Development Of A High-throughput DNA Microarray For Drug-resistant Genes And Pathogens Detection And Its Preliminary Application

Infectious diseases caused by pathogens lead huge damage to human health. With the advances in diagnostic technology, excessive synthesis and unreasonable use of different antibiotics leads to the emergence of multiple drug-resistant bacteria. For example, superbugs appear in recent years. The current situation brings difficult to the clinical treatment and poses a serious public health problem. Because of the increasingly serious environmental pollution, some new diseases result in a new round of threats to human being. Traditional methods of pathogen detection suffer from several limitations:long detection time required for bacterial culture; one by one detection of samples, which is increasingly unable to meet clinical detection needs. Therefore, the development of rapid, accurate, sensitive and specific detection method of pathogens has been one goal.Since1990s, gene chip technology as a high-tech molecular biology has developed rapidly. Because of its high-throughput, large-scale and parallel processing features, the gene chip provide strong technical conditions for the molecular diagnosis of pathogens. Gene chip can achieve the level of parallel high-throughput identification of multiple target genes, and detect a variety of pathogens, drug resistance and virulence factor genes, which is a very important prerequisite for prevention and control of infectious diseases. The purpose of this study is to establish a high-throughput DNA microarray for drug-resistant genes and pathogens detection, in order to provide the basis for the rapid detection of pathogens and clinical therapy. To design a microarray representing the most comprehensive set of drug-resistant genes, pathogens species-specific genes and virulence genes, all of the genes were searched in the NCBI (http://www.ncbi.nlm.nih.gov/) GenBank database and published literatures. A total of17classes of drug-resistant genes included extended-spectrum β-lactamase-resistant genes, cephalosporinase-resistant genes, carbapenemase-resistant genes, mecA gene, tetracycline resistance family genes, aminoglycoside-resistant genes, antiseptic resistance genes, erythromycin-resistant genes, macrolide efflux genes, vancomycin-resistant genes, multidrug efflux pump genes, mupirocin-resistant genes, sulfanilamide-resistant genes, tylosin-resistant genes, fluoroquinolone-resistant genes, integration enzyme genes, and resistance genes commonly used in genetic engineering were selected;8classes of pathogens species-specific genes of burkholderia, burkholderia coli, melioidosis burkholderia coli, brucella, salmonella, yersinia pestis, bacillus anthracis, tula francis bacteria, shigella and invasive escherichia coli (EIEC), vibrio cholerae;7classes of virulence genes of diphtheria toxin, shiga toxin, botulinum toxin, ricin, tetanus toxin, staphylococcal enterotoxin and cholera toxin. According to the probe design’s principles, we selected and designed a total of172probes, and established drug-resistant gene chip and comprehensive chip (including the drug-resistant genes, the pathogen species-specific genes and virulence genes probes).We compared the nucleic acid extraction method, optimized the amplification marker system of the whole genome DNA and explore the Explored the impact of probe concentration, point-like temperature and humidity, the composition and concentration of the hybridization solution, hybridization temperature and time on the microarray hybridization results. Then we filtered out the optimal conditions for hybridization. On this basis, we evaluate the chip sensitivity, specificity and reproducibility. Reference strains and clinical isolates were used to validate further the performance of microarray.In this study, E.coli DH5a/pET28is selected. CTAB/NaCl method is the appropriate method to extract genomic DNA. The optimized hybridization condition was as follows:the probe was immobilized onto the slides in a concentration of40pmol/μL at25℃with55%humidity. Hybridization was carried out using2×hybridization solution (50%formamide,5×SSC,0.1%SDS, and0.5μg/μL salmon sperm DNA) at42℃for4h. The microarray test sensitivity was20ng/μL DNA. The performance of the drug-resistant gene chip was validated using reference strains and clinical isolates, and showed its good specificity and accuracy. The comprehensive chip was preliminarily used in detecting Vibrio cholerae, Salmonella, Shigella, Staphylococcus aureus and genetic engineering bacteria and got the correct test results.42clinically suspected resistant bacteria were detected, in which there ara six Multi-drug resistant bacteria, and the results were consistent with drug susceptibility tests.The specificity and sensitivity of the DNA microarray is good. The microarray can detecte resistance patterns of bacteria and pathogen species be quickly and accurately, reducing the workload greatly. The developed DNA microarray could be used to detect and screen drug-resistant bacteria rapidly and simultaneously. Thus, the present study could be helpful in effectively using antibiotics and controlling infectious diseases. The gene chip is important in prevention and control of infectious disease.