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.

The Past, Present, and Future in the Diagnosis of a Neglected Sexually Transmitted Infection: Trichomoniasis

“…In addition to the cleared systems mentioned above, different RT-PCR assays for the simultaneous detection of more than one STI pathogen (including T. vaginalis ) have also been published. Recently, different research groups have designed an in-house multiplex RT-PCR for the diagnosis of up to nine pathogens ( C. trachomatis , N. gonorrhoeae , T. vaginalis , C. albicans , Mycoplasma hominis , M. genitalium , Ureaplasma urealyticum , U. parvum , Gardnerella vaginalis, and human herpes viruses) with sensitivity and specificity values > 91% and an estimated cost per test of nearly USD 3 [ 138 , 139 ]. Also, different groups have developed loop-mediated isothermal amplification (LAMP) assays for the detection of T. vaginalis .…”

The Past, Present and Future in the Diagnosis of a Neglected Sexually Transmitted Infection: Trichomoniasis

“…In addition to the cleared systems mentioned above, different RT-PCR assays for the simultaneously detection of more the one STI pathogen (including T. vaginalis) have also been published. Recently, different research groups have designed an in-house multiplex RT-PCR for the diagnosis of up to nine pathogens (C. trachomatis, N. gonorrhoeae, T. vaginalis, C. albicans, Mycoplasma hominis, M. genitalium, Ureaplasma urealyticum, U. parvum, Gardnerella vaginalis and human herpes viruses) with sensitivity and specificity values > 91% and an estimated cost per test of near $3 [139,140]. Also, different groups have developed loop-mediated isothermal amplification (LAMP) assays for detection of T. vaginalis.…”