fast real time pcr hepatitis b virus detection kit (fluorescence pcr)...
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Fast Real Time PCR Hepatitis B Virus Detection Kit (Fluorescence PCR) Handbook
48 (Catalog No.101A0221EY)
Quantitative In Vitro Diagnostics
October 2019--Version 1
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[Proprietary Name]
SensTech® Fast RT-PCR HBV Test Kit
[Common or Usual Name]
HBV Fast RT-PCR Kit
[Intended use]
Fast RT-PCR HBV Test Kit, performed on SensTech®
QR S-200 Instrument is designed for the amplification
of hepatitis B virus (HBV) DNA in human serum or
plasma (EDTA) from HBV-infected individuals. The
test utilizes automated reverse transcriptase
polymerase chain reaction (RT-PCR) using
fluorescence probe to detect the DNA of interest.
Fast RT-PCR HBV test quantifies HBV over the range
of 15 to 100,000,000 IU/mL. SensTech® HBV Fast
RT-PCR test is intended for use as an aid in the
management of HBV infected patients undergoing
antiviral therapy. The test measures HBV DNA levels
at baseline and during treatment and can be utilized
to predict sustained and non-sustained virological
responses to antiviral therapy.
Results from SensTech® HBV Fast RT-PCR test may
also be used to confirm HBV infection in anti-HBV
positive individuals. In anti-HBV positive individuals
who test negative for HBV DNA, use of another anti-
HBV serological assay may be considered for
distinction between true HBV exposure and biological
false positivity.
Repeat HBV DNA testing may be indicated in cases
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that have HBV exposure in the last 6 months or have
clinical evidence of HBV disease.
[SensTech® QR S-200 Description]
SensTech® QR S-200 Instrument automate and
integrate specimen purification, nucleic acid
amplification, and detection of the target sequence in
simple or complex specimens using real-time reverse
transcriptase (RT- PCR) which uses fluorescence
probe to detect the DNA of interest. The systems
consist of fully automated instrument and preloaded
software for running tests and viewing the results. The
systems require the use of single- use disposable Fast
RT-PCR Test cartridges that hold the RT-PCR
reagents and host the RT-PCR processes. Because
the cartridges are self-contained, cross-
contamination between samples is minimized.
[Purpose of Usage]
This kit can be used for the quantitative detection of
Hepatitis B virus DNA (genotypes A-H) in plasma or
serum. The results are only for clinical
[Testing Principle]
This kit, includes RT-PCR tests cartridges developed
by using two pairs of specific primers and two pairs of
specific fluorescence probes, prepared with
components of PCR reaction solution, DNA
polymerases, and five kinds of nucleotide monomers
(d NTPs), adopts PCR amplification in vitro to detect
Hepatitis B virus DNA quantitatively.
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[Kit Composition]
FAST RT-PCR HBV TEST KIT 48 Tests
Fast RT-PCR Load cartridges with
integrated reaction tubes
48
Disposable 1 mL transfer pipettes 1 Box of 48 Fast RT-PCR
Tests Per Kit
Insert (includes instructions for use) 1
[Instrumentation]
PRODUCT NAME PRODUCT CODE
SensTech QR S-200 QR S-200
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[Storage]
Store at -20±5℃ temperature with a period of validity
of 12 months. [Applicable Instrument]
These kits are only dedicated for SensTech QR-S 200 machine
[Specimen collection, storage and transportation]
1 Applicable specimen type
Plasma (EDTA anticoagulant) or serum
2 Specimen collection
Plasma-Centrifuge the anticoagulant blood at 1000g
for 15min, and transfer supernatant (Plasma) to a new
tube.
Serum-MixPBScollectioncartridgebyinverting5-8times
immediately after blood collection, then let stand for
30 minutes, centrifuge at 1500g for10min.
3 Specimen Storage Serum and plasma samples may be frozen and thawed up to three times without loss of HBV. 4 Transportation Ship whole blood, plasma or serum specimens at 2–8 °C. Transportation of whole blood, plasma or serum specimens must comply with country, federal, state and local regulations for the transportation of etiologic agents
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[Values of Standards]
Values of Standards
S1 1,000 103
S2 10,000 104
S3 100,000 105
S4 10,000,000 107
[Detection steps]
1. Editing the PCR procedure
[SensTech QR-S 200 Operation](3.1-3.3)
1.1 SensTech QR-S 200 software. Click “New” to
create a new file. The file name will be
automatically created for the new experiment, or
user can edit it.
Select the “HBV” in the “projects” frame, select the
“HBV” in the “temple” frame. Click the “Finish” to
set up a new Thermal Cycling Protocol.
1.2 Click the “Plate” tab, edit the sample names.
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Select the sample type “standard” for HBV S1-S4,
and input the quantity numbers.
1.3 Save the PCR procedure when the start button
turn to green ( ), click it to run the test.
1.4 Create a new experiment
1.5 Select the FAM channel for HBV.
Select the VIC/HEX channel for internal control.
1.6 Enter the reaction volume (26μL).Edit Samples, enter the sample name, Type (Standard, Unknown, Positive, Negative), and Concentration of Standard in the “sample edit window”, save the file, run the program.
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[Result Analysis] SensTech QR-S 200 Operation (step 1-4) 1 When the test runs finished (about 50 min), the window
of SensTech QR-S 200 software will convert automatically.
2 Set Threshold value to 0.02. 3 Select sample well and click the “Result” tab. Select the
“medical” option in the “data” Frame. Then the result data will be displayed.
4 In the “Result” table, the “sample name” column will display the sample names (which has been selected and named in the step 3.2), the “item” column will display the detection target (HBV), the “test result” column will display the qualitative results, and the “quantity” column will display the quantity of the samples.
5 Analysis condition setting: According to the image after analysis to adjust the start value, end value of Baseline and Threshold value (the user can adjust according
To the actual situation, start value can be set at 1~10, end value can be set at 5~20, Threshold value can be selected at the range of 0.01 to0.2).
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6 Select Analyze in the Analysis menu for automatic analysis results.
7 Enter Report window. Record the values (Ct) and the quantitative results of unknown samples:
7.1 If the growth curve doesn't show as Steep curve or Ct value is ≥30, then judging the total content of HBV DNA of sample is less than lower limit of detection.
7.2 If the growth curve shows as S type curve and Ct value1E+8 IU/mL, then the total
content of HBV DNA of this sample is>1E+8IU/mL.
If you need accurate quantitative results, samples
after extracting can be diluted to the linear range
before testing.
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8.3 If quantitative result is
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[Quality Control]
Each test includes a Sample Volume Adequacy (SVA), Internal Quantitative Standard High and Low (IQS-H and IQS-L, also acts a specimen processing control [SPC]) and Probe Check Control (PCC).
Sample Volume Adequacy (SVA) – Ensures the sample was correctly added to the cartridge. The SVA verifies that the correct volume of sample has been added in the sample chamber. The SVA passes if it meets the validated acceptance criteria. If the SVA does not pass, an ERROR 2096 will be displayed if there is no sample or an ERROR 2097 will be displayed if there is not enough sample. The system will prevent the user from resuming the test.
Internal Quantitative Standard High and Low (IQS-H and IQS-L) – IQS-H and IQS-L are two Armored RNA® constructs in the form of a dry bead that goes through the whole assay process. The IQS-H and IQS-L are standards calibrated against the WHO 4th International standard for HBV. They are used for quantification by using lot specific parameters for the calculation of HBV RNA concentration in the sample. Additionally IQS-H and IQS-L detect specimen-associated inhibition of the RT-PCR reaction. The IQS-H and IQS-L pass if they meet the validated acceptance criteria.
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Probe Check Control (PCC) – Before the start of the PCR reaction, the SensTech Instrument System measures the fluorescence signal from the probes to monitor bead rehydration, reaction tube filling, probe integrity and dye stability. The PCC passes if it meets the validated acceptance criteria.
External Controls – External controls, not available in the kit, should be used in accordance with local, state, and federal accrediting organizations’ requirements as applicable.
All of the samples should show typical growth curve
of internal control (VIC channel).
These requirements need to be met in the same test at the same time. Otherwise, this experiment is invalid and should be repeated.
[Sensitivity of Detection]
According to clinical assessment results, the sensitivity of processed sample to kit is 15 IU/mL.
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[Precautions]
1 Please carefully read this instruction before experiment.
2 Laboratory management shall be strictly in accordance
with management standard of PCR gene amplification
laboratory, the experimenter shall undergo professional
training, experimental process shall perform under
strict partitions (reagent preparation area ,samples
preparation area, amplification and product analyzing
area), consumables used shall be disposable after
sterilization, each stage of experimental operation shall
use exclusive instrument and equipment, articles of
each area and each stage shall not be of cross using.
3 Provide biohazard safety equipment for reagent and
specimen preparation stage, one must wear coveralls
and disposable gloves, and use self-discharging tube
pipette during test.
4 Conduct quality control for experiment every time.
5 The PCR reagent shall be fully thawed before use,
but avoiding repeated freeze-cycles.
6 Extracting the substance insoluble in water in kit DNA,
sample injectors needed to mix uniformly for suction
when sampling. If failing to suction occurs due to the
pipette mouth is too thin or pipette blocking appears
after sampling, clean and non-polluting scissor can be
used to cut a section of the pipette mouth. Suction
heads used in specimen preparation area shall be
thrown into containers with disinfectant, and can be
discarded after being sterilized with wastes
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7 It shall use 10% hypochlorous acid or 75% alcohol
or ultraviolet to dispose the bench and pipette.
8 The positive quality control product in this kit shall be
deemed as having infectious substances, and the
operation and treatment shall both conform to the
requirements of relevant laws and regulations:
Ministry of Health General bio-safety standard for
microbiological and biomedical laboratories and
Regulations on the management of medical waste.
[Explanation of Symbols]
Expiration date
Batch code
Catalog number
Date of manufacture
Temperature limitation
Do not reuse
In vitro diagnostic medical device
∑
Contains sufficient for tests
Consult instructions for use
Do not use if package is damaged
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[Manufacturer’s Information]
Manufacturer’s Name
Senstech Diagnostic UK Limited
Address Peterborough, Cambridgeshire, United Kingdom
Web www.senstechdiagnostic.com