Abstract
This study established a multiplex PCR-based microarray to detect simultaneously a diverse panel of 17 sexually transmitted diseases (STDs)-associated pathogens including Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma, Herpes simplex virus (HSV) types 1 and 2, and Human papillomavirus (HPV) types 6, 11, 16, 18, 31, 33, 35, 39, 54 and 58. The target genes are 16S rRNA gene for N. gonorrhoeae, M. genitalium, M. hominism, and Ureaplasma, the major outer membrane protein gene (ompA) for C. trachomatis, the glycoprotein B gene (gB) for HSV; and the L1 gene for HPV. A total of 34 probes were selected for the microarray including 31 specific probes, one as positive control, one as negative control, and one as positional control probe for printing reference. The microarray is specific as the commensal and pathogenic microbes (and closely related organisms) in the genitourinary tract did not cross-react with the microarray probes. The microarray is 10 times more sensitive than that of the multiplex PCR. Among the 158 suspected HPV specimens examined, the microarray showed that 49 samples contained HPV, 21 samples contained Ureaplasma, 15 contained M. hominis, four contained C. trachomatis, and one contained N. gonorrhoeae. This work reports the development of the first high through-put detection system that identifies common pathogens associated with STDs from clinical samples, and paves the way for establishing a time-saving, accurate and high-throughput diagnostic tool for STDs.
Highlights
The major causative agents of sexually transmitted diseases (STDs) are Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma, Herpes simplex virus (HSV), Human papillomavirus (HPV), Human immunodeficiency virus (HIV), PLOS ONE | DOI:10.1371/journal.pone.0133927 July 24, 2015DNA Microarray for STDs Pathogen Detection
We introduce a multiplex, PCR-based oligonucleotide microarray to detect N. gonorrhoeae, C. trachomatis, M. hominis, M. genitalium, Ureaplasma, HSV, and HPV simultaneously, and different serotypes of HSV types 1 and 2, and HPV types 6, 11, 16, 18, 31, 33, 35, 39, 54 and 58
Specific primer pairs for amplification of target genes were determined, including wl-5744 and wl-5839 for 16S rRNA sequence of N. gonorrhoeae, wl-5967 and wl-5968 for 16S rRNA sequence of M. genitalium, M. hominis, and Ureaplasma, wl-5963 and wl-9516 for ompA gene of C. trachomati, wl-9940 and wl-10219 for gB gene of HSV, wl-10247 and wl-10248 for L1 gene of HPV, and wl-9502 and wl-9503 for the positive control β-globin gene, respectively
Summary
The major causative agents of sexually transmitted diseases (STDs) are Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma, Herpes simplex virus (HSV), Human papillomavirus (HPV), Human immunodeficiency virus (HIV), PLOS ONE | DOI:10.1371/journal.pone.0133927 July 24, 2015. Other strategies used in the detection of STD-associated pathogens include immunological assays and DNA amplification or hybridization [15,16] Serological assays such as enzyme immunoassays (EIA) and/or direct immunofluorescence assays (DFA) provide rapid results and eliminate the need for cell culture-based assays, but these approaches sometimes do not rule out cross-reactive epitopes or distinguish between ongoing or past infections since antibody responses to antigens that resulted from past infections [17]. Several systematic studies of microarray [19,20,21] have been conducted on the pathogens responsible for STDs, none of the studies is able to examine the most common clinical pathogens N. gonorrhoeae, C. trachomatis, Ureaplasma, M. hominis, M.genitalium, HSV and HPV simultaneously. The microarray can be used as a high throughput tool for screening sexually transmitted diseases
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