Improving gonorrhoea molecular diagnostics: Genome mining-based identification of identical multi-repeat sequences (IMRS) in Neisseria gonorrhoeae
Abstract
<b>Background</b> Curable sexually transmitted infections (STIs), such as <i>Neisseria gonorrhoeae</i> (<i>N. gonorrhoeae</i>), are a major cause of poor pregnancy outcomes. The infection is often asymptomatic in pregnant women, and a syndrome-based approach of testing leads to a missed diagnosis. Culture followed by microscopy is inadequate and time-consuming. The gold standard nucleic acid amplification tests require advanced infrastructure settings, whereas 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.
<b>Methods</b> We identified new diagnostic target biomarker regions for <i>N. gonorrhoeae</i> 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 (100 pg/μL to 1×10<sup>−3</sup> pg/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 were resolved on 1% agarose gel.
<b>Results</b> Our newly developed <i>N. gonorrhoeae</i> IMRS-PCR assay had an analytical sensitivity of 6 fg/μL representing better sensitivity than the 16S rRNA PCR assay with an analytical sensitivity of 4.3096 pg/μL. The assay was also successfully validated using clinical urethral swab samples. We further advanced this technique by developing an isothermal IMRS, which was both reliable and sensitive for detecting cultured <i>N. gonorrhoeae</i> isolates at a concentration of 38 ng/μL. Combining isothermal IMRS with a low-cost lateral flow assay, we were able to detect <i>N. gonorrhoeae</i> amplicons at a starting concentration of 100 pg/μL.
<b>Conclusion</b> 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.