Multi-repeat sequences identification using genome mining techniques for developing highly sensitive molecular...

Area of Science:

• Microbiology
• Molecular Diagnostics
• Infectious Diseases

Background:

• Chlamydia trachomatis (C. trachomatis) is a prevalent sexually transmitted infection (STI) with a high rate of asymptomatic cases, leading to undetected infections.
• Undetected C. trachomatis infections contribute to significant preventable adverse health outcomes, particularly in women and children.
• There is a critical need for developing more efficient and sensitive diagnostic methods for C. trachomatis.

Purpose of the Study:

• To develop a novel diagnostic assay for Chlamydia trachomatis (C. trachomatis) using genome-mining approaches.
• To design and validate a primer pair targeting identical multi-repeat sequences (IMRS) within the C. trachomatis genome.
• To assess the analytical sensitivity of the developed assay compared to a standard method.

Main Methods:

• Genome-mining was employed to identify identical multi-repeat sequences (IMRS) in the C. trachomatis genome.
• A specific primer pair targeting IMRS was designed for PCR-based detection.
• Genomic DNA was serially diluted and used as a template for PCR, with results compared to 16S rRNA PCR.

Main Results:

• The novel C. trachomatis IMRS-PCR assay demonstrated an analytical sensitivity of 4.31 pg/µL.
• This sensitivity is superior to the 16S rRNA PCR method, which had a sensitivity of 9.5 fg/µL.
• Lateral flow and isothermal assays were successfully developed for C. trachomatis DNA detection.

Conclusions:

• The study successfully developed a sensitive and efficient PCR-based assay for detecting Chlamydia trachomatis (C. trachomatis) DNA.
• The novel IMRS-PCR assay shows potential for improved diagnostics, especially in resource-limited or field settings.
• The developed technology can be integrated into miniaturized, isothermal, microfluidic, and lab-on-a-chip devices for point-of-care testing.