSTEM”

VMMC MOLECULAR/COVID TESTING LABORATORY

OBJECTIVES
• To define biosafety, biosecurity, and biorisk management
• Learn proper donning and doffing procedures
• Overview of VMMC Molecular Laboratory workflow
• To learn the basic principle of PCR testing
• To have an overview of RNA extraction, reagent preparation, and amplification
LABORATORY BIOSAFETY AND LABORATORY BIOSECURITY
LABORATORY BIOSAFETY • Practices implemented to prevent accidental/
unintentional release/exposure to pathogens. (ex: spills,
laboratory acquired infection [LAI])
• Includes the use of PPE and ventilated enclosures
LABORATORY BIOSECURITY • Institutional/personal security measures designed to
prevent loss, theft, misuse, diversion, or intentional
release of pathogens (ex: Bioterrorism)
• Includes the use of CCTV and window grills
BIOSAFETY “protecting people from bad bugs”
BIOSECURITY “protecting bad bugs from bad people”
BIORISK MANAGEMENT • Effective management of risks posed by working with
infectious agents and toxins in laboratories
• The goal of biorisk management is to identify and reduce
risks before they evolve into nar misses or incidents such
as improper use of PPE or LAI
BIOSAFETY + BIOSECURITY = BIORISK
MANAGEMENT
3 components: [AMP]
1. Assessment → hazard identification, risk
assessment
2. Mitigation → biorisk control measure
3. Performance → processes, quality assurance,
objectives
PROPER DONNING AND DOFFING OF PPE
PPE • designed to protect the wearer’s body from
injury or infection
• Includes hairnet, face shield, medical masks, googles,
latex gloves, lab coat
Donning and Doffing • Highly important procedures that requires significant
care. A mistake can lead to exposure to pathogens and
may result to sickness
• PPE must be worn, removed, and discard properly
Donning to put something “on”
Doffing to remove or to put something “off”

VMMC procedure for DONNING 1. Hand hygiene (soap and water/alcohol)

2. Shoe cover
3. Hand hygiene (alcohol based substance)
4. Inner gown (washable/disposable)
5. Inner gloves
6. N95 mask (must be fitted)
 7. Head cap
8. Outer gown
9. Face shield
10. Outer gloves
VMMC procedure for DOFFING 1. Outer gloves (most contaminated)
2. Outer gown
3. Shoe cover
4. Face shield
5. Head cap
6. Inner gloves
7. Inner gown
8. Hand hygiene (soap and water/)
9. N95 mask (touch the strap only)
10. Hand hygiene

RECEIVING OF SPECIMEN
Requirements: • A duly completed CIF with additional details such as
category, ID number, date, and time of collection, admitted
(ward)/outpatient specimen should be triple packaged
If the said requirements are incomplete, the specimen is
rejected.
If the requirements are met, the specimen is received and
given a succession number or specimen number
The specimen container is decontaminated and is sent
directly to the specimen proocessing area/extraction area
through a passbox

EQUIPMENT USED
Biorefrigerator and ultra-low freezers For reagent and specimen storage
PCR Cabinet (Reagent Preparation used for preparing mastermix, provides product protection
Room) by filtering the air going inside the cabinet
Biosafety Cabinet Class II A2 (Extraction used for RNA extraction and template addition, provides
Room) personnel, product, and environmental protection
Mechanism: filtering the air going inside and outside the
biosafety cabinet
Centrifuge and microcentrifuge
Vortex mixer
Autoclave (doffing area) used for treatment of biohazard wastes. Uses steam
sterilization. “steam under pressure”
Thermal cycler (amplification room) Used to run PCR
PCR plate centrifuge
Micropipette
Other supplies needed • PPE
 • Paper towel
• Adsorbent pad
• Microcentrifuge tube (MCT)
• Microcentrifuge Rack
• Specimen rack
• Spin column
• Pipette tips
• Disinfectants: 70% ethanol, 10% bleach
• Reagents: Viral extraction kit, PCR reagent
• PCR plate & PCR plate film
• Cold plate
• Biohazard Bags
• Pentel pen
WORKFLOW
-Unidirectional workflow (no backtracking)
- Clean to dirty work environment
PERSONNEL WORKFLOW Reagent Preparation And Sample Processing

Reception Area
w/out PPE
Donning Area

Reagent Preparation Area Wear your biosafety 2+

level PPE: Respirator,
(area where your perform or N95 mask and double
prepare your mastermixes) gown, double gloves

Sample Preparation Area

(area where you perform your RNA
extraction and inactvaion and also
the template addition
Doffing Area

Receiving of specimen

Reception Area

Donning Area
Wear your biosafety level 1
PPE: surgical mask and Sample Intake Area
gown
SAMPLE WORKFLOW
Reception Area

The spx container is

Sample Intake Area
decontaminated
Pass box
Sample Preparation/Extraction Area The spx container is
purified and inactivated
REAGENT WORKFLOW Reagent Preparation Area

Where we perform
template adding (mixing
Sample Preparation Area
the rgt with purified RNA)

2 TYPES OF WASTE GENERATED BY
MOLECULAR LAABORATORY
DNA and RNA
Deoxyribonucleic acid (DNA)
Ribonucleic acid (RNA)
These macromolecules
comprised of many
nucleotides
DNA REPLICATION
Doffing Area
Pass box
PCR amplification Area
1. Biohazardous wastes → reagent preparation area,
sample preparation/extraction area
2. General waste
- Waste generated by the laboratory must be properly
segregated and disposed. All biohazardous material must
be pretreated by autoclaving before disposal.
contains hereditary information
Each nucleotide is composed of 3 parts:
• Nitrogenous bases (adenine, thymine, guanine,
cytosine, uracil) -information
• A 5-carbon sugar
• At least 1 phosphate group

1. Parent DNA strand are separated

2. The addition of nucleic acid bases prodcues two
identical daugter strands (base pairing rule)
ENZYMES AT WORK ON DNA REPLICATION
DNA Helicases unwinds DNA
RNA Primase synthesis of short RNA primers
produces short RNA primer that will atach to one strand of
the DNA
DNA Polymerase III adds nucleotides to the primers, extends the strand
continuous the work of the RMA primase based on base
pairing of nitrogenoud base on parent strand
DNA Polymerase I degrades the RNA primer and replaces with DNA
After DNA polymerase III adds on the RNA primer, DNA
polymerase I degrades RNA primer and replaces it with
DNA
DNA Ligase connect two strands of DNA
PROTEIN SYNTHESIS
Transcription synthesis of RNA (mRNA) through DNA by RNA
polymerase
Produces one strand of RNA based on base pairing rule in
the parent strand of DNA. Instead of DNA polymerase,
 RNA polymerase will be used so that RNA strand will be
produced. The difference between these two is the
nitrogenous base (Thymine is replaced with Uracil).
Translation synthesis of polypeptide/protein through RNA (mRNA),
happen in the ribosomes

POLYMERASE CHAIN REACTION (PCR)

- Laboratory technique to make million/billion copies of a specific region of DNA to make enough copies
that it can analyzed
- It utilizes the enzyme DNA polymerase
Advantages: Disadvantages:
- High sensitivity and specificity (specific in their - Expensive
genes: SARS-COV2 – N gene or Orf1ab gene) - Contamination
- Can process multiple samples simultaneously - Cannot differentiate if the virus is dead or alive
- Relatively safe
- Results can be produced within the day (4hrs)
COMPONENTS OF PCR
Taq DNA Thermostable DNA polymerase
polymerase Isolated and named from the bacterium which it is isolated
“Thermus aquaticus” – thermophilic bacteria
Active in high temperature 70°C

Primer Short sequence of nucleotides that provides a start point

for DNA synthesis
Specific sequences
Deoxynucleotide triphosphates (dNTPs) provide building blocks for DNA synthesis
Buffer provides optimal condition for robust enzyme activity
has a pH of 8.0 - 9.5
common components of buffer are: K (promotes
annealing), (NH₄)₂SO₄ (enhances specificity), MgCl₂
Template DNA/RNA extracted DNA/RNA at a suitable concentration
Additional: • Reverse transcriptase (RT-PCR)
• Probe (qPCR, RT-PCR)
RT-PCR o Used to detect viral RNA
2 additional components:
 o Uses the enzyme reverse transcriptase, converts RNA
to complementary DNA (cDNA)
o Probe (made up of fluorophore [fluorescent chemical
compound that re-emit light upon light excitation] and
quencher [suppresses fluorescence by absorbing the light
emitted by the fluorophore])
STEPS IN RT-PCR AND TEMPERATURE REQS
Reverse 50°C – In this temperature, reverse transcriptase enzyme
transcription is active and converts RNA to DNA or cDNA. This step is
important because Taq polymerase is not active on RNA
or viral RNA.

Denaturation 95°C – It is the part where reaction components are

exposed to high temperature at 95 degcel. Sepratares the
two strands of the cDNA and exposes its nitrogenous
bases. It also inactovate the reverse transcriptase
enzyme.

Annealing 50°C – Around 50-60 degcel. It cools down the reaction

components so that the primer and the probe van bind to
the complementary sequences on the single stranded
DNA template. The primer binds to the flanking section of
the target sequence while the probe binds or hybridizes
near the middle of the target sequence. In this step, NO
flourscene is yet to be detected by the spectrophotometer
because the quencher is near proximity to the flourophore
absorbing its flourscence.
Extension 50°C – Taq polymerase activaes after primer
binding,adding complementary bases to build a new copy
strand. When polymerase reach the probe, it cleaves the
pprobe separating the flourophore from the quencher.
Once the flourophore is no longer near proximity to the
quencher, its flourescence can be read by the
spectrophotometer.
Collect Data 50°C - In this step, the spectrophotometer reads the
flourscence of the free flourophore or flourscent material in
the solution and the absorbance is provided in the
quantitative value

Repeat at step 2 to step 5 39 cycles

Each cyele the number of free flourescent material is
doubled and the signal detected by the spectrophotometer
should also increase.
NUCLEIC ACID EXTRACTION
- RNA extraction is done to separate the template RNA from other components of the specimen
- To inactivate enzymes such as nucleases that break down chains of nucleic acid to smaller pieces
- To remove substances that inhibit PCR (ex. blood, mucus)
Cell lysis breaking of the cell membrane to release all the contents
of the cell including nucleic acids
 Uses chemical methods to lyse the cell. It disrupts the
cells and do not harm the nucleic acids.
Common components of chemical lysis are:
• Cheotrophic salts - disrupts weak bond between
molecules. It solubilizes hydrophobic regions in
lipid bilayer and denatires proteins by disruting
tertiary protein structure. Then, nucleic acids under
go precipitation by adding ethanol. This process
allow the nucleic acid to better adhere to the filter
in the binding process. Addition of carrier RNA is
used if:
Small amounts of nucleic acids are expected
Carrier RNA catalyzes precipitation and prevents
nucleic acid from being permanently bound to the
filter.
• proteases enzyme
Binding RNA attachment to filter
Uses a buffer
The precipitated nucleic acids binds to the silica and filter
of a spin column when:
✓ the binding buffer has a lower pH and has a lower
concnetration that the filter
Nucleic acid is bound the the filter material due to the
concentrations. When the concentration is changed the
nucleic acid is released from the filter.
Washing removal of impurities
It uses wash buffers – the wash buffers allows the
contaminants to flow through the filter and leave the
nucleic acid bound to the filter. Then, the contaminants in
the filtrate is discarded.
Elution addition of nuclease free water to change pH thus,
producing purified RNA/DNA
Nucelase free water - lower salt concentration than the
filter. It releases bound nucleic acid and allows the nucleic
acid to flow through the filter thus producing a purified
RNA.

MASTERMIX PREPARATION
- Mixing of different components for RT-PCR reaction provided by PCR kit
- It is done inside a PCR cabinet in the reagent preparation room
- The work area must be clean and free from contaminants (amplicons)
- It is also the phase wherein you mix the mastermix with the negative template control
TEMPLATE ADDITION
- Mixing of mastermix and template RNA
- It may be done in a PCR / biosafety cabinet located in the extraction room
- The work area must be decontaminated before use. Actually the work area and all items that will be
used must be decontaminated before and after use.
- In this phase, you mix the mastermix with the positive template control
 POST ANALYTICAL
- Results produced by the machine is presented in an amplification chart

4 PHASES IN PCR
Baseline phase theoretical doubling of product, no change in fluorescence
signal
Exponential phase theoretical doubling of product at every cycle, exponential
signal growth
Linear phase components become limiting, reaction efficiency falling
Plateau fluorescence level may exceed instrument sensitivity,
phase reaction components exhausted, can no longer generate
more fluorescence

INTERPRETATION OF RESULT
RT-PCR - Qualitative test
- Tests if the target sequence is detected or not in a
sample
- It requires a positive and negative template control
Most PCR detection kit for COVID detect ORF1ab and N genes of
SARS-CoV2

CY5 – internal control; used to detect if the amplification is successful or if there is an inhibitor present in
the sample; it should be amplified or positive to demontrate successful amplification. If CY5 is negative, it
only means that there is an inhibitor present in the sample and you need to repeat the whole procedure.
FAM – specific for ORF1ab gene
ROX – specific for N gene (kahit isa lang mag-amplify sa kanila is considered positive pero magdepende
parin kayo sa bigay ng PCR reagents niyo)
KEYWORDS
Cycle threshold a point when sample fluorescence rises above
background fluorescence. It is adjusted above the
background
Ct value is the number of cycles it takes to cross the cycle
threshold
Background nonspecific fluorescence in the reaction (ex: inefficient
quenching of the florophore)