Prosser J I, Killham K, Glover L A, Rattray E A
Department of Molecular and Cell Biology, University of Aberdeen, Marischal College, Scotland.
Crit Rev Biotechnol. 1996;16(2):157-83. doi: 10.3109/07388559609147420.
The development of techniques for detection and tracking of microorganisms in natural environments has been accelerated by the requirement for assessment of the risks associated with environmental release of genetically engineered microbial inocula. Molecular marker systems are particularly appropriate for such studies and luminescence-based markers have the broadest range of applications, involving the introduction of prokaryotic (lux) or eukaryotic (luc) genes for the enzyme luciferase. Lux or luc genes can be detected on the basis of unique DNA sequences by gene probing and PCR amplification, but the major advantage of luminescence-based systems is the ability to detect light emitted by marked organisms or by luciferase activity in cell-free extracts. Luminescent colonies can be detected by eye, providing distinction from colonies of indigenous organisms, and the sensitivity of plate counting can be increased greatly by CCD imaging. Single cells or microcolonies of luminescent organisms can also be detected in environmental samples by CCD image-enhanced microscopy, facilitating study of their spatial distribution. The metabolic activity of luminescence-marked populations can be quantified by luminometry and does not require extraction of cells or laboratory growth. Metabolic activity, and potential activity, of marked organisms therefore can be measured during colonization of soil particles and plant material in real time without disturbing the colonization process. In comparison with traditional activity techniques, luminometry provides significant increases in sensitivity, accuracy, and, most importantly, selectivity, as activity can be measured in the presence of indigenous microbial communities. The sensitivity, speed, and convenience of luminescence measurements make this a powerful technique that is being applied to the study of an increasingly wide range of ecological problems. These include microbial survival and recovery, microbial predation, plant pathogenicity, phylloplane and rhizosphere colonization and reporting of gene expression in environmental samples.
对与基因工程微生物接种体环境释放相关风险进行评估的需求，加速了自然环境中微生物检测和追踪技术的发展。分子标记系统特别适用于此类研究，基于发光的标记具有最广泛的应用范围，涉及引入用于荧光素酶的原核（lux）或真核（luc）基因。可以通过基因探测和PCR扩增，基于独特的DNA序列检测Lux或luc基因，但基于发光系统的主要优势在于能够检测标记生物体发出的光或无细胞提取物中的荧光素酶活性。发光菌落可用肉眼检测，从而与本地生物体的菌落区分开来，并且通过电荷耦合器件（CCD）成像可大大提高平板计数的灵敏度。通过CCD图像增强显微镜，还可以在环境样品中检测发光生物体的单细胞或微菌落，便于研究它们的空间分布。发光标记群体的代谢活性可以通过发光测定法定量，并且不需要提取细胞或在实验室培养。因此，在土壤颗粒和植物材料定殖过程中，可以实时测量标记生物体的代谢活性和潜在活性，而不会干扰定殖过程。与传统活性技术相比，发光测定法在灵敏度、准确性，以及最重要的选择性方面都有显著提高，因为可以在本地微生物群落存在的情况下测量活性。发光测量的灵敏度、速度和便利性使其成为一种强大的技术，正被应用于研究越来越广泛的生态问题。这些问题包括微生物的存活和恢复、微生物捕食、植物致病性、叶表面和根际定殖，以及环境样品中基因表达的报告。
