9.

Procedure
9.1 RNA-Extraction
Different brands of RNA extraction kits are available. You may use your own extraction systems or
Revision No.: ZJ0008 the commercial kit based on the yield. For RNA extraction kit, please comply with manufacturer’s
Issue Date: Jul 1st, 2015 instructions. The recommended extraction kit is as follows:
HCV Genotype Real Time RT-PCR Kit User Manual Nucleic Acid Isolation Kit Cat. Number Manufacturer
For Research Use Only RNA Isolation Kit ME-0010/ME-0012 ZJ Biotech
QIAamp Viral RNA Mini Extraction Kit (50) 52904 QIAGEN
HR-0009-02
For use with ABI Prism®7000/7300/7500/7900/Step One Plus; iCycler iQ™4/iQ™5; 9.2 RT-PCR Protocol
Smart Cycler II;Bio-Rad CFX 96;Rotor Gene™6000; Mx3000P/3005P;MJ-Option2/Chromo4; The Master Mix volume for each reaction should be pipetted as follows:
LightCycler®480 Instrument

Shanghai ZJ Bio-Tech Co., Ltd.

www.liferiver.com.cn Tel: +86-21-34680596
[email protected] Fax: +86-21-34680595
2nd floor,No.15 Building,No.188 Xinjunhuan road,
PuJiang Hi-tech Park Shanghai China

1. Intended Use
HCV genotype real time RT-PCR kit is used for the detection of HCV genotype 1 in blood serum by
using real time PCR systems.
2. Principle of Real-Time PCR
The principle of the real-time detection is based on the fluorogenic 5’nuclease assay. During the PCR
reaction, the DNA polymerase cleaves the probe at the 5’ end and separates the reporter dye from the
quencher dye only when the probe hybridizes to the target DNA. This cleavage results in the
fluorescent signal generated by the cleaved reporter dye, which is monitored real-time by the PCR 1）The volumes of Super Mix per reaction multiply with the number of samples, which includes the
detection system. The PCR cycle at which an increase in the fluorescence signal is detected initially number of controls, standards, and sample prepared. Molecular Grade Water is used as the negative
(Ct) is proportional to the amount of the specific PCR product. Monitoring the fluorescence intensities control. For reasons of unprecise pipetting, always add an extra virtual sample. Mix completely then
during Real Time allows the detection of the accumulating product without having to re-open the spin down briefly in a centrifuge.
reaction tube after the amplification. 2) Pipet 20μl Super Mix with micropipets of sterile filter tips to several Real time PCR reaction
3. Product Description tubes. Separately add 5μl RNA sample，positive control A, positive control B and negative control to
Hepatitis C virus has at least six forms or genotypes. HCV genotypes and subtypes are distributed different reaction tubes. Immediately close the tubes to avoid contamination.
variously in different parts of the world. Genotypes 1-3 are widely distributed throughout the world. 3） Spin down briefly in order to collect the Master Mix in the bottom of the reaction tubes.
Subtype 1a is prevalent in North and South America, Europe, and Australia. Subtype 1b is common in 4) Perform the following protocol in the instrument:
North America and Europe, and is also found in parts of Asia. Genotype 2 exists in most developed
45°C for 10min 1cycle Selection of fluorescence channels
countries, and is less common than genotype 1. Some studies suggest that different types of HCV may
95°C for 15min 1cycle Reaction Mix A：FAM HCV
be related to different transmission routes. HCV genotype 1 is significantly associated with human
immunodeficiency virus. Genotype 1 is related to a poor response to treatment. Genotyping can help 95°C for 15sec,58°C for 1min Reaction Mix B：FAM HCV genotype I
( Fluorescence measured at 58°C) 40cycles
doctor determine an appropriate hepatitis C treatment and how long treatment should be given.
HCV genotype real time RT-PCR kit contains a specific ready-to-use system for the detection of HCV
genotype 1 by Reverse Transcription Polymerase Chain Reaction (RT-PCR) in the real-time PCR
5) If you use ABI Prism® system, please choose “none” as passive reference and quencher.
system. The master contains Super Mix for the specific amplification of HCV RNA and HCV
genotype1 RNA. Super Mix A is specific for HCV RNA; Super Mix B is specific for HCV genotype1
10. Threshold setting: just above the maximum level of molecular grade water.
RNA. The reaction is done in one step real time RT-PCR. The first step is a reverse transcription (RT):
HCV RNA is transcribed into cDNA. Then, a thermostable DNA polymerase is used to amplify the
specific gene fragments by polymerase chain reaction (PCR).Fluorescence is emitted and measured by 11.Quality control:
Negative control and positive control must be performed correctly, otherwise the sample results is
the real time systems´ optical unit during PCR. The detection of amplified HCV DNA fragment is
invalid.
performed in fluorimeter channel FAM with the fluorescent quencher BHQ1.
4. Kit Contents Super Mix Ct value
Control Super Mix A Super Mix B
Ref. Type of reagent Presentation 25rxns
1 HCV Super Mix A 1 vial, 480μl Molecular Grade Water UNDET UNDET
2 HCV Super Mix B 1 vial, 480μl HCV positive control A ≤35 UNDET
3 RT-PCR Enzyme Mix 1 vial, 54μl HCV positive control B ≤35 ≤35
4 Molecular Grade Water 1 vial, 400μl 12. Data Analysis and Interpretation
5 HCV Positive control A 1 vial, 60μl Negative or positive judgement：
6 HCV Positive control B 1 vial, 60μl Ct Value Negative or Positive
Analysis sensitivity: 5×103IU/ml； LOQ：1×104～1×108copies/ml 1 UNDET Negative“—”
Note: Analysis sensitivity depends on the sample volume, elution volume, nucleic acid extraction 2 ≤38 Positive “+”
methods and other factors .If you use the RNA extraction kits recommended, the analysis sensitivity is 3 38～40 Re-test; If it is still 38~40, then Negative“—”
the same as it declares. However, when the sample volume is dozens or even hundreds of times greater
The following results are possible:
than elution volume by some concentrating method, it can be much higher.
5. Storage
Results Super Mix A Super Mix B ︱Ct Value of Super
• All reagents should be stored at -20°C. Storage at +4°C is not recommended. Mix A－Ct Value of Conclusion
• All reagents can be used until the expiration date indicated on the kit label. Super Mix B︱
• Repeated thawing and freezing (>3x) should be avoided, as this may reduce the sensitivity of the 1 — — － HCV Negative
assay. 2 + + ≤3.5 HCV Positive
• Cool all reagents during the working steps. and Genotype I
• Super Mix should be stored in the dark. 3 + + >3.5 HCV Positive but
6. Additionally Required Materials and Devices not Genotype I
• Biological cabinet • Real time PCR system 4 + — － HCV Positive but
• Vortex mixer • Real time PCR reaction tubes/plates not Genotype I
• Cryo-container • Pipets (0.5μl – 1000μl)
• Sterile filter tips for micro pipets • Sterile microtubes
• Disposable gloves, powderless • Biohazard waste container For further questions or problems，please contact our technical support at [email protected]
• Refrigerator and Freezer • Tube racks
• Desktop microcentrifuge for “eppendorf” type tubes (RCF max. 16,000 x g)
7. Warnings and Precaution
• Carefully read this instruction before starting the procedure.
• For in vitro diagnostic use only.
• This assay needs to be carried out by skilled personnel.
• Clinical samples should be regarded as potentially infectious materials and
should be prepared in a laminar flow hood.
• This assay needs to be run according to Good Laboratory Practice.
• Do not use the kit after its expiration date.
• Avoid repeated thawing and freezing of the reagents, this may reduce the sensitivity of the test.
• Once the reagents have been thawed, vortex and centrifuge briefly the tubes before use.
• Prepare quickly the Reaction mix on ice or in the cooling block.
• Set up two separate working areas: 1) Isolation of the RNA/ DNA and 2) Amplification/
detection of amplification products.
• Pipets, vials and other working materials should not circulate among working units.
• Use always sterile pipette tips with filters.
• Wear separate coats and gloves in each area.
• Do not pipette by mouth. Do not eat, drink, and smoke in laboratory.
• Avoid aerosols
8. Sample Collection, Storage and Transport
• Collected samples in sterile tubes;
• Specimens can be extracted immediately or frozen at -20°C to -80°C.
• Transportation of clinical specimens must comply with local regulations for the transport of
etiologic agents.