Genomics & Informatics

Korea Genome Organization (한국유전체학회)
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Comparison of microbial molecular diagnosis efficiency within unstable template metagenomic DNA samples between qRT-PCR and chip-based digital PCR platforms

  • Dongwan Kim (Department of Microbiology, College of Science & Technology, Dankook University) ;
  • Junhyeon Jeon (Department of Microbiology, College of Science & Technology, Dankook University) ;
  • Minseo Kim (Department of Microbiology, College of Science & Technology, Dankook University) ;
  • Jinuk Jeong (Department of Bioconvergence Engineering, Dankook University) ;
  • Young Mok Heo (R&I Center, COSMAX BTI) ;
  • Dong-Geol Lee (Department of Microbiology, College of Science & Technology, Dankook University) ;
  • Dong Keon Yon (Center for Digital Health, Medical Science Research Institute, Kyung Hee University Medical Center, Kyung Hee University College of Medicine) ;
  • Kyudong Han (Department of Microbiology, College of Science & Technology, Dankook University)
  • Received : 2023.08.17
  • Accepted : 2023.10.11
  • Published : 2023.12.31
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Abstract

Accurate and efficient microbial diagnosis is crucial for effective molecular diagnostics, especially in the field of human healthcare. The gold standard equipment widely employed for detecting specific microorganisms in molecular diagnosis is quantitative real-time polymerase chain reaction (qRT-PCR). However, its limitations in low metagenomic DNA yield samples necessitate exploring alternative approaches. Digital PCR, by quantifying the number of copies of the target sequence, provides absolute quantification results for the bacterial strain. In this study, we compared the diagnostic efficiency of qRT-PCR and digital PCR in detecting a particular bacterial strain (Staphylococcus aureus), focusing on skin-derived DNA samples. Experimentally, specific primer for S. aureus were designed at transcription elongation factor (greA) gene and the target amplicon were cloned and sequenced to validate efficiency of specificity to the greA gene of S. aureus. To quantify the absolute amount of microorganisms present on the skin, the variable region 5 (V5) of the 16S rRNA gene was used, and primers for S. aureus identification were used to relative their amount in the subject's skin. The findings demonstrate the absolute convenience and efficiency of digital PCR in microbial diagnostics. We suggest that the high sensitivity and precise quantification provided by digital PCR could be a promising tool for detecting specific microorganisms, especially in skin-derived DNA samples with low metagenomic DNA yields, and that further research and implementation is needed to improve medical practice and diagnosis.

Keywords

Acknowledgement

This research was supported by Basic Science Research Capacity Enhancement Project through Korea Basic Science Institute (National Research Facilities and Equipment Center) grant funded by the Ministry of Education (Grant No. 2019R1A6C1010033). This study has been done with the participation of undergraduate students working at total-omics analysis research institute of Dankook University. The research institute has been supported by the VIP system as a part of Support Program for University Development 2023 of Dankook University. Following are results of a study on the "Leaders in INdustry-university Cooperation 3.0" Project, supported by the Ministry of Education and National Research Foundation of Korea.

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