Objectives This study presents the standardized protocols developed by the Clinical-Based Human Microbiome Research and Development Project (cHMP) in the Republic of Korea.
Methods It addresses clinical metadata collection, specimen handling, DNA extraction, sequencing methods, and quality control measures for microbiome research.
Results The cHMP involves collecting samples from healthy individuals and patients across various body sites, including the gastrointestinal tract, oral cavity, respiratory system, urogenital tract, and skin. These standardized procedures ensure consistent data quality through controlled specimen collection, storage, transportation, DNA extraction, and sequencing. Sequencing encompasses both amplicon and whole metagenome methods, followed by stringent quality checks. The protocols conform to international guidelines, ensuring that the data generated are both reliable and comparable across microbiome studies.
Conclusion The cHMP underscores the importance of methodological standardization in enhancing data integrity, reproducibility, and advancing microbiome-based research with potential applications for improving human health outcomes.
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<sec>
<title>Objectives</title>
<p>Carbapenem resistance is a serious clinical and public health threat. Carbapenemase can confer carbapenem resistance, and most carbapenemase genes are plasmid encoded so resistance can easily spread. In this study, we aimed to develop a novel system based on the TaqMan platform for the rapid detection of 6 clinically prevalent carbapenemase genes: <italic>Klebsiella pneumoniae</italic> carbapenemase, New Delhi metallo-β-lactamase, oxacillinase, imipenem-hydrolyzing, Verona integron-encoded metallo-β-lactamase, and Guiana extended-spectrum β-lactamase.</p></sec>
<sec>
<title>Methods</title>
<p>The triplex assay was verified by testing genomic DNA of 6 carbapenemase-producing <italic>Klebsiella pneumoniae</italic>. It was validated with a blinded panel of 310 Enterobacteriaceae isolates, including 225 carbapenemase-producers and 85 non-producers, by direct colony triplex real-time polymerase chain reaction (PCR). The real-time PCR was performed using the ABI 7500 fast instrument (Applied Biosystems, CA, USA) and specific primers for each carbapenemase target were designed to include modified peptide-nucleic acid oligonucleotides.</p></sec>
<sec>
<title>Results</title>
<p>No amplification was detected among the negative samples. The result showed 100% concordance with the genotypes previously identified. The entire assay, including DNA extraction and real-time PCR, was completed within 2 hours.</p></sec>
<sec>
<title>Conclusion</title>
<p>The newly developed triplex real-time PCR assay was useful for the rapid, accurate and simultaneous detection of 6 carbapenemase genes in Enterobacteriaceae, suggesting its potential to allow an early decision on the appropriate treatment, management, and prevention of the spread of resistant infections in hospitals.</p></sec>
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