A Capillary Electrophoresis Method for Analyzing the Diversity of Vaginal Microbiome

Abstract

16S ribosomal RNA (rRNA) gene sequence analysis is the common method to identify the bacteria in human vaginal flora. While specific DNA primers were designed to target 16S rRNA gene sequences, DNA fragment analysis using capillary electrophoresis can obtain more accurate qualitative and quantitative information on the microbiome. This study aimed to assess the ability of capillary electrophoresis method to analyze the diversity of vaginal microbiome and provide a theoretical basis for the accurate gene detection of vaginal flora. We collected 75 vaginal secretion samples from female outpatients aged 25 - 50, who had undergone rou-tine gynecologic examinations in Fujian provincial hospital from March 2021 to April 2021. Clinical diagnosis was based on the results of microscopic examination of Gram-stained specimens and biochemical tests of bacteria (pH value, catalase, leukocyte esterase, sialidases, β-glucuronidase, and acetylglucosaminidase). Vaginal secretion samples were collected and then total bacterial DNA was extracted. We used six pairs of fluorescent dye tagged specific primers that were designed based on the 16S rRNA genes of four Lactobacillus species (L. iners, L. crispatus, L. jensenii, L. gasseri), Gardnerella vaginalis, and Atopobium vaginae. PCR products of six species of bacteria were detected and analyzed by a 3130 Genetic Analyzer. Seventy-five samples were divided into two groups according to the vaginal microbiome evaluation, including 50 cases which had vaginal bacteria balance and 25 cases which had vaginal bacteria disorder. PCR amplification of 16S ribosomal RNA genes of L. iners, L. crispatus, L. jensenii, L. gasseri, Gardnerella vaginalis, and Atopobium vaginae was successfully performed on the DNA extracted from vaginal secretion samples. Four Lac-tobacillus species were detected in 4 - 33 cases of "Balance" group, and Gardnerella vaginalis was detected in 23 cases of "Disorder" group and, simultaneously, Atopobium vaginae was detected in 20 cases. Based on the technique of DNA fragment analysis using capillary electrophoresis method, the most common vaginal bacteria in Chinese healthy women are L. iners and L. crispatus. Gardnerella vaginalis and Atopobium vaginae are the most common pathogenic bacteria detected in the patients who had vaginal bacteria disorder. Using capillary electrophoresis method to detect the vaginal bacteria will be useful for accurate identifica-tion of vaginal microbiome. There will be an application value to find out the composition of the vaginal microbiome rapidly and detect specific gene markers to identify potential pathogenic bacteria when women are at risk of serious illness before they develop obvious symptoms.