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Development and Validation of Quantitative Synthetic Molecular Standards for Chlamydia trachomatis Lymphogranuloma Venereum (LGV) Serovars L1, L2, and L3

Poster
Transparent bubble of Chlamydia bacteria.

ASM Microbe 2022

Washington, DC, United States

June 11, 2022

Abstract

Chlamydia trachomatis is a Gram-negative, obligate intracellular pathogen responsible for the majority of bacterial sexually transmitted infections. There are currently 15 serovars of C. trachomatis, which are defined by their tissue tropism; serovars A, B, Ba, and C-K are generally restricted to mucosal tissues, while serovars L1, L2, and L3 infect lymphatic tissues. Serovars L1, L2, and L3 are associated with a long-term infection of the lymphatic system known as lymphogranuloma venereum (LGV). Without proper treatment, LGV can result in the obstruction of lymph flow and chronic swelling of genital tissues. Therefore, the rapid and accurate diagnosis of this disease during the early stages of infection is essential for timely and proper treatment.

While culture-based approaches can be used in the diagnosis of LGV, they are typically time consuming and labor intensive. PCR-based methods provide a highly sensitive and rapid alternative screening approach; however, the development and validation of these assays are dependent on the availability of high-quality reference materials. To address this need, ATCC designed and developed quantitative synthetic molecular standards for C. trachomatis serovars L1, L2, and L3. Here, we used a proprietary strategy to incorporate genes typically targeted in various assays for bacterial detection and identification. The synthetic construct for each serovar contains genes most relevant to that particular strain, and may encode for a variety of membrane proteins, virulence factors, or a variety of other sequences. Each synthetic standard sequence was validated via next-generation sequencing, and precise DNA copy numbers (copies/µL) were generated using Droplet Digital™ PCR (Bio-Rad). The sufficiency of each standard was tested using an independent, publicly available qPCR assay, verifying the efficacy of the design against relevant assays. Here, ten-fold dilutions were used to create standard curves, with DNA concentrations ranging from approximately 1.0×102 to 1×106 copies/µL. Within these data sets, the L1, L2, and L3 standards had R2 values of 1.000, 0.997, and 0.998, respectively, indicating a high degree of reproducibility. Likewise, these standards display high efficiency and amplification, with slopes ranging from M =-3.456 to -3.581. Overall, these data demonstrate the applicability of the C. trachomatis LGV synthetic standards as controls for the development and validation of molecular-based detection and quantification assays.

 

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Presenter

Britany Tang, headshot.

Britany Tang, BS

Biologist, Microbiology R&D, ATCC

Britany Tang is a Biologist at ATCC Microbiology Research and Development. She has extensive experience in the fields of molecular biology and microbiology. Her current work focuses on designing synthetic DNA and RNA molecular standards for molecular diagnostic assays and the development of mock microbial communities for microbiome research. Prior to joining ATCC, her research was centered on arboviral replication and pathogenesis. Britany holds a bachelor’s degree in Biology from George Mason University.