Life Sciences Commons^™

Gram Stains: A Resource For Retrospective Analysis Of Bacterial Pathogens In Clinical Studies, Usha Srinivasan, Sreelatha Ponnalurl, Lisa Villareal, Brenda Gillespie, Ai Wen, Arianna Miles, Brigette Bucholz, Carl F. Marrs, Ram K. Iyer, Dawn Misra, Betsy Foxman

Faculty Publications

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 105cfu/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 …