gms | German Medical Science

Background: Implementation of respiratory pathogen diagnostics into daily routine is hindered by time consuming and expensive technologies. Especially the time to results is of major importance for optimal treatment. On site pathogen diagnostic is extremly valuable, as it can help reducing false and unnecessary antibiotic prescriptions and thus avoiding 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 clinically relevant respiratory pathogens. Detected viruses are influenza A and B, parainfluenza virus 1 - 4, adenovirus, enterovirus, rhinovirus, meta-pneumovirus, respiratory syncytial virus, coronavirus OC43, 229E, HKU1 and NL63 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 layout of the chip has a 21 x 21 matrix spotted with a distance of 160 µm.

Biotin-modified primers labelled all fragments during PCR. 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 being visualised on an agarose gel electrophoresis 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. Each target specific probe resulted in organism specific signals only present when the individual target was present in the sample.

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.