Development Of DNA Microarray Assays For Detection And Identification Of Acute Pathogenic Bacteria

Objective: The infectious diseases induced by pathogenic bacteria are primary factors threatening human health and resulting in society panic and are main reasons inducing human death. Furthermore, the terrorism groups have paid attention to biological warfare weapons for a long time. Many bioterrorism affairs indicated that bioterrorism attack had become an actual threat. The outbreak and prevalence of infectious diseases will be a severe threat to people's health and interfere with the development and cooperation of politics, economy and civilization. The rapid, specific, sensitive detection of pathogenic bacteria was very important prerequisite for prevention and control of the epidemic of infectious diseases. In this paper, the rapid, accurate, high-throughout DNA microarray technology was developed for detection and identification of common intestinal pathogens, acute pathogenic bacteria and bacterial warfare agents. These studies provided effective diagnosis tools for clinical diagnosis, surveillance of food and public health, control of infectious diseases and protection of bioterrorism attack.Methods: The 16S rDNAå’Œ23S rDNA genes were chosen simultaneously as targets for screening of specific probes and designing universal primers to detect and identify fifteen intestinal pathogens. The duplex PCR reaction condition was optimized. Furthermore, we studied that the factors, such as concentration ratio of asymmetric PCR primers, Mg²⁺ concentration, annealing temperature, annealing time, hybridization temperature, components of hybridization buffer had effect on hybridization reaction. DNA microarray assay for simultaneously detecting and identifying the presence of nine acute pathogenic bacteria was developed. The specific primers and probes were designed and evaluated. Six series of duplex PCR reaction condition were optimized respectively. Moreover, we investigated that the factors, such as concentration ratio of asymmetric PCR primers, Mg²⁺ concentration, dNTPs concentration, DNA polymerase concentration, hybridization temperature, components of hybridization buffer had effect on hybridization reaction. The condition of tyramide signal amplification reaction was optimized, and then the sensitivity obtained by tyramide signal amplification was compared with that of end labeling method. On the basis of above experiments the cutoff values according to two DNA microarray assays were confirmed respectively. Furthermore, sensitivity, specificity and reproducibility of two DNA microarray assays were determined respecvtively.Results: 1. Establishment of detection and identification of fifteen intestinal pathogens using DNA microarray: the results of the first part: The development of DNA microarray assay to detect the presence of fifteen intestinal pathogens was described. The target pathogens were Listeria monocytogenes, Vibrio parahaemolyticus, Campylobacter jejuni, Yersinia enterocolitica, Staphylococcus aureus, Clostridium botulinum, Bacillus cereus group, Salmonella spp, Shigella spp. Clostridium perfringens, Proteus spp, Brucella spp, Escherichia coli O157 :H7,Yersinia spp and Vibrio cholera. The suitable method for extracting genomic DNA was boiling assay. The template of Clostridium botulinum was constructed successfully. The optimal conditions of PCR reaction were as followed: the concentration ratio of forward primer to reverse primers labeled with Cy3 was 1:10, and the final concentration of 16S-F1, 23S-F3, 16S-R, 23S-R4 was 0.05μmol/L, 0.15μmol/L, 0.5μmol/L, 1.5μmol/L accordingly. The concentration of Mg²⁺ was 2.0mmol/L, annealing temperature and time was 56℃and 30s respectively. The suitable conditions of hybridization reaction were as followed: hybridization temperature was 50℃, and components of hybridization buffer were 5×SSC, 0.2%SDS, 2.5% formamide. This DNA microarray assay was applied to detect 86 species or genera of standard strains of intestinal bacteria. All of the bacterial strains belonging to targets on this array tested are positively detected and none of the other non-target bacteria was detected. The limit of detection was approximately 10³CFU/mL for one species of pathogen and 10⁵CFU/mL for six species pathogens existing simultaneously in PCR reaction. The reproducibility of the assay was also investigated, and it showed the coefficient of variation of intra-assay and inter-assay was less than 15 %. When DNA microarray was used to screen 99 clinical specimens, the results compared with that of conventional methods showed that the accuracy rate was 80.8% including four clinical specimens with mixed pathogens. The results indicated that the DNA microarray assay was a reproducible, specific and sensitive method for the detection of fifteen intestinal pathogens in clinical specimens.2. Establishment of detection and identification of nine acute pathogenic bacteria using DNA microarray: the results of the second part: The development of DNA microarray assay to detect the presence of nine acute pathogenic bacteria was described. The target pathogenic bacteria were Bacillus anthracis, Yersinia pestis, Bacillus tularensis, Brucella spp, Salmonella typhi, E. coli O157:H7, Shigella spp, Vibrio cholerae, Yersinia enterocolitica. The target genes chosen corresponding to above pathogens were pag, 3 a, fopA, BCSP, vipR, rfbE and fliC for E. coli O157:H7, ipaH, ctxA and LPSgt for Vibrio cholerae, ail accordingly. Meanwhile, LPSgt gene was applied to distinguish Vibrio cholera serotype 01 from O139, and furthermore rfbE gene was specific to serotype 0157 and fliC gene was specific to serotype H7. 16S rDNA was used as an internal control. The six series duplex PCR reactions, 3a and fliC, vipR and BCSP, pag and fopA, rfbE and 16S, ctxA and ipaH, LPSgt and ail. The magnetic bead method was established for enriching and concentrating PCR products. The optimal conditions of hybridization reaction were as followed: hybridization temperature was 50℃, and components of hybridization buffer were 5×SSC, 0.2%SDS, 2.5% formamide. The suitable conditions of coloration reaction were as followed: 1:1500 diluted streptavidin-horseradish peroxidase, incubation for 30min at 37℃; 1:1000 diluted TSA-Cy3, incubation for 30min at 37℃. The specificity of DNA microarray assay for nine acute pathogens was evaluated using 28 standard strains of various species or genera of bacteria. All targets belonging to the array tested was positively identified while all other species were not detected by the assay. The limit of detection was approximately 10³CFU/mL for Bacillus anthracis, Yersinia pestis, Bacillus tularensis, Brucella spp and Vibrio cholerae O139 respectively. The sensitivity of TSA-Cy3 labeling method was 10 times more sensitive than that of Cy3 end labeling assay. The limit for detecting mock samples such as flour and soil was 10⁴CFU/mL/0.1g. The reproducibility of the assay was also investigated, and then it showed the coefficient of variation of intra-assay and inter-assay was less than 15 %. When DNA microarray was used to screen 10 clinical specimens and 20 double-blind samples, the results were in accordance with that of conventional methods. The results indicated that the DNA microarray assay was a reproducible, specific and sensitive method for the detection of nine acute pathogens in clinical specimens.Conclusions: DNA microarray for detection of fifteen intestinal pathogens is high-throughout, rapid, sensitive, specific assay having a very good reproducibility and specificity. This assay was applied to food safety, bacteria taxonomy and clinical diagnosis as a preliminary screening tool. Nine acute pathogenic bacteria were detected and identified using DNA microarray combined with TSA technology. Furthermore, this assay could distinguish Vibrio cholera serotype 01 from 0139, and O157:H7 from 0157: non-H7. DNA microarray for nine acute pathogenic bacteria was a rapid, accurate, effective diagnosis tool applied to clinical diagnosis, surveillance of public health, food safety, control of infectious diseases and anti-bioterrorism. This assay will be sure to not only be helpful for controlling and preventing outbreak of infectious diseases, but also be valuable for enhancing the diagnosis level and the capability for anti-bioterrorism and protecting bioterrorism attack.