Dunbar Sherry A, Vander Zee Coe A, Oliver Kerry G, Karem Kevin L, Jacobson James W
Luminex Corporation, 12212 Technology Blvd., Austin, TX 78727, USA.
J Microbiol Methods. 2003 May;53(2):245-52. doi: 10.1016/s0167-7012(03)00028-9.
Escherichia coli, Salmonella, Listeria monocytogenes and Campylobacter jejuni are bacterial pathogens commonly implicated in foodborne illnesses. Generally used detection methods (i.e., culture, biochemical testing, ELISA and nucleic acid amplification) can be laborious, time-consuming and require multiple tests to detect all of the pathogens. Our objective was to develop rapid assays to simultaneously detect these four organisms through the presence of antigen or DNA using the Luminex LabMAP system. For nucleic acid detection, organism-specific capture probes corresponding to the 23S ribosomal RNA gene (rrl) were coupled covalently to LabMAP microspheres. Target molecules included synthetic complementary oligonucleotides and genomic DNA isolated from ATCC type strains or other well-characterized strains of each organism. Universal PCR primers were designed to amplify variable regions of bacterial 23S ribosomal DNA, yielding biotinylated amplicons of 86 to 109 bp in length. Varying quantities of targets were hybridized to the combined microsphere sets, labeled with streptavidin-R-phycoerythrin and analyzed on the Luminex(100) system. Results of nucleic acid detection assays, obtained in 30 to 40 min following amplification, correctly and specifically identified each bacterial species with a detection sensitivity of 10(3) to 10(5) genome copies. Capture-sandwich immunoassays were developed with organism-specific antibodies coupled to different microsphere sets. Microspheres were incubated with organism-specific standards and reactivity was assessed with biotinylated detection antibodies and streptavidin-R-phycoerythrin. In the immunoassays, microsphere-associated fluorescence was organism concentration dependent with detectable response at < or = 1000 organisms/ml and with no apparent cross-reactivity. We have demonstrated that the Luminex LabMAP system is a rapid, flexible platform capable of simultaneous, sensitive and specific detection of pathogens. The practical significance of this multiplexing approach would be to provide more timely, economical and comprehensive information than is available with conventional isolation and identification methodologies.
大肠杆菌、沙门氏菌、单核细胞增生李斯特菌和空肠弯曲菌是通常与食源性疾病有关的细菌病原体。常用的检测方法（即培养、生化检测、酶联免疫吸附测定和核酸扩增）可能费力、耗时，并且需要多次检测才能检测出所有病原体。我们的目标是开发快速检测方法，通过使用Luminex LabMAP系统检测抗原或DNA的存在来同时检测这四种微生物。对于核酸检测，将与23S核糖体RNA基因（rrl）对应的生物体特异性捕获探针共价偶联到LabMAP微球上。靶分子包括合成的互补寡核苷酸以及从每种生物体的ATCC标准菌株或其他特征明确的菌株中分离的基因组DNA。设计通用PCR引物以扩增细菌23S核糖体DNA的可变区域，产生长度为86至109 bp的生物素化扩增子。将不同数量的靶标与组合的微球组杂交，用链霉亲和素-R-藻红蛋白标记，并在Luminex(100)系统上进行分析。扩增后30至40分钟获得的核酸检测分析结果正确且特异性地鉴定了每种细菌物种，检测灵敏度为10(3)至10(5)个基因组拷贝。用偶联到不同微球组的生物体特异性抗体开发了捕获夹心免疫测定法。将微球与生物体特异性标准品一起孵育，并用生物素化的检测抗体和链霉亲和素-R-藻红蛋白评估反应性。在免疫测定中，微球相关荧光与生物体浓度相关，在≤1000个生物体/ml时可检测到反应，且无明显交叉反应。我们已经证明，Luminex LabMAP系统是一个快速、灵活的平台，能够同时、灵敏且特异性地检测病原体。这种多重检测方法实际的意义在于，与传统的分离和鉴定方法相比，它能提供更及时、经济和全面的信息。
