Hepatitis B Virus (HBV) PCR Test Procedure (Cobas TAqman By Roche)

Principle:
This is based on the two major processes: (1) manual specimen preparation to extract HBV DNA ;( 2) automated PCR amplification of target DNA using HBV specific complementary primers and detection of cleaved dual fluorescent dye-labeled oligonucleotide probes that permit quantitation of HBV target amplified product (amplicon) and HBV Quantitation Standard DNA, which is processed, amplified and detected simultaneously with the specimen.

Precautions:

• The use of sterile disposable pipets and D-nase free pipet tips is recommended. 
• Do not pool reagents from different lots or from different bottles of the same lot.
• Wear eye protection, laboratory coats and disposable gloves when handling any reagent. Avoid contact of these materials with the skin, eyes, or mucous membranes. If spills of these reagents occur, dilute with water before wiping dry.

Procedure:

A. Specimen Preparation:

Centrifuge whole blood for 20 minutes at 800-1,600 x g within two (2) hours & transfer the plasma into  sterile 2 ml polypropylene microcentrifuge tube.

B. Reagent Preparation :

• Pipette 20 ml of 100% Ethanol to Inhibitor Removal Buffer (IRB). Mix by inverting 5-10 times. Thisis enough for 48 tests. Transfer 5 ml into clean, sterile tube per each 12 samples.
• Pipette 80 ml of 100% Ethanol to Wash Buffer (WASH). Mix by inverting 5-10 times. This is enough for 48 tests. Transfer 20 ml into clean, sterile tube per each 12 samples
• Prewarm ELUTION BUFFER (EB) at 70⁰C in 2 ml microcen tube. 

          12 tests= 2.0 ml EB; 24 tests= 4.0 ml EB 

• Pipette 5 ml (12 tests)/or 10 ml (24 tests) isopropanol into a clean tube 
• Prepare 8.25 ml Inhibitor Removal Buffer (IRB)  + 5 ml 100% Ethanol for 12 tests/33 ml of Inhibitor Removal Buffer (IRB) + 20 ml 100% Ethanol
• Prepare LYSIS/BINDING WORKING SOLUTION as follows: 

Reagent	12 Test	24 Test
Lysis/Binding Buffer (12 tests)	7 ml	14 ml
Carrier RNA	140 ul	280 ul
HBV C	39 ul	78 ul
Proteinase K	1.4 ml	2.8  ml

C. DNA Extraction 

• Pipette 625 ul of prepared Lysis binding/carrier RNA QS/Proteinase Working Solution to each well of Lysis rack (transparent). 
• Pipette 500 ul of sample (plasma). Vortex for 10 sec to mix. 
• Incubate rack at 50 0C for 10 minutes 
• Centrifuge for 10-20 sec 
• Pipette 250 ul Isopropanol to each well. 
• Mix by inverting the rack 3 X, then vortex rack for 10 sec 
• Centrifuge for 10-20 seconds 
• Place filter tube rack (yellow) onto a waste container (white) & mark position 1 on the rack for orientation. 
• Transfer 750 ul of lysate to the wells of the filter tube rack. 
• Centrifuge for 2 min at 4600 g. 
• Transfer remaining lysate (750 ul) to the corresponding well of the filter tube rack. Discard lysis rack appropriately. Centrifuge for 2 min at 4600 g. 
• Remove filter tube rack from waste container & replace with a new waste. Discard waste container.
• Pipette 400 ul of IRB to each well. Push lids. 
• Centrifuge for 2 min at 4600 g. 
• Pipette 700 ul Wash Buffer, push lids, centrifuge for 2 mins at 4600 g. 
• Remove filter tube rack from waste container & replace with a new waste rack. 
• Pipette 700 ul of Wash buffer, push lids, centrifuge for 3 min at 4600 g. 
• Remove the filter tube rack from waste container & discard waste rack. 
• Place filter tube rack onto the elution rack (blue) and snap both racks.
• Pipette 75 ul of pre-warmed Elution Buffer onto the center of each filter. 
• Incubate elution rack at room temp for a minimum of 3 min after adding the Elution Buffer to last well. Centrifuge for 3 mins at 4600 g Remove the filter tube rack from the elution rack & discard filter rack.
• Place the cover rack (IIIB blue) onto the elution rack (IIIA blue). Close lids.
• The processed specimens and controls are now ready for PCR.

Note: Add the process specimens and controls to the Working Master Mix within 3 hours of preparation. Or store at 2-8⁰C for up to 24 hours, or frozen at -20⁰C for up to 1 week in Sterile 2 ml polypropylene microcentrifuge tube.

D. Amplification:

• Equilibrate HBV MMX and CTM Mn2 at ambient temperature for 30 mins. 
• Place K-carrier in a K-carrier holder. 
• Place new K-tubes in K-carrier without touching the sides of the K-tubes. 
• Prepare the working MMX as follows: For 12 tests, remove 660 ul of HBV MMX and place in a 2 ml tube. 
• Add 90 ul of CTM Mn2, cap the tube and mix well by inverting 10 times. 
• Protect the working MMX from light and use within 2 hours. 
• Pipette 50 ul of working MMX into each K-tube. 
• Add 50 ul of each processed specimen and control to the tubes containing the working MMX. Gently mix each specimen or control up and down three times without generating bubbles. Cap the tubes and ready to load in the thermal cycler.

Note:  If fewer than 24 tubes are to be run, positions 1, 2, 5, 20, 23, and 24 must be occupied to balance the K-carrier in the thermal cycler.

1	2	3	4	5
6	7	8	9	10
11	12		13	14
15	16	17	18	19
20	21	22	23	24

                 K- carrier format