robogene quantification of hepatitis c virus (hcv)...

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Complete Solutions for BIO-Quantification

RoboGene Quantification of Hepatitis C Virus (HCV) Genomes

5’ untranslated region

Rev.1

Quantification of Hepatitis C Virus (HCV) Genomes

5’ untranslated region

for use with ABI PRISM® 7700/7000/7300 SDS; Gene Amp® 5700 SDS, iCyclerTM HCV Control RNA Cat.No.: 0203000101 HCV Real-Time Reagent Mix Cat.No.: 0204000401/02 Sample RNA Strips Cat.No.: 0205000101/02

for use with Rotor-Gene (Corbett Research) HCV Control RNA Cat.No.: 0203000111 (0.1 ml tubes) 0203000141 (0.2 ml tubes) HCV Real-Time Reagent Mix Cat.No.: 0204000401/02 Sample RNA Strips Cat.No.: 0205000111/12 (0.1 ml tubes) 0205000141/42 (0.2 ml tubes)

for use with LightCyclerTM (Roche) HCV Control RNA Cat.No.: 0203000131 HCV Real-Time Reagent Mix Cat.No.: 0204000431/32 Sample RNA Strips Cat.No.: 0205000131/32

for use with SmartCycler® (Cepheid) HCV Control RNA Cat.No.: 0203000151 HCV Real-Time Reagent Mix Cat.No.: 0204000401/02 Sample RNA Strips Cat.No.: 0205000151/52

For general laboratory use and for research use only!

Trademarks Roboscreen®, RoboGene, RoboStrip®, RoboZym, Roboscreen, BetaPrion, and TRIPLEHYB® are registered marks of the AJ Roboscreen GmbH. The Roboscreen® technology of the “Intelligent tube” technology is covered by patent and patent application DE 198 40 531 and WO 0012756. The TRIPLEHYB® technology is covered by a German and US patent pending application. ABI, ABI PRISM® , and GeneAmp®, FAM , VIC and TAMRA are registered trademarks of the Applera Corporation. BHQ-1 is a registered mark of Biosearch Technologies Inc. Yakima Yellow is a registered trademark of Epoch Biosciences. iCycler™ is a registered trademark of Bio-Rad Laboratories, Rotor-Gene is a trademark of Corbett Research. LightCycler™ is a registered trademark of the Idaho Technology Inc., SmartCycler® is a registered mark of Cepheid. GeneAmp® and AmpliTaqGold® are registered marks of Roche Molecular Systems, Inc. SuperScript™ and Platinum® are registered trademarks of Invitrogen Life Technologies. BIOMEK® is a registered mark of Beckman Instruments, Inc. Some of the applications which may be performed with this product may be covered by applicable patents in certain countries. The purchase of this product does not include or provide a license to perform patented applications. Users may be required to obtain a license depending on the country and/or application. AJ Roboscreen does not encourage the unlicensed use of patented applications.

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Contents Page 1. Introduction 3 2. Specificity 3 3. General remarks 3 4. Storage and stability 4 5. Additional reagents not contained in the modules and equipment required 4 6. Sample material and controls 4 7. Experimental Procedure 6 7.1. Quantification of HCV using ABI PRISM 7700/7000/7300 SDS, Gene Amp 5700 SDS or iCycler instruments 6 7.2. Quantification of HCV using Rotor-Gene 2000 or 3000 instrument 9 7.3. Quantification of HCV using LightCycler instrument 12 7.4. Quantification of HCV using SmartCycler instrument 16 8. Initial concentrations of the ready-to-use HCV RNA controls 19 9. Preparation of data 19 10. Specificity, sensitivity and precision of the test 20 11. Trouble Shooting 21 12. Carry-over prevention using UNG (optional) 22 Warranty Notice 22 References 22 How to contact AJ Roboscreen 22 Appendix 1: Recommended amplification conditions for different 96-well format

real-time instruments 23 Appendix 2: Calculation scheme for the preparation of a HCV reaction

mix for use with ABI PRISM 7700/7000/7300 SDS; Gene Amp 5700 SDS, iCycler 24

Appendix 3: Calculation scheme for the preparation of a HCV reaction mix for use with Rotor-Gene 25

Appendix 4: Calculation scheme for the preparation of a HCV reaction mix for use with LightCycler 26

Appendix 5: Calculation scheme for the preparation of a HCV reaction mix for use with SmartCycler 27

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1. Introduction

Based on more than 10 years of experience AJ Roboscreen realized the idea to develop and applicate innovative new products for standardized automated sample preparation and high-throughput quantitative analysis of low-copy nucleic acids for medical diagnosis based on the industrial standard of the future: the “intelligent reaction vessel”. Intelligent vessels mean that commonly used glass capillaries or plastic tubes required for either standardized nucleic acid extraction or quantification are already precoated with defined amounts of distinct reference DNA or RNA, respectively, and are manufactured by a novel technology platform covered by several national patents and international patent applications. New properties like ready-to-use applicability, storage and delivery even at room temperature, highly reproducible data recovery, etc. are making this technology highly convenient even for beginners in molecular diagnostics1,2.

Meanwhile, more than 50 quantitative kits, reagents and reference materials for molecular tumor, virus and food analysis has been developed, validaded, manufactured and distributed under the RoboGene brand. In addition, AJ Roboscreen has exclusive access to novel nucleic acids purification technologies, in vitro DNA and RNA synthesis, and calibration as well as stabilization of complete sample preparation and detection reagent mixtures containing all required reaction components. The provided assay exploits the principle of quantitative real-time fluorescence PCR/RT-PCR. The increase in fluorescence is directly proportional to the specific target amplification during PCR.

2. Specificity

Hepatitis C virus infection accounts for the majority of post-transfusion and sporadic hepatitis. There is a high frequency of progressive chronic hepatitis. The RNA genome contains a highly conserved 5’ and a 3’ untranslated region exploited by most detction kits3. Different HCV isolates show a high sequence heterogeneity4. Genotypes 1,2 and 3a are being most frequently found in western countries5. The RoboGene Quantification Module for Hepatitis C virus (HCV) Genomes is designed for the in vitro quantification of HCV RNA genomes via the 5’ untranslated region and tested with the following genotypes: 1a, 1b, 2a, 2b, 2c, 2i, 3a, 4, 5a and 6 as checked with a reference panel obtained from the Referenzzentrum für Retroviren, Universitätsklinikum Essen. 3. General remarks

The test is developed for general in vitro and research use only and are optimized for ABI PRISM 7700/7000/7300 SDS, Gene Amp 5700 SDS, Rotor-Gene or LightCycler. The use of other real-time instruments like iCycler or ABI PRISM 7900 SDS real-time instruments may require additional optimization.

For running each experiment always one 8-well-ready-to-use control RNA strip and a distinct number of sample strips are required. Perform the test according to the general “Good Laboratory Practice” (GLP) guidelines as laid down e.g. by the German DIN-Norm 58967-60 or Ref.6 for diagnostical applications of PCR. Ready-to-use virus copy controls contain units of 8 tubes or capillaries each, storage-stable coated with 8 different amounts of calibrated virus control, including amplification enhancer. They are manufactured using a patented technology developed by AJ Roboscreen. HCV Real-Time Reagent Mix contains

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respective primers and probe. PCR-grade water and passive reference dye are provided with separate vials. Sample strips are units of 8 tubes or capillaries each, coated with amplification enhancer only. Always use RNA controls (PCR quantification standard) together with the corresponding sample RNA strips and reagent mixes. 4. Module storage and stability

After delivery at room temperature store AJ Roboscreen control RNA and sample strips at –20°C. Protect Real-Time reagent mix, lyophilized always from light and store at –20°C in the dark! Under these conditions reagents are usually stable for at least 6 month.

Prepared 25x or 20x reagent mixes, respectively, should be stored at 4-8°C in the dark. Under these conditions mixes are stable for at least 2 weeks.

All reagents not contained in the test kit should be stored according to the respective manufacturer’s recommendations. 5. Additional reagents not contained in the modules and equipment

required • SuperScript III Platinum One-Step Quantitative RT-PCR System (Invitrogen), Cat.No.:

11732 • Real-time PCR instrument • Suited pipetting tools: recommended is an automated pipetting workstation, e.g.

BIOMEK 2000 (Beckman Instruments) particularly for high throughput analyzes (Ref. 1,6)

• 1.5 ml reaction tubes (e.g. Eppendorf-AG, Hamburg) • Barrier tips or Mastertips (e.g. Eppendorf) for manual pipetting • Compression pad (Applied Biosystems, Cat.No.: 4312639) for the use with real-time

instrument supporting the 96-well format 6. Sample material and controls

All kinds of sample material suited for PCR amplification in terms of purity, concentration, and absence of inhibitors can be used.

Always run at least one negative control with the samples. To prepare a negative control, replace the template RNA sample with PCR-grade water.

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Recommended nucleic acids extraction kits for use together with AJ Roboscreen quantification modules:

Virus detection in blood or cell culture supernatants • INSTANT Virus RNA Kit (AJ Innuscreen GmbH, AJ Roboscreen GmbH), up to

150 µl of sample

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7. Experimental Procedure 7.1. Quantification of HCV using ABI PRISM

7700/7000/7300 SDS, Gene Amp 5700 SDS or iCycler instruments

Contents of RoboGene quantification modules Table 1: Module contents Content Preparation of the 25x

reagent mix HCV control RNA, similar to the 5`untranslated region, HCV control RNA strips, coated with 8 different amounts of HCV Control RNA and amplification enhancer

Cat.No.: 0203000101 12 strips and 2 optical covers

HCV reagent mix, lyophilized, containing HCV primers and HCV probe (5’ FAM), PCR-grade water is supplied in separate vials, for use with SuperScript III Platinum One-Step Quantitative RT-PCR System Cat.No.: 0204000401 (100 reactions) • HCV reagent mix, lyophilized

3 tubes

add 40 µl PCR-grade water per tube and incubate at 37°C for 20 min, vortex and centrifuge briefly

• 10x passive reference dye solution 1x 0.5 ml • PCR-grade water, DEPC-treated 2x 1.5 ml Cat.No.: 0204000402 (500 reactions) • HCV reagent mix, lyophilized

15 tubes


• 10x passive reference dye solution 5x 0.5 ml • PCR-grade water, DEPC-treated 10x 1.5 ml Sample RNA strips, coated with amplification enhancer only Cat.No.: 0205000101 Cat.No.: 0205000102

12 strips and 2 optical covers

5x 12 strips and 7 optical covers

Manual

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Experimental protocol To maintain full activity of the enzyme perform all pipetting steps on ice or on an ice-cold PCR rack! Preparation of reagent mixes and samples 1. For preparation of the “25x reagent mix” please refer to Table 1. 2. Remove one strip from the storage base coded as “control RNA strips” and load at a

suited reaction base for 0.2 ml reaction tubes. Always wear gloves when handling the tubes! Caution! Make sure that the strip is plugged in the correct order into the reaction base to recode highest and lowest standard concentration!

3. Remove the required number of “sample strips” from the storage base and plug the

tubes into the free positions of the reaction base. Carefully remove the adhesive aluminum lids from the strips using tweezers.

4. Preparation of final mastermix: prepare an unique mix for both the control and sample

reactions as shown in Table 2. 5. Add appropriate aliquots of the unique mastermix to each well of the control and sample

strips considering the volume of sample to be added.

Caution! Do not exceed the final volume of 25 µl. 6. After manual or automated dispensing of the mastermix, add equal aliquots of PCR-

grade water to the desired control tubes and purified RNA sample exclusively into the desired sample tubes.

7. Tailor an optical cover according to the number of used wells and seal the tubes

carefully. Centrifuge at 200 x g for 1 min (1000 rpm) in a standard benchtop centrifuge. To avoid leakage during amplification, tightly press on the cover and use a compression pad (Applied Biosystems, Cat.No.: 4312639) following the instructions of the manufacturer! Always wear gloves when handle the tubes and covers!

8. Select the wells that contain the controls (“Standards”), non-target controls (“Blank”) and

samples (“Unknown”) from the sample type pop-up menu. Enter a number or text that identifies sample or control and enter a number that represents the quantity of the controls (see Table 13). Choose the dye “FAM” from the dye layer pop-up menu.

9. Setup the amplification conditions as shown in Table 3.

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Table 2: Pipetting scheme for the preparation of mastermix, calculated to

achieve a final PCR reaction volume of 25 µl per tube.

Reagents

HCV Mastermix [µl]

1 reaction 100 reactions ➋ final conc.

PCR-grade water ➊ ➊ -

10x Pasive reference dye Solution ❹ 2.5 250

2x Reaction mix (buffer containing RT-PCR buffer, 6 mM Mg-sulfate and dNTP) ➌

12.5 1250 1x 3.0 mM Mg-sulfate

Mg-sulfate (50 mM) ➌ 1.0 100 2.0 mM

Resulting final Mg-

sulfate concentration 5.0 mM

25x reagent mix (containing primers and probe) 1.0 100 1x

SuperScript III RT/ Platinum Taq mix ➌ 1.0 100

➊ Volume depends on the intended volume of RNA sample and the usage of passive reference dye solution; supplement to achieve a final volume of 25 µl. Calculation scheme see Appendix 2. ➋ Indicated volumes sufficient to prepare one 8-well control strip run and 88 sample runs; including pipetting reserve. For calculation of reagent volumes needed to run an intermediate number of samples please use table of Appendix 2. ➌ Reagent not contained in the module; component of the SuperScript III Platinum One-Step Quantitative RT-PCR System (Invitrogen), Cat.No.: 11732 ❹ Passive reference dye solution only required for usage of ABI PRISM or GeneAmp instruments (Applera Genomics/Applied Biosystems)

Table 3: Thermal cycle program using ABI PRISM 7000 or 7300 SDS (Applied

Biosystems), for further real-time instruments application see Appendix 1.

RT HOLD CYCLE

shut off « 9600 emulation » (window « instruments »)

Temperature (°C)

Time (min:s)

Cycles

59

60:00

95

2:00

95 57

00:30 01:30

40 - 45

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7.2. Quantification of HCV using Rotor-Gene 2000 or 3000 instrument

Contents of RoboGene quantification modules Table 4: Module contents Content Preparation of the 25x

reagent mix HCV control RNA, similar to the 5`untranslated region, HCV control RNA strips*, coated with 8 different amounts of HCV control RNA and amplification enhancer Cat.No.: 0203000111 96 tubes 0.1 ml

and 96 caps

Cat.No.: 0203000141 96 tubes 0.2 ml HCV reagent mix, lyophilized, containing HCV primers and HCV probe (5’ FAM), PCR-grade water is supplied in separate vials, for use with SuperScript III Platinum One-Step Quantitative RT-PCR System Cat.No.: 0204000401 (100 reactions) • HCV reagent mix, lyophilized

3 tubes


• PCR-grade water, DEPC-treated 2x 1.5 ml Cat.No.: 0204000402 (500 reactions) • HCV reagent mix, lyophilized

15 tubes


• PCR-grade water, DEPC-treated 10x 1.5 ml Sample RNA strips*, coated with amplification enhancer only Cat.No.: 0205000111 Cat.No.: 0205000112

96 tubes 0.1 ml and 96 caps

480 tubes 0.1 ml and 480 caps

Cat.No.: 0205000141 Cat.No.: 0205000142

96 tubes 0.2 ml 480 tubes 0.2 ml

Manual *) each strip consists of eight 0.1 ml or 0.2 ml tubes

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Experimental protocol To maintain full activity of the enzyme perform all pipetting steps on ice or on an ice-cold PCR rack! Preparation of reagent mixes and samples 1. For preparation of the “25x reagent mix” please refer to Table 4. 2. Remove one strip from the storage base coded as “control RNA tubes” and load at a

suited reaction base for 0.1 ml or 0.2 ml reaction tubes, respectively. Always wear gloves when handle the tubes! Caution! Make sure that the strip is plugged in the correct sequence order into the reaction base !

3. Remove the required number of “sample tubes” from the storage base and plug the

tubes into the free positions of the reaction base. Carefully remove the adhesive aluminum lids from the tubes using tweezers.

4. Preparation of final mastermix mix: prepare an unique mix for both the control and

sample reactions as shown in Table 5. 5. Add appropriate aliquots of the unique mastermix to each control and sample tubes

considering the volume of sample to be added.



7. Seal the tubes with the provided caps. Always wear gloves when handle the tubes

and caps! 8. Select the wells that contain the controls (“Standards”), non-target controls (“Blank”) and

samples (“Unknown”) from the sample type pop-up menu. Enter a number or text that identifies samples or controls and enter the quantity of the controls (see Table 13). Choose the dye “FAM” from the dye layer pop-up menu.

9. Setup the amplification conditions as shown in Table 6.

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Table 5: Pipetting scheme for the preparation of mastermix sufficient to run one complete rotor (72-well or 36-well, respectively), calculated to achieve a final PCR reaction volume of 25 µl per tube.

HCV mastermix [µl] Reagents

1 reaction 80 reactions ➋

40 reactions ➌ final conc.

PCR-grade water ➊ ➊ ➊ -

2x Reaction mix (buffer containing RT-PCR buffer, 6 mM Mg-sulfate and dNTP) ❹

12.5 1000 500 1x 3.0 mM Mg-sulfate

Mg-sulfate (50 mM) ❹ 1.0 80 40 2.0 mM

Resulting final Mg-sulfate

concentration 5.0 mM

25x Reagent mix (containing primers and probe)

1.0 80 40 1x

SuperScript III RT/ Platinum Taq mix ❹ 1.0 80 40

➊ Volume depends on the intended volume of RNA sample; supplement to achieve a final volume of 25 µl. Calculation scheme see Appendix 3.

➋ Indicated volumes sufficient to prepare one 8-well control strip run and 64 sample runs; including pipetting reserve for use with 72-well rotor and 0.1 ml tubes. For calculation of reagent volumes needed to run an intermediate number of samples please use table of Appendix 3.

➌ Indicated volumes sufficient to prepare one 8-well control strip run and 28 sample runs; including pipetting reserve for use with 36-well rotor and 0.2 ml tubes. For calculation of reagent volumes needed to run an intermediate number of samples please use table of Appendix 3.

❹ Reagent not contained in the module; component of the SuperScript III Platinum One-Step Quantitative RT-PCR System (Invitrogen), Cat.No.: 11732

Table 6: Thermal cycle program using Rotor-Gene

RT HOLD CYCLE

Temperature (°C)

Time (min:s)

Cycles

59

60:00

95

2:00

95 57

00:15 01:00

45

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7.3. Quantification of HCV using LightCycler instrument Contents of RoboGene quantification modules Table 7: Module contents Content Preparation of the 20x

reagent mix HCV control RNA, similar to the 5`untranslated region, HCV control RNA strips*, coated with 8 different amounts of HCV control RNA and amplification enhancer Cat.No.: 0203000131 96 capillaries and

96 stoppers

HCV reagent mix, lyophilized, containing HCV primers and HCV probe (5’ FAM), PCR-grade water is supplied in separate vials, for use with SuperScript III Platinum One-Step Quantitative RT-PCR System Cat.No.: 0204000431 (100 reactions) • HCV reagent mix, lyophilized

3 tubes


• 10x BSA solution 1x 0.5 ml • PCR-grade water, DEPC-treated 2x 1.5 ml Cat.No.: 0204000432 (500 reactions) • HCV reagent mix, lyophilized

15 tubes


• 10x BSA solution 5 x 0.5 ml • PCR-grade water, DEPC-treated 10x 1.5 ml Sample RNA strips* coated with amplification enhancer only Cat.No.: 0205000131 Cat.No.: 0205000132

96 capillaries and 96 stoppers

480 capillaries and 480 stoppers

Manual *) each strip consists of eight capillaries

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Experimental protocol To maintain full activity of the enzyme perform all pipetting steps on ice or on an ice-cold PCR rack! Preparation of reagent mixes and samples 1. For preparation of the “20x reagent mix” please refer to Table 7. 2. Remove one strip from the storage base coded as “control RNA capillaries” and load

into the precooled capillary adapters. Always wear gloves when handle the capillaries! Caution! Make sure that the strip is plugged in the correct order into the adapters to recode highest and lowest standard concentration!

3. Remove the required number of “sample capillaries” from the storage base and plug the

capillaries into the free adapters. Carefully remove the adhesive aluminum lids from the strips using tweezers.

4. Preparation of final mastermix: prepare an unique mix for both the control and sample

reactions as shown in Table 8. 5. Add appropriate aliquots of the unique mastermix to each control and sample capillary

considering the volume of sample to be added.


grade water to the desired control capillaries and purified RNA sample exclusively into the desired sample capillaries.

7. Seal each capillary with a stopper and place the adapters containing the capillaries into

a standard benchtop microcentrifuge. Centrifuge at 700 x g for 5 s (3000 rpm) in a standard benchtop microcentrifuge.

8. Carefully place the capillaries in the rotor of the LightCyler instrument. Select the

positions containing the controls (“Standards”) samples (“Unknown”) and non-template control (“NTC”) from the sample type pop-up menu. Enter a number or text that identifies samples or controls and enter the quantity of the controls (see Table 13).

9. Setup the amplification conditions as shown in Table 9 and start the run.

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Table 8: Pipetting scheme for the preparation of mastermix, calculated to

achieve a final PCR reaction volume of 20 µl per capillary.




10x BSA solution 2.0 70 1x


10.0 350 1x

3.0 mM Mg-sulfate

Mg-sulfate (50 mM) ➌ 0.8 28 2.0 mM



20x reagent mix (containing primers and probe)

1.0 35 1x


➊ Volume depends on the intended volume of RNA sample; supplement to achieve a final volume of 20 µl. Calculation scheme see Appendix 4. ➋ Indicated volumes sufficient to prepare one 8-well control strip run and 24 sample runs; including pipetting reserve. For calculation of reagent volumes needed to run an intermediate number of samples please use table of Appendix 4. ➌ Reagent not contained in the module; component of the SuperScript III Platinum One-Step Quantitative RT-PCR System (Invitrogen), Cat.No.: 11732

Table 9: Thermal cycle program using LightCycler Program: Program: Reverse Transcription Type: None Cycles 1

Segment Number

Temperature Target (°C)

Hold Time (min)

Slope (°C/sec)

2° Target Temp (°C)

Step Size (°C)

Step Delay

(Cycles)

Acquisition Mode

1 59 60 20 0 0 0 None

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Program: Denaturation Type: None Cycles 1

Segment Number


Hold Time (sec)

Slope (°C/sec)


Step Size (°C)

Step Delay

(Cycles)

Acquisition Mode

1 95 120 20 0 0 0 None Program: Amplification Type: Quantification Cycles 50-60

Segment Number


Hold Time (sec)

Slope (°C/sec)


Step Size (°C)

Step Delay

(Cycles)

Acquisition Mode

1 95 10 20 0 0 0 None 2 57 40 20 0 0 0 Single

Program: Cooling Type: None Cycles 1

Segment Number


Hold Time (sec)

Slope (°C/sec)


Step Size (°C)

Step Delay

(Cycles)

Acquisition Mode

1 40 30 20 0 0 0 None Fluorescence Settings LED Power CALIB Display Mode 3.5 Compatible Color N/A Compensation N/A Car. Movement Continous Quantification Settings Channel Setting F1 Program Name: Amplification

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7.4. Quantification of HCV using SmartCycler instrument Contents of RoboGene quantification modules Table 10: Module contents Content Preparation of the 25x

reagent mix HCV control RNA, similar to the 5`untranslated region, HCV control RNA strips*, coated with 4 different amounts of HCV control RNA and amplification enhancer Cat.No.: 0203000151 96 tubes 25 µl HCV reagent mix, lyophilized, containing HCV primers and HCV probe (5’ FAM), PCR-grade water is supplied in separate vials, for use with SuperScript III Platinum One-Step Quantitative RT-PCR System Cat.No.: 0204000401 (100 reactions) • HCV reagent mix, lyophilized

3 tubes


• 10x BSA solution 1x 0.5 ml • PCR-grade water, DEPC-treated 2x 1.5 ml Cat.No.: 0204000402 (500 reactions) • HCV reagent mix, lyophilized

15 tubes


• 10x BSA solution 5 x 0.5 ml • PCR-grade water, DEPC-treated 10x 1.5 ml Sample RNA strips* coated with amplification enhancer only Cat.No.: 0205000151 Cat.No.: 0205000152

96 tubes 25 µl 480 tubes 25 µl

Manual *) each strip consists of four 25 µl tubes Experimental protocol To maintain full activity of the enzyme perform all pipetting steps on ice or on an ice-cold PCR rack! Preparation of reagent mixes and samples 1. For preparation of the “25x reagent mix” please refer to Table 10.

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2. Remove one strip from the storage base coded as “control RNA tubes” and load into a SmartCycler tube rack. Always wear gloves when handle the tubes! Caution! Make sure that the tubes are plugged in the correct order into the rack to recode highest and lowest standard concentration!

3. Remove the required number of “sample tubes” from the storage base and plug the

tubes into the free positions of the tube rack. Carefully open the tubes! 4. Preparation of final mastermix: prepare an unique mix for both the control and sample

reactions as shown in Table 11. 5. Add appropriate aliquots of the unique mastermix to each tube of the control and

sample tubes considering the volume of sample to be added.



7. Seal the tubes with the caps and centrifuge briefly. Always wear gloves when handle

the tubes and caps! 8. Carefully place the tubes in the block of the SmartCyler instrument. Select the positions

containing the controls (“Standards”) samples (“Unknown”) and non-template control (“NTC”) from the sample type pop-up menu. Enter a number or text that identifies samples or controls and enter the quantity of the controls (see Table 13). Choose the dye set “FCTC 25”.

9. Setup the amplification conditions as shown in Table 12 and start the run.

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Table 11: Pipetting scheme for the preparation of mastermix, calculated to achieve a final PCR reaction volume of 25 µl per tube.





12.5 225

1x 3.0 mM Mg-

sulfate

Mg-sulfate (50 mM) ➌ 1.0 18 2.0 mM



10x BSA solution 2.5 45 1x

25x reagent mix (containing primers and probe)

1.0 18 1x


➊ Volume depends on the intended volume of RNA sample; supplement to achieve a final volume of 25 µl. Calculation scheme see Appendix 5. ➋ Indicated volumes sufficient to prepare one 4-tube control strip run and 12 sample runs; including pipetting reserve. For calculation of reagent volumes needed to run an intermediate number of samples please use table of Appendix 5. ➌ Reagent not contained in the module; component of the SuperScript III Platinum One-Step Quantitative RT-PCR System (Invitrogen), Cat.No.: 11732

Table 12: Thermal cycle program using SmartCycler instrument

RT HOLD CYCLE

Temperature (°C)

Time (min:s)

Cycles

Ramping

59

60:00

95

2:00

95 57

00:15 0:30

50

2.5 °C/sec

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8. Initial concentration of the HCV RNA controls Table 13: Initial concentrations of the ready-to-use HCV RNA controls

Column A: for use with ABI PRISM 7700/7000 SDS; Gene Amp 5700 SDS,iCycler, Rotor-Gene instrument, Column B: for use with LightCycler instrument, Column C: for use with SmartCycler instrument To express the final results in IU per ml enter standard values from column E and to express copies per ml enter standard values from column F. These data are only valid if extraction of 150 µl patient samples using the INSTANT Virus RNA Kit (AJ Innuscreen & AJ Roboscreen) and elution with 60 µl elution buffer is performed. 5 µl of eluted HCV sample RNA have to be used per PCR run.

Control Tube No. or Colour Coding Initial HCV control RNA per tube [molecules]

HCV control RNA

[IU/ml]

HCV control RNA

[copies /ml] A B C D E F 1 1/Green (single label) 10,000,000 6,000,000,000 12,000,000,000 2 2/Green (double label) 1 1,000,000 600,000,000 1,200,000,000 3 3/Black (single label) 100,000 60,000,000 120,000,000 4 4/Black (double label) 2 10,000 6,000,000 12,000,000 5 5/Red (single label) 1,000 600,000 1,200,000 6 6/Red (double label) 3 100 60,000 120,000 7 7/Blue (single label) 50 30,000 60,000 8 8/Blue (double label) 4 10 6,000 12,000

9. Preparation of data Please follow the recommendations of the manufacturer of the real-time instrument.

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10. Sensitivity and precision of the test, quality assessment1
Figure 1: Genotype detection of the RoboGene H
7000 SDS instrument.

c

a

Extraction of 200 µl of HCV genotype reUniversitätsklinikum Essen, each 25,000 IU pKit (AJ Innuscreen & AJ Roboscreen) and esample RNA was used per RT-PCR run (amwith a ready-to-use HCV RNA control (6x 106

540,000108,000

21,600

4,35

540,000108,000

21,600

4,35

Figure 2: Sensitivity of HCV genome quantification. HCV positive serum (Genotype 1, kindly prov

Luxembourg; 2.7 x 106 IU per ml, quantificaassay) was diluted in normal serum yielding 865 and 172 IU per ml, respectively. 200 µl of60 µl and 5 µl were used in duplicate per RT-PResulting Sensitivity at least 172 IU / ml

1 determined according to the ICH-Guidelines “Validation of ana

6x106 IU

2a
1 1b 2i 2b
2

CV Quantification module us

ference plasma (Referenzzenter ml) was performed using the luted with 60 µl elution buffer.plification conditions see TableIU/ml).

0

865

< 50

0

865

< 50

172172

Saturation curves. ided by Dr. M. Opp, Laboratoiretion was performed by the Rsamples with 540,000 , 108,00 each sample was extracted in CR run.

lytical methods: Methodology”, IC

6x106 IU

ing an ABI

rum für RetINSTANT Vi 5 µl of elutes 2 and 3) t

National deoche Amplic0 , 21,600 ,triplicate, elu

H Topic Q2B

3a
6
4
5a
PRISM

roviren, rus Mini d HCV

ogether

Sante, or HCV 4,350 , ted with

20

10

15

20

25

30

35

40

1 100 10.000 1.000.000 100.000.000

Molecules Control RNA per assay

Thre

shol

d C

ycle

(CT)

HCV reference curve, n=10

Slope: -3.321 ± 0.137 Intercept: 36.133 ± 1.247Fit R: 0.996 ± 0.003

Figure 3: Mean reference curves and SD of individual data points obtained from 10 separate experiments performed with the AJ Roboscreen “Hepatitis C Virus (HCV) Control cRNA” using an ABI PRISM 7000 SDS. The threshold cycle (CT-value) represents the PCR cycle at which an increase in reporter fluorescence above the baseline signal can first be detected. The CV values of both the slopes β and the intercepts of the individual reference curve are usually <5% from the mean values. The correlation coefficients of the calculated linear fits are usually >0.99.

Table 14: Quality assurance of HCV reference curves. Values obtained from 10

independent experiments (4 different lots of control RNA strips).

Control-Tube No.

Mean CT-value

SD CT-value

Range of CT- value*

1 12.76 0.70 11.98-14.10 2 16.49 0.46 15.89-17.18 3 19.19 0.84 18.01-20.21 4 23.01 0.45 22.27-23.51 5 25.66 1.18 24.17-27.55 6 29.97 0.53 29.06-30.65 7 30.26 1.29 28.45-32.11 8 32.60 1.01 31.14-34.19

Fit R 0.996 0.003 0.992-1.000 Slope -3.321 0.137 -3.564-(-3.173)

Intercept 36.133 1.247 34.173-37.590 *Note that the CT-values mainly depend on setting of the threshold line. Main criterion for judging the analytical quality of the run is the slope of the reference curve! 11. Trouble Shooting

Always run at least 1 negative control (i.e. PCR-grade water) and 1 positive control (HCV positive RNA sample). „Positive“ evaluated negative controls indicate latent contamination. Such runs have to be completely repeated. Clinical samples should be measured at least by duplicate or triplicate experiments.

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Since control RNA or amplicons do not have to be any longer diluted and dispensed by the purchaser, the major source of carry-over is removed. Therefore, the risk of false positive results is markedly reduced. If contamination still occurs in your laboratory UNG sterilization may be required. 12. Carry-over prevention using UNG (optional)

The heat-labile uracil-DNA glycosylase (UNG) is suitable for preventing carry-over contamination between PCRs This technique relies on the incorporation of deoxyuridine triphosphate (dUTP) during all amplification reactions and the pretreatment of all successive PCR mixtures with the UNG. The UNG enzyme cleaves DNA at 37°C at any site where a dUTP residue has been incorporated. Subsequently, the resulting abasic sites are hydrolyzed due to high temperatures during the initial denaturation step, and can not serve as PCR template any longer. The heat-labile UNG is inactivated at the same time. We recommend the application of 0.2 U UDG per assay with an initial 15-min incubation step at 37°C prior to amplification2. Warranty Notice During the warranty period the AJ Roboscreen modules for quantification of HCV genomes allow precise and reproducible data recovery combined with excellent sensitivity. For data obtained by violation to the general GLP guidelines and the manufacturer’s recommendations the right to claim under guarantee is expired.

The purchase of this product does not convey any right for its use in clinical diagnostic applications. References 1. Köhler T, Lerche D, Meye A, Weisbrich C, Wagner O. Automated analysis of nucleic acids by quantitative

PCR using DNA coated ready-to-use reaction tubes. J.Lab.Med. 1999; 23:408-414. 2. Köhler T, Rost AK, Remke H. Calibration and storage of DNA competitors used for contamination-

protected competitive PCR. Biotechniques 1997; 23:722-726. 3. Bukh J, Purcell RH, Miller RH. Importance of primer selection for the detection of hepatitis C virus RNA

with the polymerase chain reaction assay. Proc.Natl.Acad.Sci.U.S.A. 1992; 89:187-191 4. Murakawa K, Esumi M, Kato T, Kambara H, Shikata T. Heterogeneity within the nonstructural protein 5-

encoding region of hepatitis C viruses from a single patient. Gene 1992; 117:229-232 5. Cooreman MP, Schoondermark-Van de Ven EM. Hepatitis C virus: biological and clinical consequences

of genetic heterogeneity. Scand.J.Gastroenterol. Suppl. 1996; 218:106-115. 6. Neumaier M, Braun A, Deufel T, Roscher A, Wagener C. Klinische Chemie und molekulare Diagnostik.

Themenhefte, Band 2: Sicherung der Qualität molekularbiologischer Methoden in der klinischen Diagnostik. Isensee Verlag Oldenburg, 1997.

How to contact AJ Roboscreen

If you are using the Internet access our web-site at: www.roboscreen.com Please contact us by e-mail at: [email protected] or by Phone/Fax at: +49-341-9725970/+49-341-9725979

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mailto:[email protected]

Appendix 1: Recommended amplification conditions for real- time instruments supporting the 96-well format

• GeneAmp 5700 SDS and ABI PRISM 7700 SDS

RT HOLD CYCLE

Temperature (°C)

Time (min:s)

Cycles

59

60:00

95

2:00

95 57

00:15 01:00

45

Seal the tubes with optical caps or appropriate plastic film e.g. ABI PRISM Optical Adhesive Covers

(Applied Biosystems, Cat.No.: 4311971)*.

• iCycler

RT HOLD CYCLE

setting the ramping rate: 2.5°C per sec

Temperature (°C)

Time (min:s)

Cycles

59

60:00

95

2:00

95 57

00:20 01:00

45

Seal the tubes with optical caps or appropriate optical film e.g. ABI PRISM Optical Adhesive Covers

(Applied Biosystems, Cat.No.: 4311971)*.

*) How to use the film: 1. Tailor the cover according to the number of used wells. 2. To avoid leakage during amplification, after placing on the wells tightly press

on the cover and use a compression pad (Applied Biosystems, Cat.No.: 4312639), follow the instructions of the manufacturer!

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Appendix 2: Calculation scheme for the preparation of a HCV mastermix for use with ABI PRISM 7700/7000/7300 SDS; Gene Amp 5700 SDS, iCycler

Calculation example: expected volume of RNA sample is 5 µl.

Preparation of 20 µl mastermix to add to the RNA sample in order to achieve a final PCR reaction volume of 25 µl.

Reagents HCV mastermix for

1 reaction

Mastermix for 100 reactions

RNA sample volume / PCR-grade water* 5.0 µl

Volume of mastermix for a final volume of 25 µl 20.0 µl

PCR-grade water 2.0 200 µl

2x Reaction mix 12.5 µl 1250 µl 10x passive reference dye

solution 2.5 µl 250 µl

Mg-sulfate (50 mM) 1.0 µl 100 µl 25x reagent mix 1.0 µl 100 µl

SuperScript III RT/ Platinum Taq mix 1.0 µl 100 µl

For your individual calculations:

Reagents HCV mastermix for 1 reaction

Mastermix for x reactions


RNA sample volume / PCR-grade water*

Volume of mastermix

PCR-grade water

2x Reaction mix 12.5 µl 10x passive reference

dye solution 2.5 µl

Mg-sulfate (50 mM) 1.0 µl

25x reagent mix 1.0 µl

SuperScript III RT/ Platinum Taq mix 1.0 µl

* For control runs (quantification controls, NTCs) use PCR-grade water

instead of RNA sample!

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Appendix 3: Calculation scheme for the preparation of a HCV mastermix for use with Rotor-Gene

Calculation example: expected volume of RNA sample is 5 µl.


Reagents HCV

mastermix for 1 reaction




PCR-grade water 4.5 µl 360 µl 2x Reaction mix 12.5 µl 1000 µl

Mg-sulfate (50 mM) 1.0 µl 80 µl 25x reagent mix 1.0 µl 80 µl







Volume of mastermix

PCR-grade water

2x Reaction mix 12.5 µl






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Appendix 4: Calculation scheme for the preparation of a HCV mastermix for use with LightCycler Calculation example: expected volume of RNA sample is 5 µl.


Reagents HCV






Mg-sulfate (50 mM) 0.8 µl 28 µl 10x BSA solution 2.0 µl 70 µl 20x reagent mix 1.0 µl 35 µl







Volume of mastermix

PCR-grade water



10x BSA solution 2.0 µl





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Appendix 5: Calculation scheme for the preparation of a HCV mastermix for use with SmartCycler Calculation example: expected volume of RNA sample is 5 µl.


Reagents HCV






Mg-sulfate (50 mM) 1.0 µl 18 µl 10x BSA solution 2.5 µl 45 µl 25x reagent mix 1.0 µl 18 µl







Volume of mastermix

PCR-grade water



10x BSA solution 2.5 µl





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AJ Roboscreen GmbH Delitzscher Straße 135, Laborgebäude D-04129 Leipzig (Germany)

Phone +49-341-9725970 Fax +49-341-9725979

Email [email protected] Internet www.roboscreen.com

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