Construction And Clinical Application Of Gene Chip For Detection Of The Important Genitourinary Tract Pathogen

ObjectiveSince 1980s, the incidence of sexually transmitted diseases (STD) has gradually increased, and gonococcal urethritis and non-gonococcal urethritis (NGU) are of higher morbidity. The former is caused by Neisseria gonorrhoeae (Ng), and the latter mainly by Ureaplasma urealyticum (Uu), Mycoplasma hominis (Mh) or other Pathogens infections. The cause of the genitourinary tract infection pathogens is complex and diverse, which make clinical treatment difficult. Thus, it is urgent to find a pathogenic detection method with strong specificity, high sensitivity, time saving, and low expenditure, it will lay an experimental basis for the clinical treatment of etiology.At present some pathogen detection methods, such as culture, immunohistochemical techniques, PCR technology, are clinically used for genitourinary tract infections. The above technologies have played a tremendous role in the clinical diagnosis. However, there are still some shortcomings in different methods. For example, culture is cumbersome and time consuming; and specific antisera are needed in immune tests. The advantages of PCR technology itself are understandable, but cross-contamination and false positive results appear if to be used improperly. Moreover, some false negative results may occur if reaction conditions are not strictly controlled. These shortcomings need to overcome by new technology.In recent years, newly developed gene chip technology provides a powerful diagnostic tool for genitourinary tract infection etiology. Characteristics of fast, efficient, high-throughput, parallel detection of gene chip in etiological diagnosis is of the unique advantage. It has been made that considerable progress in applied research of gene chip on etiological diagnosis. Unfortunately, no practical gene chip is used in clinical detection for genitourinary tract infection pathogens to this day, and little is known about this research field in our country. To provide a high-throughput and parallel detection method for clinical diagnosis of genitourinary tract infections, we prepared a multiple-purposed gene chip in this project. This newly developed chip could simultaneously detect three kinds of pathogens, i.e. Ng, Uu and Mh that cause gonococcal urethritis and non-gonococcal urethritis, respectively. Our works will lay a foundation in both theories and technologies for the improvement of increasingly serious situation about STD prevention and control work in our country.Materials and Methods MaterialsStandard strains: Neisseria gonorrhoeae ATCC 29106 was purchased in National Institute for the Control of Pharmaceutical and Biological Produce; Ureaplasma urealyticum ATCC 33697, Mycoplasma hominis ATCC 23114 were purchased in the Capital Institute of Pediatric.Clinical samples were collected from Department of dermatonosis and cypridopathy, The First Hospital affiliated China Medical University.Mycoplasma basal medium PPLO was purchased in Beijing Zeping-Biotechnology Institute, Neisseria gonorrhoeae agar medium drugs was purchased in National Institute for the Control of Pharmaceutical and Biological Produce, and Mh-Uu selective identification solid medium (two in one-medium) was kindly provided by Shanghai Enkang Biological Technology.PCR Kit (TaKaRa).Primers and probes (Shanghai Public Health Synthesis). PCR Amplification (Biometra Personal PCR system, Germany), biochips plotter (Micro Grind II 600, Biorobtics Ltd, England), confocal laser scanner (Gene TACTMLS IV, Gnomic Solutions Inc. USA.Methods1. Two standard strains of Mh and Uu were cultured in solid and liquid culture medium, respectively. And standard strain of Ng was cultured with solid medium in the candle-cylinder.2. Clinical samples (secretions) were collected from male urethra and female cervix, using sterile cotton swab.3. A pair of universal primers and 3 specific probes were designed with bioinformatics software, and their rationality was analyzed.4. Pathogen DNA templates were extracted using the phenol-chloroform.5. Preparation of sample target sequences using asymmetric PCR.6. Preparation of pathogen detection gene chips: after film-processing, probes were added on the chip using biochips plotter according to pre-designed micro-array matrix, then the chips were hydrated and fixed.7. Repeatability and validity were detected through repeated hybridizations, sequencing analyses, and comparisons to clinical report results.Results1. Two standard strains grew well in both solid and liquid mediums. Following template DNA extraction, PCR amplification polyacrylamide gel electrophoresis and silver-staining, expected product bands with designed length were clearly observed.2. Probe design was reasonable, and had no homology compared with other viruses or bacteria. There was high specificity among different probes of detected pathogens.3. When chip hybridization temperature was 60℃for 1h, the hybridization signal was significant. 4. The results of chip hybridizations repeated well. The reproducibility rate of probe detection was 97.5%, and coincidence with out-patient reports was 96%. Analyzed by software BLAST, the results of sequencing were identical with that of chip detection. The above findings proved that chip is validity and practical.Conclusion1. The design of gene chip for urogenital tract pathogen detection is reasonable, and results are ideal.2. The chip is of good repeatability and reliability, which will lay an experimental foundation for the development of new techniques to detect clinical urogenital tract infection pathogens.