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Polymerase Chain Reaction   ( PCR )


Molecular pathology and Molecular techniques have revolutionized  in many areas of laboratory Diagnostics and have  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.


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    ( PLAIN VACUTAINER)

HSV – VIRAL LOAD

    SERUM / CSF/ SWAB FROM INFECTED SITE

CMV– VIRAL LOAD

     USE EDTA VACCUTAINER  -BUFFY COAT

DENGUE

(QUALITATIVE )

    PLASMA /SERUM (USE VACUTAINER)

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




To obtain Plasma For HIV  Quantitative ( Viral load ) estimation .


  • 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 )

  • Blood containing vacutainer  must be gently inverted seven – eight times to ensure proper mixing of specimen and the anticoagulant .

  • The vacutainer tube must be suitably labeled  indicating Name of The Patient ,
  •       age /sex and Identity  No .

  • Centrifuge the specimen at 1200g ( 3,500 rpm ) for 10- 15 mts .

  • 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  .

  • Transfer separated plasma to this vial with the help  of a plastic transfer pipette , after ensuring specimen is free from hemolysis and turbidity .

  • This plasma must be frozen and stored in the freezer compartment of the refrigerator  until time of packing and dispatch .

  • 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’DiagnosticCenter– 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.



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 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  was  developed  to increase both sensitivity  and Specificity of PCR .  It employs   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 .


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 .



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