Abstract
Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are the main pathogenic microorganisms causing sexually transmitted infections. In this study, a multiplex thermostable recombinase polymerase amplification-lateral flow detection (RPA-LFD) assay was established, and the reaction conditions such as the ratio of primer concentration, magnesium ion concentration, amplification time and template DNA concentration in the multiplex RPA reaction were optimized. The optimized multiplex RPA-LFD method was used to detect both CT and NG positive control plasmids, and it was found that the LFD could be used to obtain visible results when the plasmid copy number was only 200. The sensitivity of the multiplex RPA-LFD method used for clinical samples was 85.62 (95% CI at 53.66–97.29) for NG detection and 90.90 (95% CI at 57.12–99.52) for CT detection.
Highlights
Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are the most common sexually transmitted pathogens and are the main contributors to sexually transmitted infections (STIs)
The results showed that the genomic DNA of CT or NG strains could be amplified by Recombinase polymerase amplification (RPA) with the corresponding primers used in this study, whereas the amplified products could not be obtained by the same experimental method using the other four strains or human genomic DNA as templates, as shown in Fig 1A and 1B, suggesting a high specificity of the primers used in this study
The results showed that the amplification products could be obtained by PCR and RPA respectively using primers corresponding to the positive control plasmids, while the amplification products could not be obtained when the primers did not correspond to the positive control plasmids, suggesting that the positive control plasmids were successfully constructed
Summary
Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are the most common sexually transmitted pathogens and are the main contributors to sexually transmitted infections (STIs). CT infections are prevalent among young people under the age of 25 and pose a major threat to the reproductive health of the population [4] Both of these pathogens are often widely recognized as sexually transmitted diseases, women who develop either of these sexually transmitted infections during pregnancy are at increased risk of ectopic pregnancy and can lead to adverse pregnancy outcomes such as premature rupture of membranes, preterm delivery and low birth weight of the newborn [5,6,7]. RPA amplification products can be detected by various methods, such as flocculation assay detection, lateral flow assay detection (LFD), electrochemical detection, chemiluminescent detection, and silicon microring resonator (SMR)-based photonic detection Among these methods, the lateral flow immunoassay is widely used because of its rapid, sensitive, specific, and cost-effective nature. We optimized the reaction conditions for RPA and developed a multiplex thermostatic RPA-LFD assay, which was successfully used to detect CT and NG in clinical samples
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