Improving Gonorrhoea Molecular Diagnostics:Genome Mining-Based Identification of Identical Multi-Repeat Sequences (IMRS) in Neisseria gonorrhoeae Genome

Abstract

Purpose Curable sexually transmitted infections (STIs) such as Neisseria gonorrhoeae (N. gonorrhoeae) is a major cause of poor pregnancy outcome. The infection is often asymptomatic in pregnant women and a syndrome-based approach of testing leads to missed diagnosis. Culture followed by microscopy is inadequate and time-consuming. The gold standard Nucleic Acid Amplification Tests (NAATs) require advanced infrastructure settings whilst point of care tests are limited to immunoassays with sensitivities and specificities insufficient to accurately diagnose asymptomatic cases. This necessitates the development and validation of assays that are fit for purpose.

Materials and methods Here, we have identified new diagnostic target biomarker regions for N. gonorrhoeae using an algorithm for genome mining of identical multi repeat sequences (IMRS). These were then developed as DNA amplification primers to design better diagnostic assays. To test the primer pair, genomic DNA was 10-fold serially diluted (100pg/μL to 1×10-3pg/μL) and used as DNA template for PCR reactions. The gold standard PCR using 16S rRNA primers was also run as a comparative test, and both assay products resolved on 1% agarose gel.

Results Our newly developed N. gonorrhoeae IMRS-PCR assay had an analytical sensitivity of 6 fg/μL representing better sensitivity compared to the 16S rRNA PCR assay with analytical sensitivity of 4.3096 pg/μL. The assay was also successfully validated with clinical urethral swab samples. We further advanced this technique by developing an iso-thermal IMRS, which was both reliable and sensitive for detecting cultured N. gonorrhoeae isolates at a concentration of 38 ng/μL. Combining the iso-thermal IMRS with a low-cost Lateral Flow Assay, we were able to detect N. gonorrhoeae amplicons at a starting concentration of 100 pg/μL.

Conclusion Therefore, there is a potential to implement this concept within miniaturized, isothermal, microfluidic platforms, and laboratory-on-a-chip diagnostic devices for highly reliable point-of-care testing.