One-step Detection Kit for Hepatitis B virus (QPCR-Probe)
This kit is used for the qualitative detection of Hepatitis B Virus
(HBV) in whole blood and serum samples. It offers auxiliary means
for the diagnosis of the HBV infected patients without nucleic acid
extraction and purification. Test results should be combined with
This kit adopts PCR method combined with fluorescence probe in
vitro amplification technology. In this method, the HBV probe
contains a fluorescent reporter dye FAM at the 5'end of the probe
and a quencher dye BHQ at the 3'end of the probe. When the probe is
intact, the proximity of the reporter dye to the quencher dye
suppresses the reporter fluorescence. Probe cleavage during the PCR
reaction spatially separates the reporter dye from the quencher
thereby allowing detection of the reporter dye fluorescence. The
fragments of reporter dye are displaced from the target, resulting
in an increase in fluorescence. This step, which enables the
fluorescence signal accumulation and PCR products formed
synchronous, thus to achieve qualitative detection the HBV in the
infective patient in serum samples, which provide auxiliarymeans
for the HBV infection in the treatment of the patient.
HBV-PCR Rection Mix: Specific premiers and fluorescent probe of
HBV, qRT-PCR Master mix.
HBV Positive Control: Pseudovirus with HBV NP gene established in
vitro, Internal recombinant plasmid
Sample Dilution Buffer: PBS buffer
Note: Different batches in the kit components should not be
Analysis sensitivity: 20 copies/rxn.
• All reagents should be stored at below -20°C. Storage at +4°C is
• All reagents are valid for 6 months, can be used until the
expiration date indicated on the kit label.
• Repeated thawing and freezing (>4x) should be avoided, as this
may reduce the sensitivity of the assay.
• Cool all reagents during the working steps.
• Super Mix should be stored in the dark.
0.2 mL PCR Tube:
(1)Axygen® 0.2mL Polypropylene PCR Tube Strips (Product
#PCR-0208-C) with PCR 1 x 8 Strip Flat Caps (Product #PCR-2CP-RT-C)
/ (Product #PCR-02-FCP-C).
(2)Axygen® 0.2mL Thin Wall PCR Tubes with Flat Cap(Product
#PCR-02-L-C)/ (Product #PCR-02-C)
1. Sample Preparation/Treatment
Whole blood sample (Sodium citrate anticoagulation) or serum sample
2. Preparation of amplification reagent
Take out the HBV-PCR Rection Mix, Positive Control and Negative
control, centrifuge the sample after thawing. Add 48μl HBV-PCR Mix
into each reaction tube.
3. Adding samples and controls to the reaction tubes
• Separately add the samples from step 1, positive control and
negative control to different tubes:
1) Separately add the 2μL serum sample or 0.5 μL whole blood sample
from step1 to HBV reaction tube.
2) Separately add 2μL positive control to another HBV reaction
tubes positive control.
3) Separately add 2μL negative control to another HBV reaction tube
as negative control.
Close the tube, mix thoroughly and spin down the mixture. Operation
should be performed on the ice-bath. 2000rmp spin for 10sec. Put
the reaction tube on the test instrument.
Note: to avoid the difference from the serum sample, it is
recommend to use the serum sample which is clear. For the cloudy
sample, if the first test result is negative, please dilute the
sample to 4-10 folds using the dilution buffer provided in the kit
to detect again.
4. PCR Amplification
Perform the following protocol in the instrument:
Selection of fluorescence channels:
530nm channel (ReporterFAM): HBV
Please refer to the instrument manual for specific channel set
If the Ct value of positive control sample≤25, and the Ct of the
negative control is 0 or>28, this test is valid,or the test is
a.If the Ct of sample ≤28, report positive for the HBV;
b.If the Ct of sample 30>ct>28, please test again. If the
duplicate detection Ct of sample>28, negative control Ct value
is none, reported positive for the HBV. If the duplicate detection
Ct of sample is 0 or none, reported negative for the HBV; c. If the
Ct of sample is no value or>28, report negative for the HBV.
[Limitations of the assay]
1. Detection result of this kit is only for clinical reference,
clinical diagnosis and treatment to patients should be considered
other factors as symptoms, medical history, other laboratory tests
and therapeutic reaction.
2. False positive result is easy to caused by the contamination of
amplification product and cross-contamination of specimens.
3. Negative result could not means the patient is non-infected, the
details should be combined with other clinical findings to
determine specific diagnosis. The reason lead to false negative
① Unreasonable sample collection, transportation and treatment, low
viral titer in a sample
② Variation of virus detection target sequences
③ Unauthenticated other factors such as taking antiviral drugs
④ Infections caused by other viruses or bacteria.