704 a reservoir of the STI [1-4]. There are limited studies about Ureaplasma ure- alyticum and Mycoplasma hominis in asymptomatic people. Generally, Ureaplasma urealyticum is not known as a clinical pathogen but it can be a cause of urethritis, es- pecially in patients resistant to routine treatment [5]. Mycoplasma hominis also does not play a great role in the pathogenesis of urethritis, but it can be a heavy pathogen in immunocompromised patients [6]. Recently, many re- ports have revealed that microorganisms such as Urea- plasma urealyticum and Mycoplasma hominis can contrib- ute not only to lower genitourinary infection but also to in- fertility [7]. Therefore, identification of the incidence of asympto- matic STI such as urethritis or cervicitis and determi- nation of the prevalence of these 6 species in asymptomatic people is very important. Accordingly, we conducted a screening test for STI by using multiplex polymerase chain reaction (PCR) in asymptomatic people. MATERIALS AND METHODS This study was a point prevalence study. The institutional review board of The Catholic University of Korea, College of Medicine, approved the study protocol, and all patients provided written informed consent to participate in the study (IRB no: SC10SNSI0091). 1. Sample collection From July 2010 to December 2010, 802 persons who came to the hospital for a general checkup participated volun- tarily and a brief questionnaire was obtained. Sexually ac- tive, asymptomatic people aged between 20 and 60 years were enrolled. We defined ‘sexually active’ as having had sexual intercourse in the preceding 3 months. People who had a recently cured genitourinary infectious disease with- in 3 months were excluded. People with genitourinary symptoms such as urethral or cervical discharge, dysuria, or itching in the genital area were also excluded. Finally, 709 persons were assigned to be tested. Among these, 430 were men and 279 were women. In men, first-voided urine specimens were collected in sterile 50 ml screw-cap plastic bottles. In women, specimens were obtained via endocer- vical swabs by one gynecologist. 2. Pretreatment of clinical specimens and DNA extraction The specimens from the 709 men and women were equili- brated to room temperature and centrifuged at 5000xg for 15 minutes. The supernatant was discarded, and the pellet was resuspended in 1 ml 1xPBS before DNA extraction. Genomic DNA was extracted from the pretreated speci- mens (swab or urine) by using the QIAampⓇ DNA mini kit (Qiagen, Hilden, Germany) according to the manufactur- er's instructions. 3. Multiplex polymerase chain reaction assay PCR amplification was performed with the SeeplexⓇ STD6 Korean J Urol 2011;52:703-708 Kim et al ACE detection kit (Seegene, Seoul, Korea) according to the manufacturer's instructions. The kit contains sets of pri- mers that were specifically designed from highly conserved regions of genetic sequences for the 6 organisms (Chlamy- dia trachomatis, Neisseria gonorrhoeae, Mycoplasma geni- talium, Ureaplasma urealyticum, Mycoplasma hominis, and Trichomonas vaginalis) by use of Seegene DPO tech- nology [8]. An internal control was included in the PCR mix to detect the presence of PCR inhibitors. For the negative control, sterile deionized water was used as the PCR tem- plate instead of nucleic acid. The positive control contained in the kit was assayed to check the integrity of the primers used in the PCR assay. Amplified fragments were sepa- rated by use of the LabChipⓇ DX SeeplexⓇ Assay system (Caliper, Hopkinton, MA, USA). 4. LabChipⓇ DX SeeplexⓇ assay system Amplified PCR products were separated and detected by automated gel electrophoresis with the LabChipⓇ DX SeeplexⓇ assay system (Caliper, Hopkinton, MA, USA). The PCR products from each sample (20 μl) were trans- ferred to 96-well plates and placed in the LabChipⓇ DX instrument. The samples were loaded automatically on the SeeplexⓇ Chip and detected sequentially according to the sample order. Analysis was performed with designated software (Seegene viewer) that presents each of the sam- ples and identifies the fragments that yield a positive read- out for the bands of interest in the presented results. A tabu- lated matching matrix provides a simple readout, identify- ing matching bands to the type of STI pathogens. A text file is automatically generated and saved for each tape. RESULTS The mean age in this study was 45.4±8.1 years. Among the 709 persons, 229 (32.3%) had at least one microorganism in his or her genitourinary tract. The prevalences of Chlamydia trachomatis, Neisseria gonorrhoeae, Myco- plasma genitalium, Ureaplasma urealyticum, Mycoplas- ma hominis, and Trichomonas vaginalis in asymptomatic people were 5.6% (40/709), 0.4% (3/709), 0.3% (2/709), 22.1% (157/709), 11.6% (82/709), and 1.1% (8/709) re- spectively (Table 1). Among all persons, 50 (7.1%) had pathogens of STI in their urinary tract (Table 2). Of these patients, the number of patients with a single pathogen was 47 (6.6%), for which the number with isolated Chlamydia trachomatis, Neisseria gonorrhoeae, Myco- plasma genitalium, or Trichomonas vaginalis was 37, 1, 1, and 8, respectively. Three double-infections, i.e., samples containing 2 or more pathogens, were identified, and each sample had Chlamydia trachomatis. When Trichomonas vaginalis was identified, it was either isolated alone or co-infected with Mycoplasma hominis only and no other species. The total number of people who had at least two microorganisms in their genitourinary tract was 55 (7.5%). Among these 55 persons, 48 (6.8%) had two different spe- cies whether they were pathogens or not, 6 (0.8%) persons