gram stains a resource for retrospective analysis of bacterial pathogens in clinical studies革兰染色资源回顾性分析细菌病原体的临床研究.pdfVIP

Gram Stains: A Resource for Retrospective Analysis of Bacterial Pathogens in Clinical Studies 1 1 2 3 1 1 Usha Srinivasan *, Sreelatha Ponnaluri , Lisa Villareal , Brenda Gillespie , Ai Wen , Arianna Miles , 1 1 2 4 1 Brigette Bucholz , Carl F. Marrs , Ram K. Iyer , Dawn Misra , Betsy Foxman 1 Department of Epidemiology, School of Public Health, University of Michigan, Ann Arbor, Michigan, United States of America, 2 Molecular Genetics Laboratory, Michigan Medical Genetics Laboratories, University of Michigan Medical Center, Ann Arbor, Michigan, United States of America, 3 Department of Biostatistics, School of Public Health, University of Michigan, Ann Arbor, Michigan, United States of America, 4 Department of Family Medicine, Wayne State University, Detroit, Michigan, United States of America Abstract We demonstrate the feasibility of using qPCR on DNA extracted from vaginal Gram stain slides to estimate the presence and relative abundance of specific bacterial pathogens. We first tested Gram stained slides spiked with a mix of 108 cfu/ml of Escherichia coli and 105 cfu/ml of Lactobacillus acidophilus. Primers were designed for amplification of total and species- specific bacterial DNA based on 16S ribosomal gene regions. Sample DNA was pre-amplified with nearly full length 16S rDNA ribosomal gene fragment, followed by quantitative PCR with genera and species-specific 16S rDNA primers. Pre- amplification PCR increased the bacterial amounts; relative proportions of Escherichia coli and Lactobacillus recovered from spiked slides remained unchanged. We applied this method to forty two archived Gram stained