|Pro HBV Mix
|HBV *QS1(1×105 IU/µl)
|HBV *QS2(1×104 IU/µl)
|HBV *QS3(1×103 IU/µl)
|HBV *QS4(1×102 IU/µl)
|Water for Molecular Biology
Molecular diagnostic tests based on nucleic acids utilizing polymerase chain reaction are high- sensitive and high-specific methods for detecting Hepatitis B virus in blood samples. Before PCR reaction, HBV antigen and present antibody can be identified by ELISA. Either in positive case or possible false-negative results, then, quantitative PCR test will be necessary, as all diagnostic accuracy, drug resistance, and illness severity depend on the accurate and effective estimation of blood virus load. Real Time-PCR is the most convenient method for following estimation of isolated DNA and RNA concentration based on external standard control standard curve, which allows the quantification of HBV-specific DNA in a sample. Accordingly, a blank sample (here conserved sequence of HBV genome) is estimated based on a standard curve of homologous DNA 9 with distinct concentrations (here amplified HBV). The main advantage of the provided kit is coverage of a wide range of concentrations, as high concentrated HBV is assessable without necessary elution. Thus, HBV diagnostic kit should be able to diagnose HBV virus specifically and detect various strains of hepatitis virus efficiently. Viga Quantitative HBV Molecular Diagnostic Kit is based on Real Time-PCR technology, utilizing polymerase chain reaction (PCR) amplify of HBV specific target sequences and fluorescently labeled target-specific probes to amplify DNA. In addition to the HBV DNA specific amplification and detection system, inter/intra assay, linear range, and standard control, the present kit contains oligonucleotides for the amplification and detection of the IC, which restrict probable inhibitions during PCR reaction. This item can be added either at the begging of the purification procedure to control the whole process (involving isolation and PCR) or just in the duration of the PCR reaction, which can knock down PCR inhibitors. Noticeably, parallel control test has neither any cross-reaction nor effect on HBV virus or human genome or other types of common virus existing in human blood.
Importantly, Viga Quantitative HBV Molecular Diagnostic Kit can be operated in vitro hepatitis B virus DNA amplification and then the quantitative determination of DNA content. This kit is designed to be operated in Rotor-Gene 3000, Rotor-Gene 6000 and Rotor-Gene Q.
- Reliable HBV DNA detection and quantitation with high sensitivity and specificity across all genotypes A–H.
- Highly sensitive detection of as few as 40 IU/ml.
- Broad linear range from 100 to more than 1,000,000,000 IU/ml.
- Accurate quantitation using the 5 standards supplied.
- Non-competitive internal control to monitor extraction and PCR efficiency
- Specificity, no cross reaction with HAV, HCV, HEV and HIV
Storage and sample transportation
- Transport samples under specific precaution procedure for pathogens and make sure transportation should not exceed six hours.
- All samples must be transported at 2 – 8 °C and plasma samples at -20°C.
- Whole blood should be separated into plasma and cellular components by centrifugation at 1200-1600 rpm for 20 min. Transfer extracted plasma into sterile Eppendorf.
- Avoid freezing blood samples as the assay’s sensitivity can be reduced if you freeze the samples as a matter of routine or store them for a longer period of time.
- After extraction, the isolated Hepatitis B virus encapsulated DNA is stable for up to 14 days if stored at +4°C, for 12 weeks if stored at -20°C, and up to one year when stored at -70°C.
Take out each component from the kit and place them on bench top. Allow the reagents to equilibrate to room temperature, then briefly vortex each tube for later use.
For viral nucleic acid isolation, use DNall VirAll Kit (REF: DN983053), DNjia Virus DNA kit DN983056 ) or other kits with confirmation of ministry health.
Take out each component from the kit and place them on bench top. Allow the reagents to equilibrate to room temperature, then briefly vortex each tube for later use. The volume of isolated sample in this test should be 10µl. Prepare PCR reaction refer to Table 1 and then perform Realtime PCR refer to Table 2.
Table 1: Regent’s preparation per one single reaction (DNA isolation efficiency and PCR inhibition is controlled by adding internal control in purification stage).
|Pro HBV Mix
Table 2: PCR program for HBV
Annealing and Extension
Annealing and Extension and acquisition on channel Green and Yellow
Interpretation of results
Quantification Standards in Viga Quantitative HBV Molecular Diagnostic Kit use a standard panel consisting of identified concentrations of HBV DNA. In preparation of both sample and standard concentrations, 10µl of isolated DNA should be added to the 15µl Master Mix. Quantification Standards are used to generate the standard curve, which allows the quantification of HBV-specific DNA concentration in the sample. Enter qualification standard in Rotor Gene-specific software just in IU/ml. Follow provided formula to convert IU/µl by which kit contents are supplied.
Result (IU/ml) =
If the volume of whole plasma were 200µl and elution 50µl, the first standard would be 2.5×107 IU/ml entered in Rotor Gene-specific software.
Validity of a Diagnostic PCR Run
A diagnostic PCR Run is valid if met the following control condition:
Table3: Control conditions for a valid PCR Run
|VIC™ (Internal Control)
|FAM™ (HBV target)
(Std / Pos)
|NTC negative control
- Notice that all reagents may exclusively be used in in-vitro diagnostics.
- The product is to be used by personnel specially instructed and trained in the in-vitro diagnostics only.
- Strict compliance with the user manual is required for optimal PCR results.
- Attention should be paid to expiration dates printed on the box and labels of all components. Do not use expired components.
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