A New Multiplex Genetic Detection Assay Method for the Rapid Semi-Quantitative Detection of Six Common Curable Sexually Transmitted Pathogens From the Genital Tract

Abstract

BackgroundSexually transmitted infections (STIs) are some of the most common communicable conditions and exert impact on the health and lives of many hundreds of millions of people across the world every year. Screening high-risk populations and conducting comprehensive detection tests would lead to a significant improvement in preventing the transmission of STIs and help us to provide rapid treatment to those affected. Here, we successfully established and validated a novel high-throughput multiplex gene detection system (HMGS) for the simultaneous and semiquantitative detection of six important curable sexually transmitted pathogens in a single reaction from secretions samples.MethodFluorescently labeled primers were designed to target specific conserved and single-copy gene fragments of Ureaplasma urealyticum (U. urealyticum), Mycoplasma hominis (M. hominis), Chlamydia trachomatis (C. trachomatis), Neisseria gonorrhoeae (N. gonorrhoeae), Trichomonas vaginalis (T. vaginalis), and Treponema pallidum (T. pallidum). The specificity and sensitivity of the STI-HMGS was validated and optimized using plasmids and quantitative genomic DNA. Next, we validated the performances of the STI-HMGS for clinical application by testing samples of clinical secretions collected from patients who visited the gynecology and urology outpatient clinics of our reproductive medicine center. Results derived from the STI-HMGS were then compared with three approved commercialized kits that used to detect U. urealyticum, C. trachomatis and N. gonorrhoeae, respectively, followed by further validation with Sanger sequencing for all pathogens. Finally, a comprehensive analysis of epidemiology was performed among different subgroups to investigate the association between infection rates and clinically-relevant information.ResultsThe sensitivity of STI-HMGS for six target genes was 10 copies/µL. Data derived from the detection of 381 clinical secretions demonstrated that the STI-HMGS exhibited high concordance rate compared with approved commercialized kits and almost 100% sensitivity and specificity for the detection of six sexually transmitted pathogens when validated by Sanger sequencing. Semi-quantitative analysis found that STIs caused by N. gonorrhoeae had a significantly higher (P<0.05) pathogen load than the other pathogens. Infections caused by C. trachomatis were significantly more common in younger individuals (P<0.05). We also found that U. urealyticum infections were more likely to happen in females; while the males were more affected by N. gonorrhoeae (P<0.05).ConclusionsSTI-HMGS proved to be an efficient method for the semi-quantitative detection of six important curable sexually transmitted pathogens and therefore represents an alternative method for the clinical detection and monitoring of STIs.

Rapid and simultaneous detection of multiple pathogens in the lower reproductive tract during pregnancy based on loop-mediated isothermal amplification-microfluidic chip

“…Multi-target LAMP detection has been relatively widely used in the detection of the foodborne pathogens [ 33 , 39 , 45 ], but the detection of multiple pathogens in the reproductive tract during pregnancy is rather rare, largely because of the difficulty of obtaining samples from pregnant women for method validation and evaluation. Sun et al [ 46 ] proved that high-throughput multiplex gene detection system of Sexually Transmitted Infections (STI-HMGS) was an efficient approach for the semi-quantitative detection of six important curable sexually transmitted pathogens by discriminating the length of target fragments in the products. In this study, the LAMP-microfluidic chip could simultaneously and rapidly detect and distinguish all five target pathogens in clinic samples through primer fixation and fluorescence real-time detection, and report the results within 90 min.…”

Copan Walk Away Specimen Processor (WASP) Automated System for Pathogen Detection in Female Reproductive Tract Specimens

“…PCR was performed in triplicate in 20 μl mixtures containing 2 μl of polymerase and 18 μl of reaction liquid (Jiangsu Bioperfectus Technologies Co. Ltd. and BioChain Co. Ltd.). PCRs were performed on an ABI 7500 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) using the following cycling parameters: 37°C for 5 min with uracil-DNA glycosylase, 95°C for 5 min, followed by 40 cycles at 95°C for 10s and 55°C for 40s ( Sun et al., 2021 ). The products were analyzed on the Cobas z480 Analyzer (F. Hoffmann-La Roche Ltd., USA).…”

Detection of Ureaplasma urealyticum by Catalytic Hairpin Assembly Combined with a Lateral Flow Immunoassay Strip

“…New detection methods are also being developed, such as loop-mediated isothermal amplification (LAMP) method, 11 droplet digital PCR, 12 and high-throughput multiple gene detection system. 13 …”

“…New detection methods are also being developed, such as loop-mediated isothermal amplification (LAMP) method, 11 T h i s c o n t e n t i s droplet digital PCR, 12 and high-throughput multiple gene detection system. 13 Catalytic hairpin assembly (CHA) is an isothermal nonenzyme nucleic acid signal amplification system. It was originally reported by YIN.…”

“…The reported specificity and sensitivity were 85 and 94%, respectively. , The PCR method has high sensitivity and specificity, but it needs special technical personnel and specific requirements for instruments, so it cannot be popularized in grass-roots units and remote areas. New detection methods are also being developed, such as loop-mediated isothermal amplification (LAMP) method, droplet digital PCR, and high-throughput multiple gene detection system …”