PCR FOR HBV &

HCV
TOPIC WE NEED TO KNOW
What is pcr?
Pcr machine- thermocycler
Pcr components – reaction mix (target DNA, primers,
dNTPs)
Mechanism of pcr-steps
Advantages of pcr
Disadvantages of pcr
WHAT IS PCR?
PCR MACHINE - THERMOCYCLER
COMPONENTS OF PCR
 TYPES OF PCR

COLONY PCR

NESTED PCR

REVERSE TRANSCRIPTASE PCR

REAL TIME PCR

INVERSE PCR
 Colony PCR is used for Pick a bacterial colony
the screening of with autoclaved
bacterial(E.coli) or yeast toothpick, swirl it into 25
clones for correct micron l of the TE
ligation or plasmid autoclaved dh2O in an
products. microphage tube.

Heat the mix in a boiling Spin sample for 2

COLONY PCR water bath (90-100C) for
2 min.
minutes high speed in
centrifuge.

Take 1-2 micron l of the

Transfer 20 micron l of
supernatant as template
the supernatant into a
in a 25 micron l PCR
new microfuge tube.
reaction.
 ▪ Reverse transcription polymerase
chain reaction is a laboratory
technique combining reverse
REVERSE transcription of RNA into DNA and
TRANSCRIPTASE amplification of specific DNA
PCR targets using polymerase chain
reaction. It is primarily used to
measure the amount of a specific
RNA.
 ▪ Inverse polymerase chain
reaction (Inverse PCR) is a variant
of the polymerase chain
reaction that is used to amplify
INVERSE PCR DNA with only one known
sequence.
▪ Inverse PCR is especially useful
for the determination of insert
locations.
 ▪ A real-time polymerase chain
reaction, also known as
quantitative polymerase chain
reaction, is a laboratory technique
of molecular biology based on the
REAL TIME PCR polymerase chain reaction. It
monitors the amplification of a
targeted DNA molecule during the
PCR, i.e. in real-time, and not at its
end, as in conventional PCR
SYBR GREEN REAL TIME PCR
TAQMAN-PROBE REAL TIME PCR
 ▪ INRODUCTION
HCV RNA This is an in vitro
diagnostic test for the qualification
QUALITATIVE detection of human hepatitis C virus
RNA in human serum or plasma. It is
FLUORESCEN intended for use as an aid in the
diagnosis of an HCV infection.
CE
DIAGNOSIS
 TEST PRINCIPLE
▪ Magnetic bead technology to extract HCV-RNA from human serum or plasma.
▪ By applying real-time fluorescence quantitative PCR technology, this test used a pair o
specific primers which are designed to target a conserved sequence of HCV-RNA and
specific fluorescence probe, accompanied with other ingredients in PCR-Mastermix, t
achieve qualitative detection of HCV-RNA through fluorescent signal changes.
storage:
Extraction reagents should be stored at 2-8C and the amplification
reaction reagents at-20+5C.
 COMPATIBLE INSTRUMENT
▪ The product applies to fluorescence PCR instrument such as ABI7500, Stratagene Mx3000P and
Roche LightCycler 480, Sansure Slan, etc.
Specimen requirements:
1. Applicable specimen type: human serum or plasma.
2. Collection of specimen
I. Collection of serum: use a sterile syringe to draw 2 ml of venous blood from the
subject and injected it into a sterile tube, hold it at room temperature for up to 4
hours to separate serum it self
II. Collection of plasma: Use a sterile syringe to dram 2 ml of venous blood from the
subject and inject it into a sterile tube with EDTA or citric acid salt.
3. Storage and transport of specimens
Human serum or plasma specimens collected via
the above mentioned method can be used for immediate detection, or stored at -20+5 C
for 3 months or stored below -70C for a long term.
 TEST METHOD

1. Preparation of reagent.
2. Processing of specimens.

PCR AMPLIFICATION:-
1. Place PCR reaction tube into the specimen wells of the amplification device. Set up the HCV
negative control, HCV Positive control, HCV low positive control and unknown samples in the
corresponding sequence and input sample information.
2. Select PCR test channel.
 RESULTS
▪ Result will be saved automatically.
▪ Analyze the HCV curve and the HCV internal control curve respectively.
▪ After analysis, adjust start, End and Threshold values of baseline of the graph.

Quality control:-
The test result is treated as valid if all the conditions in
the table bellow are met for the same test.
Otherwise the test result is treated as invalid and needs to be re-tested.
 REFERENCE RANGE
▪ Trough the research on reference values, the positive reference rang is
determine to be Ct<42, and the Ct reference value of internal control is
determined to be 38.

Explanation of detection result:

1. For specimens which are detected with Ct<42 and the amplification curve is
shaped like S the test results can be reported positive
2. For specimens which are detected with Ct>42 or no Ct and internal control is
detected positive, the test result can be reported as negative.
3. If the internal control is off normal , then the specimens detection result is
invalid. An investigated should be performed to find out reason and then
retest the specimens.
 Introduction:
The promotor nucleic
acid(DNA) extraction kit is intended
for use in conjunction with the
HBV DNA promotor NES-32 nucleic acid
extraction system to extract DNA
from various specimens, such as
EXTRACTI serum, plasma, swab, feces, urine,
etc.

ON PCR Extracted DNA can

be used in biological applications
and clinical molecular diagnosis,
such as, nucleic acid testing, gene
cloning and sequencing, PCR,
molecular hybridization, etc
 PRINCIPLE
▪ Magnetic isolation method, specifically using beads, is an efficient and effective
method to extract nucleic acid from specimens.
▪ Magnetic carriers with immobilized ligands or biopolymers with an affinity to bind to the
target nucleic acid are used.
▪ These magnetic materials are produced from particles such as synthetic polymers,
biopolymers, porous glass, or magnetic particles made from inorganic magnetic
materials.
▪ Spherical objects such as beads, are often used because of their large surface area
preferred for binding nucleic acid.
▪ Macromolecule functional groups are added to the beads to help facilitate the
adsorption of nucleic acid.
 PRINCIPLE (CONT…..)
▪ Amino, sulfhydryl, and epoxy groups are used to modify the macromolecule coated on
the surface of the magnetic beads.
▪ The process begins when the lysis buffer is added to the specimen to promote the
breakdown of the cell membrane and release of the nucleic acid.
▪ Magnetic beads are then added to the mixture.
▪ A magnetic rod is used to efficiently attract the nucleic acid bond to the magnetic
beads, while the supernatant is washed away.
▪ An elution buffer is added to unbind the nucleic acids from additional materials before
completing extraction for downstream applications.
 ▪ Unopened kits should be stored at 2-30C
upon receipt. All unopened reagents are

STORAGE AND
stable through the expiration date printed on
the box if stored between 2-30C. Once
opened, use the plate immediately. The

STABILITY extraction plate should be kept of its front

face up to prevent reagent sticking to
sealing film.
 SPECIMEN COLLECTION
AND PREPARATION
Specimen collection
serum or plasma
▪ Collect 5 ml of venepuncture whole blood using disposable syringe.
▪ Separate serum or plasma for blood as soon as possible to avoid haemolysis.
▪ Centrifuge the tube for 20 min at 2000-4000 rpm.
▪ Transfer serum or plasma for testing.
Faeces;
▪ collect faeces using a sterilized tube.
▪ Add 1 ml of 0.9% NaCl into a 1.5 ml centrifuge tube, then collect 200mg specimen using
a sterilized toothpick and add it to the 1.5 ml centrifuge tube.
 SPECIMEN COLLECTION
▪ Swirl and gently shake until the specimen is homogenized.
▪ Centrifuge the tube for 3 min at 3000 rpm, and then collect the supernatant.
▪ Centrifuge the supernatant for 10 min at 12000 rpm, and then discard the supernatant.
▪ Re-suspend precipitate in 200micron l of 0.9%NaCl for nucleic acid extraction.

Cotton swab
▪ Collect specimen using aseptic cotton swab and put the swab in a sterilized
plastic screw top tube.
▪ Added 1 ml of b0.9% NaCl into tube with swab cotton containing specimen and
shake it gently.
CONT….
▪ Remove the cotton swab from the tube. While removing, press the
cotton swab firmly against the inside of the tube to remove any excess
liquid that might be absorbed by cotton swab.
▪ Transfer the specimen solution into 1.5 ml centrifuge tube.
▪ Centrifuge the tube for 5 minutes at 12000 rpm.
▪ Remove and discard 8800 micron l of supernatant.
▪ Re-suspend the pellet in the remaining 200 micron l of the supernatant.
Urine
▪ Add 10 ml of the urine into a sterilized glass tube, seal and transport for
testing.
▪ Add 0.5 ml of 0.5M EDTA to 4.5 ml urine.
▪ Discard supernatant.
▪ Re-suspended the pellet in 200 micron l of 0.9%NaCl.
MATERIALS REQUIRED
▪ Anhydrous ethanol
▪ 1.5 ml centrifuge tubes
▪ Pipette tips
▪ Disposable gloves and other protective gear
▪ The promoter NES-32 nucleic acid extension system.
 PERFORMANCE
CHARACTERISTICS
Precision
Was evaluated by testing low concentration HBV sample.
Can give positive result after 10 replicate with CV value less
than 5%.
Sensitivity
was detected by testing a HBV positive sample with
concentration of 20 IU per ml after 20 replicates.
The detected person was 100%.