PCR-free method for detecting a target genome
Current analytical methodologies for the detection of pathogenic microorganisms are in vitro diagnostic methods (IVD) based on immunological tests or molecularly quantifying the genomic sequences by means of PCR-based technologies. Molecular diagnostics is preferable to immunological tests due to its ability to determine the presence of the pathogen’s genome in the sample, to identify subjects with ongoing infection. However, the molecular tests currently available require the amplification of genomic strand via PCR (polymerase chain reaction), a complex and expensive procedure, and necessarily carried out in qualified laboratories. PCR amplification can also lead to false positives and makes quantification of nucleic acids difficult.
To overcome these shortfalls, a new electrochemiluminescence analytical method for the quantification of RNA genomes of pathogenic microorganisms – such as the Covid-19 virus – without PCR amplification is presented, allowing more accurate, rapid and economical tests both for mass screening and in point-of-care diagnostics. We demonstrated that we were able to quantify Hepatitis B Virus (HBV) genome with a Limit of Detection (LoD) of 0.06 cps mL-1 for extracted HBV – lower than the one of PCR – without any analyte-amplification process. The versatility of our approach could be easily adapted to the quantification of other virus the methodology was tested in biological fluids and the specificity was demonstrated by analyzing unspecific targes such as Mycobacterium tuberculosis. This result has been obtained thanks to the combination of a surface cooperative hybridization scheme with an ECL detection strategy exploiting a large signal amplification. This latter effect is based on the intercalation of hundreds of intercalating Ru-based complexes in the whole genome that is captured at the electrode surface. TRL 4.
- Advanced molecular diagnostics
- clinical analysis for the identification of pathogenic microorganisms
- point of care (POC) diagnostics
- massive tests processing (for instance for Covid-19)
- Quick and direct determination;
- High analytical performance;
- Applicable to different target pathogens, which can also be detected during the same analytical process;
- Can be employed in point-of-care technologies;
- Possibility of detecting ds-DNA of pathogens (DNA viruses such as HBV).