2023-07-14-世界卫生组织-WHO_BS_2023.2455_WHO_1st_Reference_Reagent_for_DNA_extraction_of_gut_microbiome_63页_1mb

Summary of the WHO Collaborative Study on the WC-Gut RR (NIBSC 22/210)

Core Content

The World Health Organization (WHO) conducted a collaborative study to evaluate the suitability of NIBSC 22/210 as an International Reference Reagent (IRR) for DNA extraction of the gut microbiome. This reagent, composed of 20 inactivated bacterial strains from the human gut microbiome, was developed by the Medicines and Healthcare products Regulatory Agency (MHRA) and is intended to support the standardisation of DNA extraction methods, thereby improving reproducibility and comparability in microbiome research.

The study involved 22 participants from 14 countries across 4 continents, covering a wide range of backgrounds including academia, regulatory agencies, and the pharmaceutical industry. Participants were asked to extract DNA from the reagent using two methods of their choice and to return the results for analysis. The reagent was designed to act as a 'mock' microbial community, enabling users to identify biases in their DNA extraction procedures and assess the accuracy of their methods.

Main Points

• Purpose: To evaluate the candidate WHO International Reference Reagent (WC-Gut RR, NIBSC 22/210) and establish Minimum Quality Criteria (MQC) for DNA extraction methods of gut microbiome samples.
• Composition: The reagent is a mix of 20 inactivated bacterial strains, representing 5 phyla, 13 families, 16 genera, and 19 species.
• Methodology:
  • The reagent was tested for stability across different temperatures and timepoints.
  • DNA extraction methods were evaluated using four key reporting measures: Sensitivity, False Positive Relative Abundance (FPRA), Diversity, and Similarity.
  • Shotgun sequencing and bioinformatics tools (e.g., Bbduk and MetaPhlAn4) were used for data analysis.
• Study Design:
  • Six ampoules were sent to each participant, with five containing the reagent and one as a negative control.
  • Each participant reconstituted the reagent and performed DNA extraction using two methods, with results sent back for analysis.
  • The study aimed to identify variability in DNA extraction methods and assess the impact on microbiome composition.

Key Findings

• Variability in DNA Extraction: A large range of DNA extraction methods (44 in total) showed high variability in performance, indicating the need for standardisation.
• Stability of the Reagent: The reagent was found to be stable at various temperatures, with species composition remaining consistent over time. The similarity to the baseline (-20°C) was greater than 98%.
• Intra-laboratory Variation: Within the same laboratory, the five replicates of the reagent showed consistent DNA concentration, purity, and integrity (CV < 1), except for three methods that failed to detect certain bacterial species in some samples.
• Inter-laboratory Variation: Different labs using the same extraction kit showed variation in results, highlighting the importance of standardised protocols and quality criteria.
• Proposed MQC: The study proposed MQC to be established for users to ensure accurate and consistent DNA extraction procedures.

Recommendations

• The WC-Gut RR (NIBSC 22/210) should be established as a WHO IRR for gut microbiome DNA extraction.
• MQC should be developed based on the four reporting measures (Sensitivity, FPRA, Diversity, and Similarity) to ensure accuracy and reproducibility.
• The study recommends that users aim to meet the proposed MQC values to enhance the accuracy of their DNA extraction and microbiome analysis.
• Further stability testing is planned for additional timepoints (6 months, 12 months, 2, 3, 5, and 10 years).
• The study acknowledges the need for separate reference reagents to assess matrix bias (e.g., from stool, mucus, or blood samples).

Conclusion

The collaborative study demonstrated the importance of standardisation in microbiome research, particularly in DNA extraction. The WC-Gut RR (NIBSC 22/210) is a valuable tool for evaluating and improving the accuracy of DNA extraction methods, ensuring that microbiome studies are more reliable and comparable across different laboratories and studies. The proposed MQC and the establishment of the reagent as a WHO IRR are essential steps towards harmonising microbiome research and supporting the development of microbiome-based therapies and diagnostics.