TITLE: Molecular Diagnosis of Viral Hepatitis B Infection
AUTHORS: Frank Twum Aboagye, Maame Ekua Acquah

[DESCRIPTION]
With over 400 million HBV infections, viral hepatitis B remains a global public health concern. Diagnosis is primarily based on an immunological assay approach, which utilizes the Hepatitis B surface antigen in detection amongst other markers. This method, however, has several limitations which include the inability to detect mutation in the viral genome resulting in diagnostic escape and low antigen titers in study samples. Using an alternative approach, which is the molecular diagnostic technique such as real-time polymerase chain reaction (RT-PCR) would circumvent the limitations of immune detection. This protocol, thus, provides a step-by-step process of HBV diagnosis using RT-PCR which is a sensitive tool for diagnosis. The steps involved include sample collection and preparation, nuclei acid isolation, HBV detection and quantification using RT-PCR as well as the interpretation of results.
This protocol combines the high nuclei acid yield from isolation using Zymo Quick DNA Mini-prep Kit and Bosphore HBV Quantitative Kit for amplification of HBV DNA. The Bosphore HBV Quantitative Kit has a low detection limit of 1×101 IU/ml and with a turn-around time of less than 4 hours when combined with Zymo Quick DNA Mini-prep Kit. The nucleic acid isolated in this protocol can be amplified using the Bosphore HBV Quantitative kit on several thermocyclers, which makes the protocol robust, cost-efficient, and cost-effective in resource-scarce areas.

[BEFORE_START]
Sample Collection and Preparation
Aseptically collect venous blood from the antecubital vein of the forearm and dispense it into either a serum separator tube (SST), a plain tube (red top), or an EDTA tube (plasma).
Centrifuge the specimen at 3500 rpm for 10 minutes and transfer the serum or plasma into a sterile plain tube or a cryovial.

NB:
For samples collected into SST or plain tubes, allow the specimen to stand vertically undisturbed for at least 1 hour for the specimen to clot completely before centrifuging it to yield serum
Samples collected into EDTA tubes can be centrifuged immediately to yield plasma.
Care should be taken during sample collection and processing to avoid haemolysis of the specimen

Handling of PCR Reagents
Allow the PCR Master Mix and the other components of the Bosphore HBV Quantification kit to thaw completely at 4°C before use and avoid centrifuging to thaw.

[GUIDELINES]
Reagent Preparation for Nucleic Acid Isolation
Nucleic Acid Isolation
Detection and Quantification of HBV Nucleic Acid
Results and Interpretation
Troubleshooting

[STEPS]
SECTION: HBV Nucleic Acid Isolation
2. Transfer 200 µL of   to a sterile 1.5 mL microcentrifuge tube (pre-labeled).

SECTION: HBV Nucleic Acid Isolation
3. Add 5 µL of Internal Control (provided with the amplification kit) to each specimen and vortex for 30 s

SECTION: HBV Nucleic Acid Isolation
4. Add 400 µL of Genomic Lysis Buffer and 10 µL of Proteinase K to the sample.

SECTION: HBV Nucleic Acid Isolation
5. Vortex and incubate the sample at 56 °C for 3 - 5 hours or 300 min .

SECTION: HBV Nucleic Acid Isolation
6. Vortex the sample at   for 30 s 
SECTION: HBV Nucleic Acid Isolation
7. Transfer the entire content of the 1.5 ml microcentrifuge tube into a Zymo-Spin IIC Column in a collection tube.
SECTION: HBV Nucleic Acid Isolation
8. Centrifuge at 10000 rpm for 1 min. Discard the flow-through liquid.

SECTION: HBV Nucleic Acid Isolation
9. Transfer the Zymo-Spin IIC column into a new collection tube.
SECTION: HBV Nucleic Acid Isolation
10. Add 200 µL of DNA Pre-Wash Buffer to the spin column and centrifuge 10000 rpm for 1 min. Discard the flow-through liquid.

SECTION: HBV Nucleic Acid Isolation
11. 10.  Add 500 µL of gDNA Wash Buffer to the spin column and centrifuge at 10000 rpm for 1 min. Discard the flow-through liquid.
SECTION: HBV Nucleic Acid Isolation
12. 11.  Transfer the Zymo-Spin IIC Column into a sterile 1.5 mL microcentrifuge tube
SECTION: HBV Nucleic Acid Isolation
13. 12.  Add 100 µL of DNA Elution Buffer to the spin column and incubate at Room temperature  for 30 min

SECTION: HBV Nucleic Acid Isolation
14. 13.  Centrifuge at 13000 rpm for 30 s to elute the DNA.

SECTION: HBV Nucleic Acid Isolation
15. 14.  Store the Viral Nucleic acid at -20 °C pending further analysis.

SECTION: RT-PCR  Detection and Quantification of HBV Nuclei Acid
16. The PCR is done in a 25 µL reaction as described below:

 


SECTION: RT-PCR  Detection and Quantification of HBV Nuclei Acid
17. The reaction is done under the following cycling conditions:

 

SECTION: RT-PCR  Detection and Quantification of HBV Nuclei Acid
18. Select the appropriate fluorophore depending on the analyzer been used. For Bio-Rad CFX 1000 Series, select the FAM dye for HBV and HEX for Internal Control (IC).
 



SECTION: Results and Interpretation
21. 
Samples that cross the threshold in the FAM channel are displayed with their starting quantities and the corresponding Ct-values. Samples that do not cross the threshold are displayed as “No Ct” or “ –”

SECTION: Results and Interpretation
22. Possible outcomes from the PCR:
 

20. Initiate the protocol
SECTION: Troubleshooting
23. *If the sample has a viral load <10 IU/ml and no Internal Control amplification, it requires that the assay is repeated once. Freeze-thaw[freeze the sample (temperature ≤ -10°C) for 20 minutes and defrost at room temperature for 10 minutes]  the nucleic acid sample and dilute it with nuclease-free water in a ratio of 1:2. Use the diluted nucleic acid for repeating the assay

** Repeat the assay once taking into consideration possible pipetting errors in the first assay.

If both repeat as indicated above fail to yield the expected/better results, a new specimen should be collected from the patient and the process from nucleic acid isolation to amplification should be repeated with the new sample.


SECTION: Reagent Preparation for Nucleic Acid Isolation
1. Add 500 µL of β-mercaptoethanol to 100 mL of Genomic Lysis Buffer
 


SECTION: RT-PCR  Detection and Quantification of HBV Nuclei Acid
19. Identify unknown samples (test samples), standards, positive control, and negative control on the plate as described below. Assign the right quantitative values to the standards