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

[Manufacturer’s Information]

Manufacturer’s Name

Senstech Diagnostic UK Limited

Address Peterborough, Cambridgeshire, United Kingdom

Web www.senstechdiagnostic.com