rev 1.0 (13/07/2016)
IV. Description and content of the kit
The RT-PCR kit is ready to use for the specific detection of a target. The kit contains reagents and enzymes necessary of the amplification of a specific target, synthetic Target positif RNA (positive control) and of RNA inhibitory control (IPC) (see table 1). Fluorescence is emitted and measured individually by an optical system during PCR. The detection of the amplified fragment is usually carried out by the fluorimeter using the channels listed in table 2.
Table 1:
| Components of the kits | 48 reactions | 96 reactions | Reconstitution |
| Volume (µl) | Volume (µl) | ||
| PCR pre-mix |
1160 | 2000 | Ready to use |
| Taq polymerase Nucleis (Taq N) | 116 | 200 | Ready to use |
| Reverse transciptase Nucleis (RT N) | 58 | 100 | Ready to use |
| Target RNA Positive control | 200 | 200 | Ready to use |
| RNA IPC (inhibitory control) | 150 | 300 | Ready to use |
Table 2: Fluorophores usually used in duplex target-IPC kits.
| Target | Fluorophore | Excitation | Emission |
| Target | FAM | 499 | 519 |
| IPC | VIC | 520 | 548 |
(Please check the kit certificat for the fluorophore used in a specific kit.)
Alternatives:
-channel FAM: (ABI Systems, smartcycler II; Chromo 4/CFX96, System Agilent MX and aria, realplex), channel 530 (LC480), channel Green (RotorGene)
-channel VIC: Channel VIC (ABI systems, Biorad CFX96, Agilent Mx and aria, realplex), channel Alexa 532 (Smart cyclerII), Channel 560 (LC 480); channel Yellow (RotorGene)
Required material and reagents not provided
- Elution buffer
- A RNA extraction and purification kit
- Biological hood
- qPCR instrument
- Micro centrifuge
- Vortex
- Plates / tubes for qPCR
- Micropipettes
- Filtre tips for micropipettes
- Sterile microtubes
- A viral RNA extraction and purification kit
- Non powdered gloves
All reagents must be stored at -20°C. Storage at 4°C is forbidden.
All reagents can be used until the expiration date indicated on the kit
Many freezing/thawing cycles (>3x) must be avoided, could lead to decrease in sensitivity
VI. Cautions and notes
- Read carefully instructions before starting.
- The experiment must be performed by competent staff
- Instruments must have been properly installed, calibrated and maintained according to the manufacturer’s recommendations
- Clinical samples are potentially infectious and must be processed under a laminar flow hood/
- The experiment must be performed according to good laboratory practices
- Do not use this kit after expiration date
- Many freezing/thawing cycles of the reagents must be avoided, and could lead to decrease of sensitivity
- Once defrosted, spin down briefly the tubes before use
- Spin down briefly enzymes tubes to pellet the viscous content at the bottom of the tubes.
- Use of ice or cooling block is mandatory to prepare the mix and dispense reconstituted mix in wells.
- Define 3 working areas: 1)Isolation of DNA/RNA, 2) Preparation of the reaction mix and 3) Amplification/detection of amplified products.
- Pipettes, reagents and other materials must not cross each area
- Specific caution is required to preserve the purity of the reagents and reaction mixtures. The use of gloves is mandatory. Appropriate methods of preparation of DNA should be used.
- Always use filtered tips for micropipettes
- Use specific lab coat and gloves in each working area
- Do not pipette with mouth and do not eat, drink or smoke in the area
- Avoid sprays
VII. Collection of samples, transport and storage
- Collect samples in sterile tubes
- The samples should be extracted immediately or frozen from -20°C to 80°C
- Transport of clinical samples must obey local regulations for this type of infectious agents.