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   CHIC Technology

CHIC technology (Competitive Heterologous Internal Control) is our own developed internal amplification control system included in RealCycler kits.

This technology allows the use of any extraction system without losing sensibility and it monitors sample's inhibition with high accuracy at the same time. For this reason, RealCycler Monotest, RealCycler Universal and RealCycler Chic-Out kits are completely compatible with all DNA/RNA extraction systems available in the market, without the need to make any changes to the original protocols provided by the producer.

Internal control of amplification

The false negatives in the techniques for the detection of pathogens based on PCR occurr when some factor prevents the generation of amplification product, regardless the presence of the pathogen in the sample.  The common cause is the presence of the PCR's inhibitors, that could be components of the clinical sample (hemoglobin or other proteins, mucopolysaccharides, urea, etc.) as well as artificially added compounds during their handling (heparin, EDTA, proteinase K, powder of latex gloves, etc.).  Since it's a feature inherent to the samples or its processing, the inhibitions cannot be detected unless specific prevention systems are used.    

The internal control (IC) of amplification is a system for the detection of false negatives. It's based on the parallel detection of a fragment different than the one of the pathogen, regardless the presence of the pathogen in the sample and, therefore, it always has to be detected. It's composed by a mould of DNA or RNA and oligonucleotides and the probe that allow its specific detection. The IC can also be used to detect problems in the DNA extraction or its omission in the mix of its amplification. Its usage also enables the prevention of false positives due to the amplification of nonspecific products. In the absence of the pathogen agent's DNA, the IC consumes the surplus reagent, minimizing the amplification of undesired sequences and hindering the generation of a false positive. 

The CHIC internal control is an internal control system based on the CHIC technology (Competitive Heterologous Internal Control) developped by our own. This technology is based on the usage of artificial heterologous DNA constructions (composed by combined sequences of human DNA and by microrganisms) as mould for the detection of the internal control. The main feature of this technology is that the internal control's amplification finds itself in a competitive disadvantage with the pathogen. In this way it doesn't affects the sensibility of the whole method and enables the development of high sensitive systems. RealCycler products dispose of this internal control system in all its editions: RealCycler Monotest, RealCycler Universal and RealCycler Chic-Out. Additionally, this technology allows the usage of any extraction system, wihtout losing sensibility and, at the same time, it monitors sample's inhibition with high accuracy. 

CHIC features

  • Co-amplificated in the reaction mix together with the pathogens.
  • Competitive disadvantage with the pathogen = it does not decrease the pathogen detection sensitivity.
  • Regardless of the DNA/RNA extraction system = Complete compatibility with all extraction systems.  
  • It monitors inhibition with high accuracy (see examples below).

M

Negative sample
(without inhibition)

CHIC signal is detected in the channel of HEX fluorophore (green line) with a Ct = 31,75 (within the acceptance range belonging to this lot Ct CHIC < 35). Pathogen signal in the channel of FAM fluorophore (blue line) is flat and does not mark a Ct (not detected).

NOTE: this kind of signal is observed when using agua as negative control (complete absence of inhibition). While, the obtained DNA/RNA of a clinical sample usually presents a certain quantity of natural inhibitors that cause a slight delay in the Ct value of the CHIC, even though it usually stays within the acceptance range  (see next graphic).

Negative sample
(with partial inhibition)

CHIC signal is detected in the channel of HEX fluorophore (green line) with a Ct = 33,98 (within the acceptance range of this lot Ct CHIC < 35). Pathogen signal in the channel of FAM fluorophore (blue line) is flat (not detected).

NOTE: Higher Ct compared to previous case indicates a partial inhibition of the amplification reaction. Nevertheless, the result can be accepted given that the Ct value is within the acceptance range provided by the kit..

Positive sample
(NO competence pathogen-CHIC)

Pathogen signal is detected in the channel of FAM fluorophore (blue line) with a Ct = 30,78. Therefore, the sample is positive regardless of the CHIC value.

NOTE: CHIC signal is detected in the channel of HEX fluorophore (green line) with a Ct = 31,4 (within the acceptance range). This indicates there has been no competence between CHIC and pathogen, and suggests a low concentration of the pathogen.

Positive sample
(competence pathogen-CHIC)

Pathogen signal is detected in the channel of FAM fluorophore (blue line) with a Ct = 27,68. Therefore, the sample is positive regardless of the CHIC value.

NOTE: CHIC signal is detected in the channel of HEX fluorophore (green line) with a Ct = 36,38. Despite the Ct is outside the acceptance range, it does not mean inhibition but competence between pathogen and CHIC. In this case, CHIC has been displaced because of the competitive disadvantage.

Positive sample
(competence pathogen-CHIC)

Pathogen signal is detected in the channel of FAM fluorophore (blue line) with a Ct = 24,55. Therefore, the sample is positive regardless of the CHIC value.

NOTE: CHIC signal in the channel of HEX fluorophore (green line) is flat. This indicates that there has been competence between pathogen and CHIC, and CHIC could not be detected because of the competitive disadvantage. The result suggests a high concentration of the pathogen.

Sample inhibited
(result NOT ACCEPTABLE)

CHIC signal detected in the channel of FAM fluorophore (green line) is flat as well as the pathogen signal (blue line). This indicates a total inhibition of the amplification reaction and therefore the result is not acceptable.