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Brief User Manual

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HBV/4DR 9G testTM

HBV/4DR 9G testTM

Overall workflow overview

PCR1

Hybridization2

Washing3

Scan & Save result4

After PCR, detection of HBV and 4 drug-related Mutationssimultaneously in just 30 min !

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HBV/4DR 9G testTM

Molecular Genetic Assay for Identification of the Hepatitis B Virus and itsResistance to Lamivudine, Telbivudine, Adefovir and Entecavir from ClinicalSpecimens.

A. Intended Use

The HBV/4DR 9G testTM is a qualitative, PCR-based, line probe assay in-vitro diagnostic test for the identificationof the Hepatitis B Virus and its Resistance to Lamivudine, Telbivudine, Adefovir and Entecavir from clinicalspecimens. The detection of 5 most significant mutations in four codons associated the viral reverse transcriptase(RT) gene allows the identification of drug-related resistance.The HBV/4DR 9G testTM is intended for use with specimens from patients for whom there is clinical suspicion ofHBV infection. Use of the HBV/4DR 9G testTM for the detection of Hepatitis B Virus (HBV) or determination of 4drug susceptibility has not been validated for patients who are receiving treatment for HBV infection.

B. Summary and Explanation

Globally, 240 million people are infected with HBV and 780,000 people died every year from the complicationsof HBV1. Current treatment for the HBV infection consists of two major classes: Interferon-based therapy and oralnucleotide analogues. In spite of interferon’s therapeutic advantages, their severe side effects such as anxiety,depression and anemia has limited their therapeutic uses. Although nucleotide analogues have significant lowerside effects, long term treatment with treatment is required. Determining the rate of the development of resistancefrom clinical experience varies considerably depending on the agents. The one year resistance rates forlamivudine and telbivudine are 30 % and 15 %, respectively. When used individually, entecavir and adefovirhave higher barriers of resistance, with rates less than 2 % after two years. No resistance to tenofovir has yet beenreported. After five years of treatment, the rates of resistance were about 70 % for lamivudine, 28 % for adefovir,less than 1% for entecavir, 63-65 and 0 % for tenofovir and interferon2.Hepatitis B is a potentially life-threatening liver infection caused by the hepatitis B virus. It is a major global healthproblem. It can cause chronic infection and puts people at high risk of death from cirrhosis and liver cancer.However, it can be prevented by currently available safe and effective vaccine. For effective control of HBV, earlydiagnosis of infection is important for rapid and appropriate treatment.

C. Principles of the Procedure

The HBV/4DR 9G testTM is based on the line probe assay. The whole procedure is divided into three steps: [1]DNA extraction from clinical specimens - as well as aseptic fluids should be used for the PCR (-the necessaryreagents are not provided), [2] PCR amplification with Cy5-labled primers, and [3] hybridization.All reagents needed for amplification such as polymerase and primers and are optimized for this test. Themembrane strips are lined with specific probes complementary to the amplified nucleic acids. The amplified nucleicacids bind to the probes (hybridization). To obtain final results, the strip is scanned by BMT 1D scanner™, which isa lightweight portable device.The primers in the HBV/4DR 9G testTM amplify a portion of an 321-bp region of the viral RT gene corresponding tocodons rt169 to rt250, which codes for the reverse transcriptase of hepatitis B virus. The probes allowdiscrimination between the conserved wild-type sequence and 5 mutations in the four codons (rt181V, rt204V,rt204I, rt236T, and rt250V) of RT region at 25 °C in less than 30 min after PCR.

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D. Reagents and Instruments

1. Material Provided

HBV/4DR 9G testTM contains sufficient reagents to process 48 patients.Following are the contents of each kit

2. Storage and Handling• Store Reagent kit at -20 oC or below• The rest of the contents, 9G Membrane tests and contents box can be stored at 18 to 30 oC.• Store all constituents strictly separated from contaminating DNA.• Do not use the reagents beyond their expiry date.

E. Materials required but not provided

• DNA extraction kit (commercially available kits are recommended)• Disposable gloves• Disposable aerosol-resistant DNA/Dnase-free pipette tips• DNA/Dnase free microtubes• Microtube racks• Microcentrifuge (capable of carrying 1.5 ml and 2 ml microcentrifuge tubes)• Vortex mixer• 60-65 °C incubator or water bath• Isopropanol• DNA thermal cycler and equipment• Pipettes adjustable to deliver 1 - 20 ul, 20 - 200 ul, and 200 - 1000 ul• DNA/Dnase-free deionized/distilled water (PCR grade)• Timer• Discard containers• Waste containers• 15 ml sterile graduated conical tubes• Labels or marking pen

Kit Contents Amount Shelf life

A. HBV/4DR 9G testTM 1 box

A-1. HBV/4DR 9G test Strip 48 strips 12 months

A-2. Hybridization Solution 1 bottle (6 ml) 12 months

A-3. Washing Buffer 1 bottle (20 ml) 12 months

A-4. Tris Buffer 1 bottle (20 ml) 12 months

B. Reagent Kit 12 months

B-1. PCR Premix 1 kit (48 tubes) 12 months

B-2. HBV/4DR 9G test Primer set 2 tube (30 tests/tube) 12 months

C. User manual 1

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F. Laboratory cleaning and maintenance1. Materials needed

• 0.1 N sodium hydroxide solution (household bleach)• Spray bottles• Protective equipment - gowns/laboratory coats/mask/gloves• Paper towels• Discard containers for used tips, microtubes• Waste containers for used strips

2. Formulae for cleaning agentsFresh cleaning reagents should be prepared on a daily basis and should not be stored in diluted form becausetheir activity will diminish with time. Label all prepared reagents with name and date of preparation.

3. 0.1 N NaOH solutionSodium hydroxide is widely accepted for cleaning, sanitizing. The benefits of its use include efficacy, low cost,ease of detection, removal, and disposal. Sodium hydroxide has been shown to be effective in removing proteinsand nucleic acids. It is also effective for inactivating most viruses, bacteria, yeasts, and endotoxins.

4. Cleaning procedures:The laboratories should be cleaned before and after work in the manner described.a. Prepare fresh cleaning solutions.b. Put on protective clothing, mask and gloves.c. Spray 0.1 N NaOH solution on the bench tops and PCR equipment, wait at least 10 min and wipe all

surfaces with clean paper towel (All surfaces and equipment within the laboratory should be regarded as potentially infectious and should be cleaned).

d. Clean micropipette and all racks with 0.1 N NaOH solution.e. Spray gloves with 0.1 N NaOH solution before work.f. Spray all used pipette tips, microtubes and strips with 0.1 N NaOH solution right after finishing each step

and discard in a respectively designated container for biohazardous waste.h. Racks for molecular testing should be cleaned after use by immersion in 0.1 N NaOH solution. i. Mop the floor with 0.1 N NaOH solution. j. Record all cleaning and maintenance in the Laboratory Cleaning and Maintenance Logbook.k. Remove lab coats, wash hands, and do not reenter pre-amplification room after working until following

morning.

G. Warning and precautionsTreat all biological specimens, including used strips, as if capable of transmitting infectious agents. Because it is

often impossible to know which might be infectious, all biological specimens should be treated with standardprecautions. Guidelines for specimen handling are available from the U.S. Centers for Disease Control andPrevention and the Clinical and Laboratory Standards Institute (formerly National Committee for ClinicalLaboratory Standards).

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• Wear protective disposable gloves, laboratory coats and eye protection whenhandling specimens and reagents. Wash hands thoroughly after handlingspecimens and test reagents.

• Follow your institution’s safety procedures for working with chemicals andhandling biological samples.

• Each HBV/4DR 9G test strip is used to process one test. Do not reuseprocessed strips.

• Check your regional/country hazardous and medical waste disposalrequirements. If regulations (or lack thereof ) do not provide clear direction onproper disposal, biological specimens, including used strips, should be treatedas capable of transmitting infectious agents. Dispose used strips as hazardoushealth-care waste in durable waste containers.

• WHO [World Health Organization] medical waste handling and disposalguidelines.

• Dispose of used strips according to your institution’s and country’s safetyguidelines for hazardous material.

• Discard used pipette tips, microtubes immediately after use in a container forbiohazardous waste. After finishing the assay, discard all used disposables ina container for biohazardous waste.

H. Specimen Requirements

HBV-positive or -negative patient specimens such as blood and serum can be used as starting material for DNAextraction.

1. Precautions for handling specimens

Patient specimens must always be considered as potentially infectious and must be handled. Always wearsuitable protective clothing and gloves. Samples from risk patients (infected by pathogenic microorganismsincluding Hepatitis B and Human Immunodeficiency Virus (HIV)) and cultures made from those samples mustalways be labeled an handled under suitable safety conditions according to institutional guidelines.

2. Storage and transport

Store and transport specimens at 2 to 8 °C prior to processing whenever possible. The transport of specimens atcold(2 to 8 °C) temperature has to be carried out as soon as possible and should be done within 1 day. Freshblood samples can be stored at -20 °C or -80 °C for a maximum of 5 days until performing DNA extraction.

I. DNA Extraction

The working area must be free from contaminating DNA. For DNA extraction from blood, the commercially availa-ble DNA extraction kit is used according to manufacturers protocol. Please refer to the respective instructions foruse. There are many kits and processes available for the DNA Extraction from blood. You can use any of the ava-ilable for methods. However, for precise analysis, Using the smallest elution buffer is highly recommended followingprotocol DNA extraction kit. *eg.) 30 ul elution buffer for 200 ul of serum sample.

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J. Amplification

Notes before starting• In order to avoid DNA contamination, a physical

separation between the pre- and post-amplificationsteps is recommended: separate rooms, separatepipettes and other lab material, separate lab coatsand gloves (and their stock) are minimum precautionsfor prevention of contamination and as a part ofgood laboratory practice.

• The reagents should be isolated from any source ofcontaminating DNA, especially amplified DNAproducts. Also avoid microbial contamination ofreagents.

• All pipette tips and tubes used for the amplificationprocess should be autoclaved. Aerosol-resistantpipette tips are recommended.

• The reagents for amplification processes should behandled in a room free of DNA.

• Slowly release plunger of the pipet so that thesample is correctly drawn into the pipet tip.

• In order to avoid the contamination, the DNA extract-ion and the HBV/4DR test including PCR should be done in the separated rooms. The person involved in making PCR products should not be involved in the extraction step and PCR preparation step on the same day.

• Avoid any return of materials from post-amplificationroom to the pre-amplification room.

1. PreparationImportant note: Avoid unnecessary delays in thesetup of the run.

d. PCR conditions: Make sure the PCR tubes arevortexed 10 sec before positioning in the PCRmachine and amplification profile is as follow.*This protocol can be used with most commercialtypes of PCR machine.*It should take less than17~19s from denaturation (94 oC) to annealing(57 °C). If it takes longer than 21s, please useanother PCR machine.

e. After this process, use amplification products imm-ediately.

PCR condition

Temp(℃) Time(sec) Cycle

94 300 1

94 15

7053 10

72 20

72 300 1

4 ∞

Reagents Volume

HBV/4DR 9G test Primer set 10 ul

Extracted DNA sample 10 ul

PCR Premix Freeze-dried

Total mixed volume 20 ul

Volumes of Reagents Required for PCR

a. Dissolve HBV/4DR 9G test Primer set in 300 ul ofoffered Tris buffer, then vortex 4 times (each 15sec). *The dissolved Primer set can be stored at 4oC and consumed within 4 weeks. (avoidrepetitive freeze-and-thaw)

b. Transfer 10 ul of dissolved HBV/4DR 9G testPrimer set into PCR premix. *Before using primerset it should be vortexed 4 times (each 15 sec)Important note: It is very important to use thecorrect amount of each component.

c. 10 ul of DNA template is added into the PCRPremix tube with Primer then spin down brieflythe reagents. *Only one tube cap should beopened and make sure other caps are closedduring DNA transfer to avoid contamination.

The reagent volume for each reaction should be pipetted as follows:

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K. Hybridization

Note before starting• Maintain temperature at 23~26 °C and humidity of

20~60 % for hybridization and washing process.• Higher humidity over 70 % may cause false results.• Keep the washing bottle in designated area after use.• The “Test zone” should not be touched by hand.

1. Put appropriately sized aluminum foil on the table.

2. To prepare the HBV/4DR test strips (A-1) needed,tear the pouch to take out strips and then arrangethem with sample port upward as depicted in imagebellow on the aluminum foil. It should be preparedright before the use for the sake of convenience.To minimize the errors, only 24 tests should be doneat a time.

3. Marking sample ID # on the HBV/4DR 9G test strip (A-1).Open the pouches to take out the HBV/4DR test stripsand mark them according to the sample ID # on the capof the PCR tubes you are going to load on the strip.*The pouches should be opened right before useand only 24 tests should be done at a time.

4. Sample preparation (Hybridization solution + PCR prod-uct): add 110 ul of Hybridization solution in each PCRtube containing PCR products and wait for 5 min.

5. Load 110 ul of mixture of PCR product and hybridizationsolution (A-2) on the sample port of HBV/4DR 9G teststrip and wait for 20 min.

12345678

Test zone

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Mixture of PCR product and Hybridization solution

110 ul

Hybridization solution

110 ul

PCR products

3. Washing: Load 200 ul of washing buffer(A-3) slowly in washing port and wait for 8 min.

L. Scanning

1. Press the power button (button on the right) for 3 sec to turn on the BMT 1D scanner™.

2. Double click “HBV 4DR Chip Analyzer V4.2”icon on the monitor. 3. Insert the strip in the strip holder.4. Click “Start Scan” button on the program or press the scan button (the middle button) on the scanner.

*For best result, the test strip must be scanned within 4 min after washing.

BMT 1D scanner™

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Washing buffer

M. Disposal

1. The used tips, tubes should be disposed in 0.1 N NaOH solution right after the test in the plastic bag and seal

(knot) it.

a. Spray all used pipette tip with 0.1N NaOH solution right after finishing each step and dispose them in plastic bag and seal (knot) the bag.

b. Spray all used tubes with 0.1N NaOH solution right after finishing each step and dispose them in plastic bag and seal (knot) the bag.

2. Spray all used strips and aluminum foil with 0.1N NaOH solution right after finishing each step and dispose them in plastic bag and seal (knot) the bag.

1 2

1 2

1 2 3

3. Discard in designated containers for biohazardous waste. (or follow your institution’s safety procedures for working with chemicals and handling biological samples.)

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N. Results, save data & report fileFinal results will appear right after scanning process finishes.

Save data & report file

1. Making folderMake a folder named “HBV 4DR data”. In the folder,make folders named dates such as “20130506”

(yyyymmdd) to save data. (Shown as side figure)

\ HBV 4DR data \ 20130506

*Result 1. HBV positive/Negative with no drug resistance *Result 2. HBV positive with Lamivudine or telbivudine resistance

*Result 3. HBV positive with Adefovir resistance *Result 4. HBV positive with Entecavir resistance

181V 204V 204I 236T 250V

Lamivudine+

+ +

Telbivudine +

Adefovir

+

+

+ +

Entecavir + +/- +

Drug-resistant related mutation table

*Multiple mutations can be founded in one test

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HBV 4DR

HBV/4DR

ReadoutLMV

(Lamivudine)

LdT(Telbivudine)

ADV(Adefovir)

ETV(Entecavir)

TDF(Tenofovir)

204V and/or I + 250V X X O X O

2. Labeling patient listCopy the patient list from reference folder/patient listand Paste it in date folder such as 20130506 folderon the upper left. According to sample number R1~24,make a patient list with name and ID (see Patient list onthe left). Save the list such as patient list (R1~R24)below.

If you have more than 24 patients each day pleasecopy the list such as patient list (R25~48), patient list(R49~72), patient list (R73~96), patient list (G1~24)and etc. and do the same action for patient list(R1~R24). Save patient list (R1~24)

3. Making report form “R1~24” fileCopy the Sample No (R1~24) from referencefolder/Sample No and Paste it in date folder such as20130506 folder on the upper left. The file with 24identical pages is designed to put each data report ineach page.Caution: The sample No listed in each page of thereport form is to check that you are pasting the correctpatient information on each page. Please check thepatient information you are pasting is matching withthe sample No on the report form.

Patient list

R1~24 file

4. Copy of patient informationNow open both patient list file and Sample No files.Copy patient information and paste it in separatepages of Sample No file one by one.

5. After scanning, Final results will appear right afterscanning process finishes.

6. Save Graph on the report formIn order to attach the data directly on the report file,window press the “Alt” key and “prt sc” keytogether(In case of desktop) or “Alt” key and “prt sc”key and “Fn” key together together(In case of laptop)and to paste on the report form click on the positionfor the result to be passed and press the “ctrl” keyand the “V” key together. And do the same action forthe next samples.

HBV/4DR result Report HBV/4DR result Report

HBV/4DR result Report

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After the results pasted on the results form, save the file before you do the same action in the next page for thenext sample.

7. Save report fileAfter all data attached in each page file in “HBV 4DR report form”, save it.

After attachment of R1~24 data in the report formsave it and all data automatically saved on theR1~24 file.

Caution: Before you run the test train yourself for thecopy and paste the result window for 24 individualscan and save the file. Check the file is filled withcorrect data at least you are confident on thecopying and pasting the result window on thecorrect report form with correct patient information.

Check the patient information whenever you pastethe result window on the file.

HBV/4DR result Report

HBV/4DR result Report

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O. Troubleshooting Guide

Symptoms Possible Solution

Insufficient volume made for reaction mixes • Verify the correct reaction mix volumes were added to each well.

Poor DNA yield • Verify that proper procedure was followed for DNA extraction.

• Verify sufficient elution volume during DNAExtraction

• Repeat DNA extraction from the specimen.• The DNA pellet was lost during isopropanol

precipitation. Use extreme care when removing theisopropanol to avoid losing the pellet.

• The sample was too old. Best yields are obtainedwith fresh sample. Samples that have been storedat 2-8 oC.

No Target signal displays results • Be sure all samples, reagents and reaction mixesare mixed thoroughly.

• When adding reaction mix to each well, place tipsat the bottom of the well and slowly pipette up anddown

• Verify all liquid is expelled from the pipette tipduring additions.

• Verify the correct reagent was added to each well.• Verify the correct reagent volumes were added to

each well.

Unexpected or contradiction result: Suspected contamination

• Use nuclease-free aerosol barrier tips and steriletubes when making the reaction mixes.

• Wear gloves when setting up the test.• Make sure that pipette tips touch only the solution

being dispensed.• Do not touch pipette tips with hands.• Clean lab surfaces using appropriate materials• Verify proper procedure was followed for

“Laboratory cleaning and maintenance” before andafter you work.

• Only open respective tube and keep close the othertubes during the transfer of extracted DNA to PCRtube.

• PCR machine ramping time (94 oC to 60 oC) shouldtake 17~19s. If it takes longer than 21s, pleasefind another PCR machine.

• Verify proper procedure was followed for “Dispose”(refer to page 10)

False results • Higher humidity over 70 % may cause false results.

Abnormal high positive rate: Suspected contamination during sample addition.

Check Negative sample• Negative sample preparation: follow:

“Amplification” page 7, put same amount of Trisbuffer instead of DNA template.

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※ If you do not follow protocol and caution, we can not ensure right result.

In case of further problems, please contact the technical support at BMT:Email: BMTsupport@bmtchip.comTel : 82-033-258-6097Fax : 82-033-258-6099

P. Analytical Sensitivity / specificity

For determination of analytical sensitivity of the HBV/4DR 9G testTM, 24 replicates of different concentrations (105,104, 103, 200, 10 copies/test, respectively) of HBV DNA spiked into negative clinical samples using each of thesix HBV Wild, rt181V, rt204V/I, rt236T and rt250V controls, and analyzed with the HBV/4DR 9G testTM . A limitdetection was determined to 200 copy /test with this methods. For determining the specificity, samples containingother mutations such as rt169T, rt184G, rt202I, rt214A/s, rt180M, rt180A with rt181V/I, rt180M with rt181V/I,were tested with 105 copies/test. There were no cross-reactivity with the probes indicating the target-specificity of theprobes used.

Q. Referneces

1. WHO, medica centre, Hepatits B. 2015. http://www.who.int/mediacentre/factsheets/fs204/en/2. Nidhi Gupta, Milky Goyal, Catherine H. Wu and George Y. Wu. J. Clin. Tansl. Hepatology. 2014. 2, 202-211.

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