B-250 Simultaneous Detection of Twelve Pathogens of Sexually Transmitted Infections Using an Innovative All-in-One PCR Instrument

Abstract

Abstract Background Every day, nearly 1 million people are infected with sexually transmitted infections (STI) that are transmitted through sexual contact. Early diagnosis and treatment of STI are critical in reducing their transmission, and PCR is the most sensitive method among diagnostic tests. To address this need, we have developed a new STI Real-Time PCR-based diagnostic kit, which is capable of detecting twelve STI pathogens simultaneously in up to 94 specimens at once. To evaluate the analytical performance, we used the ExistationTM FA System, which automatically performs from decapping primary sample tubes followed by nucleic acid extraction to PCR analysis within 2 h. Twelve (12) pathogens of STI are described below: Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Ureaplasma urealyticum (UU), Mycoplasma genitalium (MG), Trichomonas vaginalis (TV), Mycoplasma hominis (MH), Herpes simplex virus type1 (HSV1), Herpes simplex virus type2 (HSV2), Ureaplasma parvum (UP), Treponema pallidum (TP), Candida albicans (CA), Gardnerella vaginalis (GV), from male urine and female vaginal swabs. Methods The analytical performance evaluation was performed using the new STI diagnostic kit to detect twelve pathogens of STI. The analysis was performed using culture fluids (CT, NG, UU, MG, TV, MH, HSV1, HSV2, UP, CA, GV; ATCC, USA) and TP genomic DNA (Vircell, Spain), which were spiked in urine and vaginal swab media. The limit of detection (LoD) was determined by testing multiple replicates of serial dilutions of the spiked sample around the expected LoD. The LoD was defined as the lowest dilution at which the assay detected ≥95% of 24 replicates. To evaluate precision, each sample was tested 2 times a day in duplicate per run for 5 days. To assess cross-reactivity, twenty-two bacteria and viruses related to STI were used. Results The LoD was determined 60.26 IFU/mL, 35.48 cfu/mL, 467.74 copy/mL, 45.71 cell/mL, 19.5 cells/mL, 316.23 copy/mL, 199.53 TCID50/mL, 13.18 TCID50/mL, 588.84 copy/mL, 993.25 copy/ml, 660.69 copy/mL, 389.05 copy/mL in Urine and 47.86 IFU/mL, 27.54 cfu/mL, 363.08 copy/mL, 38.90 cell/mL, 15.14 cells/mL, 245.47 copy/mL, 181.97 TCID50/mL, 8.71 TCID50/mL, 575.44 copy/mL, 741.31 copy/ml, 524.81 copy/mL, 288.40 copy/mL in vaginal swab media for CT, NG, UU, MG, TV, MH, HSV1, HSV2, UP, CA, GV, and TP, respectively. For repeatability assay, the 12 STI samples showed similar Ct value in each run, with all standard deviation (SD) were less than 2.0. In terms of cross-reactivity, there were no positive results obtained from testing bacteria and viruses using the new STI diagnostic kit. Conclusions The ExistationTM FA system is highly convenient platform for Real-Time PCR-based diagnostics. Particularly, our new STI diagnostic kit provides an accurate and efficient method for simultaneous detection of 12 STI pathogens.