Simultaneous real-time PCR detection of nine prevalent sexually transmitted infections using a predesigned double-quenched TaqMan probe panel

Abstract

Sexually transmitted diseases are major causes of infertility, ectopic pregnancy, and premature birth. Here, we developed a new multiplex real-time polymerase chain reaction (PCR) assay for the simultaneous detection of nine major sexually transmitted infections (STIs) found in Vietnamese women, including Chlamydia trachomatis, Neisseria gonorrhoeae, Gardnerella vaginalis, Trichomonas vaginalis, Candida albicans, Mycoplasma hominis, Mycoplasma genitalium, and human alphaherpesviruses 1 and 2. A panel containing three tubes × three pathogens/tube was predesigned based on double-quenched TaqMan probes to increase detection sensitivity. There was no cross-reactivity among the nine STIs and other non-targeted microorganisms. Depending on each pathogen, the agreement with commercial kits, sensitivity, specificity, repeatability and reproducibility coefficient of variation (CV), and limit of detection of the developed real-time PCR assay were 99.0%-100%, 92.9%-100%, 100%, <3%, and 8-58 copies/reaction, respectively. One assay cost only 2.34 USD. Application of the assay for the detection of the nine STIs in 535 vaginal swab samples collected from women in Vietnam yielded 532 positive cases (99.44%). Among the positive samples, 37.76% had one pathogen, with G. vaginalis (33.83%) as the most prevalent; 46.36% had two pathogens, with G. vaginalis + C. albicans as the most prevalent combination (38.13%); and 11.78%, 2.99%, and 0.56% had three, four, and five pathogens, respectively. In conclusion, the developed assay represents a sensitive and cost-effective molecular diagnostic tool for the detection of major STIs in Vietnam and is a model for the development of panel detections of common STIs in other countries.

Conflict of interest statement

The authors have declared that no competing interests exist.

Copyright: © 2023 Bui et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Figures

Fig 1. Amplification plots of “in-house” real-time PCR assay using double-quenched TaqMan probes for nine pathogens.

Standard plasmids at seven 10-fold reducing concentrations in the range of 5 × 102–5 × 108 copies/reaction (plots were presented from the left to the right).

Fig 2. Standard curves representing for the relationship between log10 plasmid copies/reaction and the threshold cycle (Ct).

Equations: y = -ax + b with R2 for amplification of nine pathogens.

Fig 3. Real-time PCR amplification curves taken from five representative vaginal swab clinical samples.

Samples include positive with one (A1–A3), two (B1–B3), three (C1–C3), four (D1–D3), and five (E1–E3) pathogens among the screened nine STIs. Positive and negative controls in the three master mixes 1/2/3 were PC 1/2/3 and NC1/2/3, respectively. Amplification curves for positive controls, negative controls, and positive clinical samples are presented in red, black, and green, respectively.