Study on a Rapid Extraction and Detection Method for 16S rRNA of Intestinal Flora
Abstract
This study established a rapid extraction method for the 16S rRNA gene of intestinal flora. Combined with next-generation sequencing (NGS) technology, this method can be applied to intestinal microbial ecology analysis. The fecal sample was mixed with lysis buffer, incubated at 90°C for 15 minutes, vortexed, and then centrifuged. The supernatant was collected, and specific primers were added for PCR amplification. The PCR products were purified, and index tags were established for the targets to obtain libraries. After library purification and quality inspection, the libraries could be loaded for sequencing. Under optimized conditions: when the number of PCR cycles was 25 and the initial amount of DNA for library construction was 12.5 ng, fewer chimeras were generated. The library size was 500–700 bp, and there were no primer dimers of approximately 120 bp. This method is rapid, accurate, and sensitive, and can be used for the analysis and detection of the 16S rRNA gene of intestinal flora.
References
Clemente J, Ursell L, Parfrey L, et al., 2012, The Impact of the Gut Microbiota on Human Health: An Integrative View. Cell, 148(6): 1258–1270.
Liao B, Wang S, Zhang J, et al., 2025, The Role of Intestinal Microbiota in Human Diseases. Chinese Journal of Gastroenterology, 20(2): 126-128.
Zhao L, 2010, Genomics: The Tale of Our Other Genome. Nature, 465(7300): 879–880.
Song G, Su D, Yang M, et al., 2019, A Detection Method for 16S rRNA Gene of Intestinal Flora and its Application, China.
Ye L, Yan Y, Chen Q, 2016, Application of High-Throughput Sequencing Technology in Metagenomics Research of Intestinal Microbiota. Journal of Chinese Institute of Food Science and Technology, 16(7): 216–223
Zhang R, Wang J, Liu P, 2016, Research Methods for Intestinal Microecology. Chinese Journal of Microecology, 28(8): 971–974.
Almeida A, Mitchell A, Boland M, et al., 2019, A New Genomic Blueprint of the Human Gut Microbiota. Nature, 2019(568): 499–504.
Viciani E, Anonye B, Kumar N, et al., 2019, A Human Gut Bacterial Genome and Culture Collection for Improved Metagenomic Analyses. Nature Biotechnology, 2019(37): 186–192.
Chen L, Zhou X, Zhou Y, et al., 2016, Application of High-Throughput Sequencing Technology in Intestinal Flora Research. Journal of Food Industry, 37(3): 269–273.
Jovel J, Patterson J, Wang W, et al., 2016, Characterization of the Gut Microbiome Using 16S or Shotgun Metagenomics. Frontiers in Microbiology, 2016(7): 459.
Johnson J, Spakowicz D, Hong B, et al., 2019, Evaluation of 16S rRNA Gene Sequencing for Species and Strain-Level Microbiome Analysis. Nature Communications, 10(5029): 1–11.
Tian X, Feng J, Wang Y, et al., 2019, A Rapid and Efficient DNA Extraction Method and Its Application in qPCR Detection of Staphylococcus aureus. Food Science and Technology, 44(2): 344–350.
Peng X, Yu K, Deng G, et al., 2013, Comparison of Direct Boiling Method with Commercial Kits for Extracting Fecal Microbiome DNA by Illumina Sequencing of 16S rRNA Tags. Journal of Microbiological Methods, 95(3): 455–462.
