Simultaneously Detection Of Bacterial Bioterrorism Agents By Multiplex PCR-DNA Microarray

Bioterrorism has been recognized for a long time, and it attracts more and more concerns after the uncovering of Iraq's biological weapons projects, and the United States'anthrax terrorist attacks recently. Anti-biological terrorism is no doubt an important part of the international fight against terrorism. A vital issue in getting prepared for bioterrorism is to detect and identify the bioterrorism agents rapidly and accurately. Developing methods for the detection of multiple agents will be of great help to deal with the possible bioterrorist attacks.DNA microarray is a common strategy for the universal detection of bacterial pathogens in the past decades. Our laboratory has also been trying to set up this platform. Normally, the conserved 16S rRNA or 23S rRNA genes are selected as targets, in which universal PCR primers and species specific probes are designed. The targeted genes of tested bacteria are amplified and labeled with universal PCR primers, and then the amplicons are hybridized with DNA microarray harboring specific probes. However, some probes targeting rRNA genes are not so good to avoid cross hybridization, and the specificity for these probes needs to be improved. This is even a more tough challenge for some bioterrorism agent. For instance, 16S rRNA genes in Bacillus anthracis and Bacillus cereus are nearly identical, while the sequences of 16S rRNA and 23S rRNA genes in Yersinia pestis and Yersinia pseudotuberculosis are also almost the same.For the time being, the most specific and sensitive assay to detect bacterial bioterrorism agents is the virulence-gene-specific PCR. Our laboratory has also set up PCR assays for some bacterial agents. However, these PCR assay can only detect one gene for certain agents at one time. Based on the experiences on both DNA microarray and PCR assays on bioterrorism agent, we propose a novel strategy for the universal detection of multiple agents simultaneously. The basic idea is quite simple, first using multiple PCR to amplify and label the specific genes for targeted agents, and then using a microarray to visualize the amplicons. This system seems to be a promising solution for detecting multiple agents rapidly, specifically and simultaneously.Objective:To establish a universal platform for the detection of bacterial bioterrorism agents, which combines the advantages of specific gene multiplex PCR and DNA microarray. With the sensitivity and specificity of PCR assays, the high throughput of DNA microarray technology, this platform is supposed to be able to avoid the possible false positive amplicons in multiple PCR, and unexpected cross hybridization in rRNA gene based microarray assay as well.Method:For eight targeted agents (Bacillus anthracis, Yersinia pestis, Brucella, Francisella tularensis, Burkholderia pseudomallei, Rickettsia prowazekii, Rickettsia rickettsii and Coxiella burnetii), two sets of specific PCR primers in each agents were selected individually. A multiplex PCR-DNA microarray assay was set up by deliberated optimizations. Then this system was carefully evaluated by serious tests, such as sensitivity tests, specificity tests, the ability to detect multiple targets, animal sample tests, unrelated bacteria spiking tests and blind sample tests.Results:1. By combing multiplex PCR with gene-specific DNA microarray, we successfully set up a system to detect bacterial bioterrorism agents in a simple and rapid way. CEL silylated slides were selected as the base for DNA microarray, on which two specific probes for each targeting agents were printed (totally 16 probes). The hybridization temperature was set as 65℃to insure the specificity, and the hybridization time was optimized to 45 minutes. The whole process will take around three hours, including sample preparation, multiplex PCR and hybridization.2. The 16 sets of PCR primers for eight agents were combined into three"cocktails", which will amplify and label the target gene fragments in three multiplex PCR reactions. Then the amplicons were submitted to DNA microarray hybridization. By testing the eight agents individually, we can get clear positive hybridization signals for the corresponding probes, which imply the efficiency of the PCR primer, probes and the whole system in the detection of target agents. 3. We randomly selected three pathogenic bacteria, Corynebacterium diphtheriae, Staphylococcus aureus and Salmonella typhi to test the specificity of the system. No hybridization signal was identified in any probes, which suggests the specificity of this system.4. Yersinia pseudotuberculosis and Bacillus cereus gave no signal to any of the probes, which suggest the ability of our system for discriminating Y. pestis and B. anthracis from their phylogeneticaly close related counterparts.5. We used Y. pestis strain EV76 and B. anthracis strain A16R to perform the sensitivity test. By serial dilution tests, Y. pestis (EV76) can be detected down to 11 CFU, and the spiked blood samples can be detected down to 110 CFU. For B. anthracis (A16R), the sensitivity of this system is 14 CFU for pure bacteria, and 140 CFU for spiked blood sample. Our system is ten times sensitive than traditional PCR assays.6. We prepared samples containing two, three, four, five, six and eight target bioterrorism agents respectively, and applied to the multiple PCR - microarray system. Each sample can get positive signals for multiple probes, which are identical to the expected hybridization patterns. The system established in this study is able to detect multiple target agents simultaneously as it is supposed to be.7. We prepared six samples (negative, one target agents and more than two targets) for blind test. The system can identify all the six blind samples correctly, which implies the ability to detect multiple targets simultaneously from any unknown samples.8. We challenged mice with Y. pestis strain 201, and harvested livers, spleens and lungs from infected mice for DNA extraction. The system established in this study can clearly detect Y. pestis from these samples, which strongly support its ability to detect animal tissue samples directly.9. Even with 10,000 times of unrelated bacteria (E. coli) interference, the multiplex PCR– DNA microarray is still able to detect trace level of Y. pestis accurately. This test implies the potential of our system to handle complex samples. Conclusion:In this study, we established a universal system for detecting eight bacterial bioterrorism agents simultaneously, which took advantage of the power of multiplex PCR and DNA microarray as well. The system tends to be both specific and sensitive. With the abilities to detect multiple agents simultaneously and handle complex samples, it is quite promising in preparation for bioterrorism attacks, unexpected pandemics, and endemic surveillance as well. This system can be upgraded to include more pathogenic bacteria, also potentially modified to detect viral or fungal pathogens.