CHIC Technology

CHIC technology (Competitive Heterologous Internal Control) is the internal amplification control system developed by Progenie Molecular that includes RealCycler kits.. 

This proprietary technology allows the use of any extraction system without loss of sensitivity, and at the same time it monitors accurately a possible sample inhibition. For this reason, RealCycler Monotest and RealCycler Universal kits are compatible with almost all DNA/RNA extraction systems available in the market, without making any changes to the original protocols provided by the manufacturer. 

Internal amplification control

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

The internal amplification control (IC) is a system for the detection of false negatives. It is based on the simultaneous detection of a fragment different from that of the pathogen, regardless the presence of the pathogen in the sample and therefore, it is always detected. It is made of a DNA or RNA template, oligonucleotides, and the probe that allows its specific detection. The IC can also be used to detect problems in the DNA extraction or its absence in the amplification mix. Its use also allows the prevention of false positives due to the amplification of unspecific products. In the absence of the pathogen's DNA, the IC consumes the surplus reagent, minimizing the amplification of undesired sequences and hindering the generation of a false positive. 

The internal control CHIC is an internal control system based on our proprietary CHIC technology (Competitive Heterologous Internal Control). This technology is based on the use of heterologous DNA artificial constructions (made of combined sequences of human and microorganisms DNA) as template for the detection of the internal control. The main feature of this technology is that the amplification of the internal control finds itself in a competitive disadvantage with the pathogen. In this way it does not affect the sensitivity of the method as a whole and it allows the development of highly sensitive systems. RealCycler products have this internal control system in all its formats: RealCycler Monotest and RealCycler Universal. Additionally, this technology allows the use of almost any extraction system without the loss of sensitivity and, at the same time, it monitors accurately a possible sample inhibition.

CHIC features

  • Co-amplified in the reaction mix together with the detected pathogens.
  • Competitive disadvantage with the pathogen = it does not decrease the sensitivity of the pathogen detection.
  • Independent of the extraction system = almost complete compatibility with all extraction systems.
  • It monitors inhibition with high accuracy (see examples below).

M

Negative sample (without inhibition)
VALID NEGATIVE RESULT

Result:
- CHIC signal is detected in the HEX channel (green line) with a Ct = 31,75
- CHIC acceptance range is Ct < 35. Therefore, in this case it is within the range.
- The pathogen signal in the FAM channel (blue line) is flat and does not show a Ct (not detected).

Interpretation:
This sample does not show any inhibition, so the result is clearly NEGATIVE.

This kind of signal is observed when using water as negative control or samples with a complete absence of inhibition.

Negative sample (with partial inhibition)
VALID NEGATIVE RESULT

Result:
- CHIC signal is detected in the HEX channel (green line) with a Ct = 33,98.
- CHIC acceptance range is Ct < 35. Therefore, in this case it is within the range.
- The pathogen signal in the FAM channel (blue line) is flat and does not show a Ct (not detected).

Interpretation:
The reaction is not inhibited given that CHIC is within the range, so the result of the sample is NEGATIVE.

CHIC Ct value is higher than in the previous case, indicating a partial PCR inhibition. However, given that it is inside the acceptance range, the result can be accepted.

Positive sample (NO competition pathogen-CHIC)
VALID POSITIVE RESULT

Result:
- Pathogen signal is detected in the FAM channel (blue line) with a Ct = 30,78.
- The CHIC Ct value = 31,1 (green line). Given that there is a signal in the FAM channel for the pathogen, the CHIC value is indifferent.

Interpretation:
Positive sample (blue line).

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

Positive sample (competition pathogen-CHIC)
VALID POSITIVE RESULT

Result:
-The pathogen signal is detected in the FAM channel (blue line) with a Ct = 27,68.

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

Positive sample (competition pathogen-CHIC)
VALID POSITIVE RESULT

Result:
- The pathogen signal is detected in the FAM channel (blue line) with a Ct = 24,55.

Interpretation:
Positive sample (regardless of the CHIC value).
CHIC signal is detected in the HEX channel (flat green line). It does not have Ct, which indicates that there has been competition between the pathogen and CHIC, and that CHIC could not be detected because of the competitive disadvantage. The result suggests a high concentration of the pathogen.

Inhibited sample
INVALID RESULT

Result:
- CHIC signal detected in the HEX channel (green line) is flat and the pathogen signal also (blue line).

Interpretation:
Invalid result.
This indicates a total inhibition of the amplification reaction and therefore the result is not acceptable.