Quantitative and Genotyping Test for Hepatitis B Virus

• In Vitro Diagnostics/ User Manual  
• Code No.: GT-NTB002

Contents

• Intended use:
• Preface:
• Name and function:
• Principle of the test:
• Reagent provided in kit,
• Storage and stability:
• Other materials required, but not provided:
• Precautions for users:
• Specimen preparation and storage:
• Collection: Transport: Preparation:
• Test procedures:
• Pre-amplification:
• Amplification:
• Post-amplification:
• Result Interpretation:
• Quantification:
• Genotype determination :
• Performance validation:
• Troubleshooting:

INTENDED USE

For in vitro detection, quantitative determination and genotype differentiate into A, C, E, G and B, D, F groups of hepatitis B viral DNA in human serum.

PREFACE

Hepatitis B is a disease caused by viral infection. The route of infection can be an improper needle puncture or blood transfusion. Hepatitis B (HB) has become a significant problem for public health management. Almost one in every ten adults, who has been infected by hepatitis B virus (HBV), develops some forms of chronic liver diseases and becomes a long-term carrier of HBV. Serological markers are commonly used as diagnostic indicators of acute or chronic HBV infection. The most common marker of HBV infection is the presence of HBV surface antigen (HBsAg), whereas HBV e antigen (HBeAg) is generally used as a secondary marker to indicate active HBV replication associated with progressive liver disease. But due to strains variance and detection sensitivity, the use of this marker to monitor HBV disease progression is limited.

The quantification of viral DNA in serum or plasma is the most direct measurement of viral infection and replication. Combined with serological test, it provides the most reliable and sensitive way to detect HBV infection.

NAME AND FUNCTION

HBV-QG is a molecular diagnostic kit used for screening and quantitative test of HBV existence in serum. Also, HBV-QG is capable of differentiating virus genotype into two genotype groups: A, C, E, G, and B, D, F. Using HBV-QG diagnostic kit to monitor the viral replication in patients during therapy, providing a valuable tool for physician to treat patient with interferon therapy and evaluate the efficacy of anti-viral drug.

PRINCIPLE OF THE TEST

The kit is intended for the in vitro detection and quantitative measurement of Hepatitis B virus DNA in human serum or plasma utilizing Polymerase Chain Reaction (PCR) nucleic acid amplification and hybridization probes technology. The Primer and Probe Mix contain primer pairs and hybridization probes specific for HBV target/standard and internal control. The HBV Standard is a non-infectious DNA molecule containing primer and probe binding sites same as HBV target. The quantification of HBV DNA is performed with an External Standard Curve of serial diluted HBV Standard generated with every run. The log value of copy number of each standard is plotted against the Crossing Point (Cp) value for each standard to generate a standard curve. HBV DNA levels in the sample specimens are determined by comparing the Cp for each specimen to the standard curve generated with each run. Using hybridization probe as detection method, the genotyping of HBV can be achieved by the melting curve analysis. A single mismatch between the hybridization probe and its target sequence can drastically change the melting temperature of the bound probe. Therefore, different HBV genotypes can be distinguished by the melting peaks at different temperatures.

The internal control (IC), which is a special designed non-infectious DNA molecule, is incorporated into each standard and specimen at known copy number and is carried through all amplification and detection steps. Therefore, IC can work as an indicator for PCR performance. If the PCR is proceeded normally, the Cp of IC should be larger than zero. Only two circumstances will result in the Cp of IC is zero, one is the PCR is blocked by inhibitor; the other is strong positive specimen. Due to the IC is competitive with target DNA in the same reaction tubes. Therefore, when specimen is strong positive, Cp of IC may be zero or large than zero; on the contrary, if specimen is negative, IC should be large than zero. The information of IC is valuable to verify the negative result is due to negative of HBV DNA (true negative) or negative of PCR amplification(false negative by inhibitor).  The fluorescence of target and IC can be determined by switching channel from F2/Back-F1 to  F3/Back-F1.

REAGENT PROVIDED IN KIT, STORAGE AND STABILITY

OTHER MATERIALS REQUIRED, BUT NOT PROVIDED

1. Latex gloves, powderless

2. Microcentrifuge

3. 1.5 ml DNase-free centrifuge tubes

4. 10µl, 20µl, 200µl micropipettes and disposable DNase-free tips with aerosol barrier

5. Viral DNA Extraction Kit (QIAamp DNA Blood Mini Kit, Cat. No. 51106, or Roche High Pure Viral Nucleic Acid Kit, Cat. No. 1 858 874, are recommended)

6. Roche LightCycler capillaries (Cat. No. 1 909 339)

7. Roche LightCycler centrifuge adapters (Cat. No. 1 909 312)

8. Roche LightCycler instrument (Cat. No. 2 011 468)

9. Roche LightCycler Color compensation set (Cat. No. 2 158 850)

PRECAUTIONS FOR USERS

1. This reagent kit is for in vitro diagnosis only.

2. This reagent must not be used after expiration date.

3. Do not pool reagents from different lots or from different bottles of the same lot.

4. Nucleic acids are very sensitive to environmental nuclease degradation. Nucleases are present on human skin and on surfaces or materials handled by human. Clean and cover work surfaces with disposable pads and wear powder-free gloves when performing all test steps.

5. To protect reagents be contaminated from microbial, the tests is suggested to be performed in PCR grade laboratory.

6. Post-amplification area should be kept as far as possible from Pre-amplification area to avoid aerosol contamination.

7. Specimens must be stored separately from reagents so as not to contaminate open reagents.

8. Positive control is made of selected partial sequence from HBV. For safety, it must be treated as infectious material.

9. Do not smoke, eat or drink in work areas, or pipet by mouth.

10. Latex gloves and lab coat must be worn when handling reagents or specimens and must be changed before leaving an area. Wash hands thoroughly afterwards.

11. To avoid cross contamination between specimens and reagents, workflow in the laboratory must proceed in a uni-directional manner from the Pre-amplification area to Post-amplification area.

12. Thoroughly clean and disinfect all work surfaces with 5% sodium hypochlorite.

13. Before disposal, all waste materials should be properly disinfected by autoclave for at least one hour at 121.1°C or incineration.

14. To performing dual-color detections with both LightCyclerRed 640-labeled and LightCyclerRed 705-labeled Hybridization Probes in a single capillary, color
compensation must be done to avoid the fluorescence residual crosstalk between channels.

SPECIMEN PREPARATION AND STORAGE

Collection

The kit is for use with serum or plasma specimens only. Serum may be collected in a Serum Separator tube. Whole blood should be collected into sterile EDTA or CITRATE blood collection tubes. Specimens anti-coagulated with heparin are unsuitable for this test. Within 6 hours of blood draw, blood collection tubes must be centrifuged at 3000 rpm for 10 minutes at room temperature to collect serum or plasma. Transfer serum or plasma to sterile polypropylene tube with evident label. The serum specimen may be stored for up to 3 days at 2-8°C. For longer-term storage, the specimen should be frozen at -20°C or colder. It is recommended that specimens be store in 250µl aliquots in sterile, 2ml polypropylenecrew cap tubes.

Transport

Transportation of whole blood, serum or plasma must be complied with country, federal, state and local regulations for the transport of etiologic agents. Whole blood must be transported at 2-25°C and processed within 6 hours of collection. Serum and plasma may be transported at 2-8°C or frozen.

Preparation

Purify target DNA from 200µl sample serum by using commercial viral DNA extraction kit, such as QIAamp DNA Blood Mini Kit or Roche High Pure Viral Nucleic Acid Kit. Please carry out the DNA extraction according the manufacturer’s instructions. Elute the HBV viral DNA by adding 50 µl of DNase-free H2O.

For the improvement of sensitivity, 500µl to 1000µl of sample serum can be utilized to purified the viral DNA according the instructions of extraction kit, and elute the viral DNA by at least 20µl of DNase-free H2O.

TEST PROCEDURES

Pre-amplification:

Before preparing the reaction mix, make sure that the Cooling Block as well as capillary adapters (accessories of the LightCycler. instrument) are pre-cooled to 4°C. All required reagents, specimens and controls need to be thawed completely prior to beginning the test.

1. Mix all reagents well. Master Mix and HBV Pb by repeated tipping for gently mix and others by brief vortexing.

2. Briefly centrifuge all the reagents.

3. Prepare the reaction mix by multiplying the amount in the “volume” column by the number of reactions to be cycled, plus one additional reaction. Please make sure that at least five quantification standard (HBV QS1~5) as well as one no template control (PCR grade water) are included per PCR run.

4. Place the desired number of LightCycler. capillaries into the adapters of the Cooling Block.

5. Mix reaction mix gently. Pipette 10 µl of the reaction mix into the plastic reservoir of each capillary.

6. Add 10 µl of the eluted sample DNA to each capillary. Correspondingly, 10 µl of the serial dilutions of HBV standards (HBV QS1~QS5) as well as 10 µl of water are added directly to each capillary as standards and negative control, respectively.

7. Close the capillaries and centrifuge at 400 x g (2000 rpm) for 5 seconds to transfer the mixture from the plastic reservoir of the capillary into the glass tube.

8. Incubate the mixture for 5 min at room temperature for UNG decontamination reaction.

Amplification:

1. Place the capillaries in the carousal of the LightCycler. instrument and close the lid.

2. Cycle the samples as described in the following.

3. When the program is completed, dispose of PCR reaction capillaries and waste in accordance with country and local regulations.

4. The program runs for approximately 1.5 hour.

Open the pre-stored color compensation file that compensates fluorimeter channels
interference by activating the Choose CCC File.

Program 1: Denaturation

Program 2: Amplification

Program 3: Melting

Program 4: Cooling

None Post-amplification:

After PCR program has finished, separate signals can be analyzed by switching channel from F2/Back-F1 for target HBV DNA to F3/Back-F1 for the internal control in data analysis screen.

Select program to amplification, then click on quantification to analysis screen. Choose Second Derivative Maximum method for quantification of HBV DNA. For genotype determination, select program to melting, then click on melting curve to analysis screen. Choose Polynomial for Calculation Method. For detailed information about data displayed procedures, please follow the instructions given in LightCycler Operator’s Manual.

RESULT INTERPRETATION

Quantification

The sensitivity of kit is 1000 copies/ml serum.

Due to specimen DNA concentration calculation depends on the starting volume of sample serum and the H2O volume user added during the extraction to elute specimen DNA, user may concentrate or dilute it by adding less or more H2O to elute, but a further calculation is needed to obtain the original concentration. (Moreover, if you use automatic machine to isolate DNA, the nucleic acid recovery rate also should be considered.) The standard procedure is to elute HBV DNA from 200 µl serum by adding 50 µl H2O, thus the final concentration is 4-fold of the original. Therefore, the original concentration of specimen can be obtained by dividing calculated concentration by 4.

Using five external standards to build up a standard curve. All 5 quantification controls should be used and defined in the Sample Loading Screen as standards with the specified concentration (copies/ml). The log value of each external standard copy number is plotted against the Cp value for each standard to generate a standard curve. HBV DNA levels in the test specimen are determined by comparing the Cp for each specimen to the standard curve generated with each run. The concentration of HBV DNA of each specimen is shown in the calculated concentration column on the left of the Quantification and Melting Curves Screen. Determination of qualitative and quantification results can follow the rules listed below:

1. While the LC-Red 640 Cp value is larger than 0 AND fluorescence value is large than 0.005, the specimen is positive. Virus copy numbers of test specimen can be determined automatically by showing in the calculated concentration column.

2. While the LC-Red 640 Cp value is 0 AND according LC-Red 705 Cp value is NOT 0, or the LC-Red 640 fluorescence value is lower than 0.005 AND according LC-Red 705 Cp value is NOT 0, the specimen is negative.

3. While the LC-Red 640 Cp value is 0 AND according LC-Red 705 Cp value is 0, the result is invalidation. Please dilute the DNA specimen and do it again. (dilution factor must be calculated)

4. If LC-Red 640 Cp value of specimen is large than 42, or less than 12, or amplification curve of specimen is not standard sigmoid curve, please do it again.

5. The dynamic range of Cp value in this kit is from 12 to 40 (5x107 to 5x101 copies/rxn, 5x1010 to 5x103 copies/ml), the quantification results are more reliable within this region.

Genotype determination

The genotype is determined by the melting peak (Tm) of each HBV specimen. The automatic Tm calculation can be provided in Peak Areas tool; also it can be obtained in Manual Tm tool. This kit is designated to differentiate HBV into two genotype groups: A, C, E, G, and B, D, F which melting peak of each group is 54.8°C ± 1.8°C and 60.9°C ± 1.8°C, respectively. The HBV standard provided in this kit is genotype B for reference.

A HBV genotype panel can be obtained by us as below

PERFORMANCE VALIDATION

The performance of each run can be validated by the information of standard curve and NTC. The standard curve and NTC should be qualified by following criteria:

1. The linearity of five external standards detected by PCR system must be larger than 0.95 (r = -0.95), or else the result is invalidation.

2. The slope is representing the overall reaction efficiency. To achieve efficiency for the standard curve between 1.75(87%) and 2.10(105%), the slope has to be between -4.1 and -3.1, or else the result is invalidation.

3. The LC-Red 640 (F2/Back-F1) Cp value of negative sample or NTC should be 0 and according IC LC-Red 705 (F3/Back-F1) Cp should be larger than 0, or else the result is invalidation.

4. In strong-positive sample, the IC LC-Red 705 (F3/Back-F1) Cp may be 0 or larger than 0.

HBV Genotype Panel (Code No: HRP-B02)

A panel consisting of 1 ml samples from 15 individuals and representing the 6 genotypes of Hepatitis B (A-F). Viral load is reported using results from both the Roche Amplicor assay and NGI SuperQuant asssay. Genotypes were determined by Consolidated laboratory Services. All samples confirmed positive for HBsAg by the Abbot Auszyme assay and confirmed negative for antibodies to HIV 1 & 2, HCV and HIV antigen by FDA licensed assays.