gms | German Medical Science

Background: Implementation of diagnostics for the detection of respiratory pathogens into daily routine medicine is hindered by time consuming and expensive technologies. On site pathogen diagnostics are extremely valuable, since they reduce false and unnecessary antibiotic prescriptions and thus avoid antimicrobial resistance. In addition, identifying the cause of infection enables measures of infection control and targeted treatment.

Objectives: The objective was to develop an easy to use and cost-effective assay for the detection of more than 95 % of the most clinically relevant respiratory pathogens. Detected viruses are influenza, parainfluenza, adenovirus, enterovirus, rhinovirus, meta-pneumovirus, respiratory syncytial virus, coronavirus and bocavirus. Legionella pneumoniae, Bordetella pertussis, Bordetella paratussis, Chlamydophila pneumonia and Mycoplasma pneumonia were also included. The main focus of R&D was to minimize time to result and hands on time.

Methods: A one-step RT PCR approach was developed allowing amplification of RNA and DNA pathogens. The macroarray chip, carrying immobilized C7-amino modified nucleic acid probes, was integrated into a common 1.5 ml reaction tube. The amplified targets were then hybridized onto the macroarray to form nucleic acid duplex structures on their respective target probes. A TMB-like substrate coupled with an HRP immuno-precipitation reaction resulted in a characteristic precipitation pattern. This was subsequently read out by an analytical software within seconds.

Results: The method was established with samples originally characterised in an in house PCR based ELISA method. It turned out that the new method was able to amplify each of the respective pathogens independently either by a singleplex amplification or by a multiplex PCR one step amplification. A first step verification occurred by fragment length comparison of the targets in comparison to length standards. A second verification was performed by hybridisation onto the chip array.

Conclusion: The described method enables an easy, fast and cost-effective pathogen typing. The use of a one-step RT PCR offers the possibility to amplify potential target DNA and RNA simultaneously without a time consuming separate PCR step. The developed analytical software enables instantaneous interpretation of reducing hands-on-time. Validation of this novel macroarray chip with an extended number of samples is currently under way.