Visual and rapid identification of Chlamydia trachomatis and Neisseria gonorrhoeae using multiplex loop-mediated isothermal amplification and a gold nanoparticle-based lateral flow biosensor
Xu Chen, Qingxue Zhou, Wei Yuan, Yuanfang Shi, Shilei Dong, Xin-Hua Luo
Abstract
Sexually transmitted chlamydia and gonorrhea infections caused by the bacteria Chlamydia trachomatis and Neisseria gonorrhoeae remain a major public health concern worldwide, particularly in less developed nations. It is crucial to use a point of care (POC) diagnostic method that is quick, specific, sensitive, and user-friendly to treat and control these infections effectively. Here, a novel molecular diagnostic assay, combining multiplex loop-mediated isothermal amplification (mLAMP) with a visual gold nanoparticles-based lateral flow biosensor (AuNPs-LFB) was devised and used for highly specific, sensitive, rapid, visual, and easy identification of C. trachomatis and N. gonorrhoeae . Two unique independent primer pairs were successful designed against the ompA and orf1 genes of C. trachomatis and N. gonorrhoeae , respectively. The optimal mLAMP-AuNPs-LFB reaction conditions were determined to be 67°C for 35 min. The detection procedure, involving crude genomic DNA extraction (~5 min), LAMP amplification (35 min), and visual results interpretation (<2 min), can be completed within 45 min. Our assay has a detection limit of 50 copies per test, and we did not observe any cross-reactivity with any other bacteria in our testing. Hence, our mLAMP-AuNPs-LFB assay can potentially be used for POC testing to detect C. trachomatis and N. gonorrhoeae in clinical settings, particularly in underdeveloped regions.