Dissertation > Biological Sciences > Microbiology > Microbiology and microbial experiments

Development of Suspension Array-based Rapid Multiplex Detection of Bacterial Pathogens

Author ZhaoJinYin
Tutor WangJingLin
School PLA Military Academy of Medical Sciences
Course Microbiology
Keywords suspension array bacterial pathogens multiplex detection
CLC Q93-3
Type Master's thesis
Year 2009
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Diseases caused by foodborne or waterborne pathogens are the important factors for threatening human health, and are the main reasons for human death and property damage. Currently, the detection of pathogens relies mainly on the culture-based assays and immunology-based assays. But they are time-consuming and labor-consuming, and they can only confirm the present or absent of one pathogen and cannot cater to all the needs of public health surveillance, clinical diagnosis and epidemiological investigation. Therefore, a multiplex, rapid and accurate detection system is needed.Suspension array as known as liquid array developed by Luminex Corp. at the middle of 1990’s is a new biochip technology. It innovatively uses the fluorescence encode microspheres as the carriers of bio-reaction, by adjusting the fluorescence intensity up to one hundred characteristic microspheres with different fluorescence spectra. Microspheres suspend in liquid environment, providing the extremely response environment for the bio-reaction, which is advantageous to maintain the native conformation of the nucleic acid and protein. It has some obvious advantages, such as high-throughput, high speed, low cost, accuracy, repeatability, sensitivity, wide linear range, easier operation. In this study, we developed a suspension array detection technology platform to detect common bacterial pathogens.Specific genes of pathogens were selected and their sequences were obtained from the GenBank. Sequences were analyzed by Clustal W to select the homologous area. Primers and probes were designed by Primer Premier 5.0. The primers and probes were validated for high specificity by Blast analysis available through the NCBI website. Each probe was incorporated an Amino-(CH212 modifier on the 5’ end to reduce steric hindrance from microspheres. Single-PCR and Multiplex-PCR were developed and PCR products were identified by suspension array. The validation of suspension was valuated by simulating environmental samples.Through bioinformatics analysis and experimental validation, fourteen sets of primer and corresponding probes were determined. One set of common primer and corresponding probe were selected as internal control to monitor PCR reaction and the hybridization system.We tested primers annealing temperature, primers concentration, Mg2+ concentration, TaqTM polymerase concentration of single and multiplex PCR reaction. Finally, the ideal annealing temperature that was compatible with 15 primer sets in the multiplex reaction was 53℃. We have made a great effort to adjust the concentration of each primer sets when a great differences in hybridization signals was present. Mg2+ concentration was followed manufacturer’s instructions at 1.5 mM. TaqTM polymerase concentration plays a great role on the hybridization signal. Increasing the concentration distinctly improves the sensitivity of multiplex PCR. We adopted 1.5 U Taq in 20μl reaction volume which was three times higher than that of manufacturer’s instruction. An asymmetric PCR at a ratio of 5:2 (Biotin-labeled primers: non-labeled primers) was employed to generate high MFI.Hybridization temperature and time of Suspension array were tested by five PCR products, Ipah, rfbE, nuc, rfbX and bont/A. Our results show that the perfect condition of hybridization was at 52℃for more than 15 min.We successfully amplified 14 targeted gene segments and one universal sequence in a single reaction tube. In each tube, we also detected Shigella flexneri, Shigella dysenteriae, Shigella boydii, Shigella sonnei, Vibrio cholerae O1, Vibrio cholerae O139, Legionella pneumophila, Clostridium botulinum type A, B and E, Staphylococcus aureus, respectively.The sensitivity of the array assay was performed with total genomic DNA extracting from cultured Shigella dysenteriae, Vibrio cholerae O1, Staphylococcus aureus. The suspension array assay coupled with single-plex PCR was able to detect 100 fg for IpaH, 100 fg for Ial, 100 fg for rfbE, 102 fg for ct, 101 fg for nuc, 101 fg for scpA, while coupled with multiplex PCR 103 fg for IpaH, 104 fg for Ial, 102 fg for rfbE, 104 fg for ct, 102 fg for nuc, 104 fg for scpA. The utility of suspension array to detect pathogens in the environmental sample was carried out with ten-fold dilutions of Shigella dysenteriae cells. The suspension array assay coupled with single-plex PCR was able to detect 2×102 CFU/100ml (theoretical equivalent to 10 cells in reaction), 2×103 CFU/100ml, 2×104 CFU/100ml and 2×106 CFU/100ml for Ial, IpaH nuc and scpA respectively. In contrast, coupled with 15-plex PCR, it can detec 2×104 CFU/100ml, 2×105 CFU/100ml, 2×106 CFU/100ml and 2×107 CFU/100ml for IpaH, Ial, nuc and scpA, respectively.The specificity was tested using 1-10 ng of genomic DNA from all involving strains in this study. All target bacteria gave positive signals, while non-target bacteria showed negative with no amplification product of appropriate size, indicating 100% specificity.We also developed another 13-plex detection system based on 16S rDNA and hsp60 gene to detect Shigella spp., Vibrio cholerae, Legionella pneumophila, Staphylococcus aureus, Clostridium perfringens, Listeria monocytogenes. The suspension array assay coupled with single-plex PCR was able to detect 101 fg-103 fg, coupled with 7-plex and 13-plex to detect 102 fg-105 fg, respectively. The utility of suspension array to detect pathogens in the environmental sample was carried out with ten-fold dilutions of Shigella dysenteriae cells. The suspension array assay coupled with 1, 7, 13-plex PCR was able to detect 2×102 CFU/ml, 2×102 CFU/ml, 2×107 CFU/ml for 16S rDNA, respectively; the array coupled with 1, 7, 13-plex PCR was able to detect 2×105 CFU/ml, 2×107 CFU/ml, 2×107 CFU/ml for hsp60, respectively.Because of PCR potentially extreme sensitivity and powerful amplification, false-positive results from carryover-contamination of PCR products have become a significant challenge for PCR-based detection systems. In our study, we employed UNG to overcome this problem. Our results revail that UNG can powerfully clean up 1011 copies for IpaH、107 copies for rfbE、1011 copies for rfbX、1011 copies for bont/A、1011 copies for nuc. UNG is a powerful tool to decrease PCR products carryover-contaminationIn this study, we builded a multiplex detection technology platform based on suspension array, with rapid, sensitive and specific capability. This method may have a wide range of applications in public health surveillance, clinical diagnosis, epidemiological investigation etc.

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