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. 2021 Aug 23:11:704037.
doi: 10.3389/fcimb.2021.704037. eCollection 2021.

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

Zhaoyang Sun  1   2   3 , Jun Meng  4 , Su Wang  1   2   3 , Feng Yang  1   2   3 , Tao Liu  1   2   3 , Xianping Zeng  5 , Dijun Zhang  5 , Haowei Zhu  5 , Wenjing Chi  1   2   3 , Yixin Liu  1   2   3 , Wenrong Jiang  1   2   3 , Li Ding  1   2   3 , Yingxin Miao  1   2   3 , Yong Wu  5 , Hu Zhao  1   2   3 , Yanmei Zhang  1   2   3
Affiliations

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

Zhaoyang Sun et al. Front Cell Infect Microbiol. .

Abstract

Background: Sexually 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.

Method: Fluorescently 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.

Results: The 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).

Conclusions: STI-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.

Keywords: High-throughput multiplex gene detection system (HMGS); Sexually transmitted infections (STIs); pathogens; positive rates; rapid; semi-quantitative detection.

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Conflict of interest statement

Authors XZ, DZ, HWZ, and YW were employed by company Ningbo HEALTH Gene Technologies Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
The sexually transmitted infection high-throughput multiplex gene detection system (STI-HMGS) assay produced specific amplification signals for 8 specific targets. The X-axis indicates the actual PCR product size while the Y-axis indicates the signal intensity. (A-H) Assay results arising from the amplification of 6 STI pathogens and two control genes: Trichomonas vaginalis (TV), Treponema pallidum (TP), Chlamydia trachomatis (CT), Ureaplasma urealyticumm (UU), Neisseria gonorrhoeae (NG), Mycoplasma hominis (MH), human internal DNA control gene (Human DNA) and systematic internal control (IC), respectively. All target genes were specifically amplified by the STI-HMGS assay without non-specific amplification.
Figure 2
Figure 2
The optimized sexually transmitted infection high-throughput multiplex gene detection system(STI-HMGS) assay exhibited high levels of sensitivity for the simultaneous detection of all six pathogens and control genes in a single PCR. The detection limits of the STI-HMGS assay when detecting pathogens were determined by amplifying plasmids (diluted by ten-fold) containing equal concentrations of 6 pathogens and 2 quality control templates at a concentration of 100 copies/µL. The six pathogen-defining DNA targets all generated specific peaks, from left to right are Treponema pallidum (TP), Trichomonas vaginalis (TV), Mycoplasma hominis (MH), Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Ureaplasma urealyticumm (UU). The human internal DNA control gene (Hum DNA) and systematic internal control (IC) produced specific peaks at 308bp and 315bp, respectively.
Figure 3
Figure 3
The sexually transmitted infection high-throughput multiplex gene detection system (STI-HMGS) assay accurately detected an individual pathogen in a mixture containing two DNA templates without interference from a dominant template over a large range of concentrations. (A) The Trichomonas vaginalis (TV) plasmid produced a signal intensity of 2200rfu. (B–G) Within the mixture of DNA templates, we increased the concentration of the Treponema pallidum (TP) plasmid from 101, 102, 103, 104, 105 to 106 copies/µL in a sequential manner while maintaining the concentration of the TV plasmid at 10 copies/µL throughout. The signal intensity for the TV plasmid showed progressive weakening from 20000, 18000, 12000, 6000, 2000, to 1000rfu. The two control genes were maintained at the same concentrations at all times; the signal intensity for these genes also showed a gradual reduction.
Figure 4
Figure 4
The detection of six pathogens from corresponding plasmids showing the quantitative standard curves obtained by 10-fold dilutions of the DNA template from 1 to 104 copies/µL (x-axis) and their corresponding sexually transmitted infections high-throughput multiplex gene detection system (STI-HMGS) peak areas (y-axis). Each concentration was tested on three independent occasions using the STI-HMGS assay. Error bars are not shown if they are shorter than the size of the symbol used to indicate the mean value of the three peak areas. (A–F) Calibration curves for Mycoplasma hominis (MH) (R2 = 0.9829), Chlamydia trachomatis (CT) (R2 = 0.9410), Neisseria gonorrhoeae (NG) (R2 = 0.9799), Ureaplasma urealyticumm (UU) (R2 = 0.9588), Trichomonas vaginalis (TV) (R2 = 0.9761) and Treponema pallidum (TP) (R2 = 0.9826), respectively.
Figure 5
Figure 5
Analyses of clinical data based on Sexually transmitted infections high-throughput multiplex gene detection system (STI-HMGS). (A) STI-HMGS assays of the 123 confirmed STI patients showed that 83.7% of patients had a single infection, 13.0% had a double infection, and 3.3% had a triple infection. (B) A comparison of the positivity rates for each pathogen and the overall rate among the three groups. (C) The relationship between positive detection rates and different age groups. (D) A comparison of the infection rate between the 21-30 years group and the 31-40years group; only infections caused by Chlamydia trachomatis (CT) were significant. (E) Comparison of infection rate between the ≤30 years group and the >30 years group across the 381 subjects; only infections caused by CT were statistically significant. *p < 0.05, **p < 0.01, ****p < 0.0001, ns, not significant; p > 0.05.
Figure 6
Figure 6
Gender ratio and infection rates across different groups. (A–D) Gender ratio across all 381 subjects, 143 patients with genital tract infections, 124 infertile patients, and 115 healthy controls. (E) The infection rates of 131 males compared with 250 females. (F) The infection rates of 82 males compared with 61 females in the group featuring 143 genital tract infections. *p < 0.05, **p < 0.01, ****p < 0.0001, ns, not significant; p > 0.05.
Figure 7
Figure 7
The distribution of peak area values for each pathogen as determined by the Sexually transmitted infections high-throughput multiplex gene detection system (STI-HMGS) assay from the 123 positive clinical samples compared to pathogen loading. (A) The peak area values for each individual pathogen, which showed a wide distribution between 0 and 550000, varied across the different clinical samples. (B) The level of the peak area values for Neisseria gonorrhoeae (NG) were significantly higher than the level of Chlamydia trachomatis (CT). (C) The level of the peak area values for NG was significantly higher than the level of Mycoplasma hominis (MH). (D) The level of peak area values for NG were significantly higher than the level for Ureaplasma urealyticumm (UU). ***p < 0.001, ****p < 0.0001; p > 0.05.
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