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Polymerase Chain Reaction ( PCR )
Molecular pathology and Molecular techniques have revolutionized
laboratory Diagnostics and have
in many areas of
vastly expanded the horizons of both academic and
community based pathology practice . The Molecular techniques are applicable to all the
sections of the laboratory . The Development of Polymerase Chain Reaction ( PCR ) by
the Californian Molecular scientists Mullis and et al was a milestone in Biotechnology and
heralded the beginning of Molecular Diagnostics . Mullis was awarded the Nobel prize in
1993.
The PCR and other Molecular techniques deal with the analysis of Nucleic acids

To Diagnose Disease ,

Predict the prognosis of Disease ,

Guide therapy , and

Evaluate susceptibility to disease before disease is evident .
These techniques have sensitivity unparalleled in Laboratory medicine and have created
new opportunities for the clinical laboratory
to improve patient care in the areas of
infectious diseases like Hepatitis , HIV, HSV, CMV, DENGUE , CHIKUNGUNYA, Typhoid,
Listeria, Mycobacterium tuberculosis , Atypical Mycobacterium ,Birds flue, Cancer , and
Genetic disorders etc.,
In addition to traditional laboratory tests , PCR and other Molecular techniques are the
additional complementary to day to day medical practices .
Nucleic acids are critical molecule of life . Deoxyribonucleic acid (DNA ) resides in the
nucleus of Eukaryotic cells and maintains all the information necessary for maintenance
of the organism and for the transfer of information to successive generations .
Ribonucleic acid ( RNA ) carries information from DNA to the cytoplasm of a cell and
directs synthesis of the proteins necessary for the functions of the organism .
The PCR is a simple , in vitro , chemical reaction that permits the synthesis of essentially
limitless quantities of a targeted Nucleic acid sequence . This accomplished through the
action of DNA Polymerase , under the right condition can copy a strand of DNA.
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A successful PCR analysis Involves 3 major steps :
1) Appropriate sampling and Preservation
2) Nucleic acid extraction
3) Polymerase Chain Reaction .
SAMPLING DETAILS FOR REAL TIME PCR TESTS
All the appropriate clinical samples for DNA analysis must be transported to the Molecular
biology lab with minimum possible delay preferably 30mts to 1 hr by maintaining the cold
chain .
RNA estimation required SERUM/ PLASMA / CSF must be transported immediately to the
Molecular biology lab in the frozen state ,using dry ice.
TESTS
SAMPLE
HCV, HIV and CHICK -V
PLASMA ( EDTA VACUTAINER )
HBV – VIRAL LOAD
SERUM
HSV – VIRAL LOAD
SERUM / CSF/ SWAB FROM INFECTED SITE
CMV– VIRAL LOAD
USE EDTA VACCUTAINER -BUFFY COAT
DENGUE
( PLAIN VACUTAINER)
PLASMA /SERUM (USE VACUTAINER)
(QUALITATIVE )
Mycobacterium species
(QUALITATIVE )
SAMPLE FROM INFECTED SITE –
( Sputum, Bronchial wash, Body fluids, CSF,
Tissue material in normal saline etc . Serum /
Plasma
are
usually
not
recommended .
Exceptional applicable .
* Never use Heparin as anti-coagulant for BLOOD & BODY FLUIDS
* SERUM / PLASMA usually not recommended sample for Mycobacterial – PCR
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To obtain Plasma For HIV Quantitative ( Viral load ) estimation .
1. Blood must be colleted in a lavender cap EDTA vacutainer to a full draw ( till
blood flow stops on its own or volume of blood collected is as stated on the
vacutainer )
2. Blood containing vacutainer must be gently inverted seven – eight times to ensure
proper mixing of specimen and the anticoagulant .
3. The vacutainer tube must be suitably labeled indicating Name of The Patient ,
age /sex and Identity No .
4. Centrifuge the specimen at 1200g ( 3,500 rpm ) for 10- 15 mts .
5. Label the plastic transfer vial specifying anticoagulant used to obtain the plasma
and time of collection . These details must match the details on the vacutainer
label .
6. Transfer separated plasma to this vial with the help of a plastic transfer pipette ,
after ensuring specimen is free from hemolysis and turbidity .
7. This plasma must be frozen and stored in the freezer compartment of the
refrigerator until time of packing and dispatch .
8. The frozen plasma –containing vial must be packed in the vacutainer packing
Styrofoam box, this box must be placed in between two layers of dry ice in dry ice
box . The quantity of dry ice should be enough to maintain the specimen in frozen
condition until it reaches Doctors’ Diagnostic Center – Trichy .
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Nucleic Acid Extraction :
The Nucleic acid extraction is one of the important step in PCR analysis .
It purely depends on the efficiency of reagents,
stability of reagents on storage and
expertise skill in extraction .
QIAGEN RNA EXTRACTION PROCEDURE FROM
(Plasma Cell free body fluids, cell culture supernatants.)
1.
Pipette 560Mul of prepared Buffer AVL containing RNA in to a 1.5ml micro centrifuge
tube .
2.
Add 140 MuL of sample to the above tube. Vortex for 15 secs.
3.
Incubate at room temperature for 10 mts.
4.
Centrifuge briefly to remove drops from the inside of the lid.
5.
Add 560MuL of ethanol (96-100 %) to the above tube and vortex for 15 Secs
6.
Centrifuge briefly to remove drops from the inside of the lid.
7.
Transfer 630Mul from the above tube to a spin column & Centrifuge at 8000 Rpm for 2 mts.
8.
Carefully remove the spin column from the collection tube and discard the contents of
the collection tube and place the spin column back in the same collection tube.
9.
Repeat the Steps 7 & 8 .
10. Add 500 Mul of Buffer AW1
11. Centrifuge at 8000 rpm for 3 mts.
12. Carefully remove the spin column from the collection tube and discard the contents of
the collection tube and place the spin column back in the same collection tube.
13. Add 500 Mul of Buffer AW2
14. Centrifuge at 8000 rpm for 1minute and 14,000 rpm for 5 minutes .
15. Place the spin column in a new 2 ml ependrof tube
16. Add 60 MuL of Buffer AVE and incubate at Room temperature for 2 minutes.
17. Centrifuge at 8000 rpm for 2 minutes
18. Now DISCARD THE SPIN COLUMN and store the Eppendroff tube containing RNA at 20deg C.
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Polymerase Chain Reaction :
A PCR cycle consists of 3 steps : Denaturation , Annealing , and extension .
At the end of each cycle , the PCR products are theoretically doubled . Thus after n PCR
cycles , the target sequence can be amplified . 2n -fold
. The whole procedure is carried
out in a programmable thermal cycler that precisely controls the temperature at which
the steps occur , length of time that reaction is held at the different temperatures, and the
number of cycles . Ideally, after 20 cycles of PCR a million fold amplification is achieved
and after 30 cycles , a billion fold . In practice, the amplification may not be completely
efficient due t o failure to optimize the reaction conditions or the presence of inhibitors
of the DNA polymerase . Different PCR techniques are described below :
Reverse Transcriptase PCR
PCR as it was originally described was a technique for DNA , amplification . Reverse
Transcriptase PCR ( RT- PCR
) was developed to amplify RNA targets . In this process
complementary DNA ( cDNA ) is first produced from RNA. As originally described .
Nested PCR
Nested PCR
employs
was
developed
to increase both sensitivity
and Specificity of PCR .
It
2 pairs of amplification primers and 2 rounds of PCR . Typically, one primer
pair is used in the first round of PCR of 15 to 30 cycles . The products of the first round
of amplification using the second set of primers that anneal to a sequence internal to
sequence amplified by the first set of primers . The increased sensitivity arises from high
total cycle number and the increased specificity arises from the annealing of the second
primer set to sequences found only in the first-round products, thus verifying the identity
of first round product .
Multiplex PCR :
In multiplex PCR , two or more primer sets, designed for amplification of different targets,
are included in the same reaction mixture .
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Real –Time PCR
All real time PCR systems rely upon the detection and quantitation of a fluorescent
reporter the signal of which increases in direct proportion to the amount of PCR product
in a reaction. The reporter is the fluorescent probe or a double stranded DNA specific dye
SYBR green which upon excitation emits light. Thus, as a PCR product accumulates,
fluorescence increases. Real time PCR assays used
for quantitation RT-PCR combine the
best attributes of both relative and competitive ( end point ). RT – PCR in that they are
accurate, precise, capable of high throughput, and relatively easy to perform.
Real time PCR methods decrease the
time required to perform nucleic acid assays
because there are no post PCR processing steps. Also, since amplification and detection
occur in the same closed tube, these methods eliminate the post amplification
manipulations that can lead to laboratory contamination with amplicon. In addition real
time PCR methods lend themselves well to quantitative applications because analysis is
performed early in the log phase of product accumulation.
Significance of PCR technology :

Helps in early diagnosis of infection , particularly It detects window period
( anti-HIV- anti body , ELISA negative state ) patients, even at the moment of
high viral invasion.

To quantitation of Viral load during anti viral therapy .

Accurate and Differential detection of Mycobacterium tuberculous , MTB complex
and Atypical Mycobacteria by Multiplex PCR primers in 2-3 hrs of time , when
traditional smear and culture tests are negative .

This PCR amplifies a specific DNA genomic sequence, whereby the presence of an
extremely small number of bacteria can be detected. The high sensitivity of PCR is
particularly useful in paucibacillary situations such as non-pulmonary tuberculosis
(TB).

To enumerate the pathological status of antigens in antibody positive status .