Developing A Large Collection of Vaginal Microbiota Assays Using TaqManTM Chemistry on High Throughput Nano-Microfluidic OpenArrayTM
Kelly Li, Sunali Patel, Nicole Fantin, Ioanna Pagani, Nitin Puri, Evan Diamond, Kamini Varma
Genetic Analysis R&D, Life Science Division, Thermo Fisher Scientific, 180 Oyster Point Blvd, South San Francisco, CA 94080
Imbalance of vaginal flora has been implicated in vaginal infections, like bacterial vaginosis (BV), vulvovaginal candidiasis (VC) and Trichomonas vaginitis. These infections are associated in pre-term birth, infertility, and increased risk of sexually transmitted diseases (STD). Studying vaginal microbiota, their dynamics and imbalance provides insights into women health. Current technologies for vaginal microbial studies are costly, lack sensitivity/specificity, or require lengthy workflows. Conventional qPCR-based detection is powerful, but lacks of target throughput. To address these unmet needs, we have developed a large collection of TaqManTM microbiota assays by leveraging TaqManTM chemistry and high throughput OpenArrayTM technology. We targeted over 30 healthy or pathogenic species in vaginal microbiota, including bacteria, fungi, protozoa and viruses. We identified unique gene targets that are species-specific and designed TaqManTM assays using proprietary design pipeline. These assays were evaluated with synthetic and ATCC genomic DNA (gDNA) controls on OpenArrayTM plate, a microscope slide with 3,072 through-holes. The assays were evaluated with each of their controls. The accuracy revealed that all the assays display expected on-target Ct values with their corresponding controls. For specificity, each assay was tested against all the rest of synthetic controls as well as inclusivity and exclusivity panels of ATCC gDNA samples. No significant cross-reactivity was observed. Linearity and analytical sensitivity studies revealed that at least 5 log linear dynamic range (with R2 >0.99) with limit of detection (LOD) down to ~100 copies for all and even lower for some of the analytes. In summary, we developed a large collection of TaqManTM vaginal microbiota assays. We have demonstrated excellent assay performance of accuracy, sensitivity, specificity, and reproducibility. In conjunction with OpenArrayTM platform, the application enables researchers to study vaginal microbiota in a single reaction with a simple workflow, fast turnaround time, and high throughput yet flexible sample/target combinations.
For Research Use Only.
Not for use in diagnostic procedures