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Published on: April 27, 2011
Development of DNA Microarray for Indication of Viral Community-Acquired Pneumonia Pathogens
N A Sakharnov1, E N Filatova2, M I Popkova2
1Senior Researcher, Laboratory of Molecular Biology and Biotechnology; Academician I.N. Blokhina Nizhny Novgorod Scientific Research Institute of Epidemiology and Microbiology of Rospotrebnadzor (Russian Federal Consumer Rights Protection and Human Health Control Service), 71 Malaya Yamskaya St., Nizhny Novgorod, 603950, Russia.
None:
The aim of the study is to develop a DNA microarray for the indication of viral pathogens causing community-acquired pneumonia.
Materials And Methods:
The study materials were swab samples from the nasopharyngeal and oropharyngeal mucous membranes of patients aged 2 months to 18 years with X-ray confirmed pneumonia. The selection of DNA probes for the specific detection of viral community-acquired pneumonia pathogens and development of the microarray design were carried out using our previously developed disprose program. The nucleotide sequences of pathogens were obtained from the NCBI Nucleotide and GISAID databases. The selected DNA probes were synthesized on CustomArray slides (USA). The optimal hybridization temperature was selected on a model pooled sample containing adenovirus DNA and SARS-CoV-2 coronavirus RNA. The selection criteria were the percentage of effective probes with a standardized hybridization signal (SHS) ≥3 Z and the excess of SHS levels of effective specific probes compared to SHS of effective non-specific probes. The DNA probes were selected for the specific detection of viral community-acquired pneumonia pathogens, characterized by an effective hybridization signal under the identified conditions. Using ROC analysis, threshold values of specific probe signals were established, the excess of which was interpreted as the evidence of the pathogen presence in a sample.
Results:
A microarray design included 544 DNA probes for the detection of adenovirus, bocavirus, respiratory syncytial virus, metapneumovirus, parainfluenza virus, rhinovirus, and coronavirus. The DNA probes were synthesized on slides. The optimal DNA hybridization temperature on microarrays was established (47°C). A list of probes for specific detection of adenovirus group B, bocavirus, parainfluenza virus type 3, respiratory syncytial virus, rhinovirus, and SARS-CoV-2 coronavirus, characterized by an effective hybridization signal under the identified conditions, was selected. The threshold values of probe signals for specific detection of these pathogens in clinical samples were determined.
Conclusion:
A DNA microarray for the indication of viral community-acquired pneumonia pathogens was developed and synthesized. The interpretation of the hybridization results corresponds to the results obtained by the PCR method. The developed microarray can be used to improve laboratory diagnostics of viral community-acquired pneumonia pathogens.
