Tani K, Kurokawa K, Nasu M
Department of Microbiology and Environmental Science, Faculty of Pharmaceutical Sciences, Osaka University, Japan.
Appl Environ Microbiol. 1998 Apr;64(4):1536-40. doi: 10.1128/AEM.64.4.1536-1540.1998.
We applied HNPP (2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate) to direct in situ PCR for the routine detection of specific bacterial cells at the single-cell level. PCR was performed on glass slides with digoxigenin-labeled dUTP. The digoxigenin-labeled PCR products were detected with alkaline phosphatase-labeled antidigoxigenin antibody and HNPP which was combined with Fast Red TR. A bright red fluorescent signal was produced from conversion to HNP (dephosphorylated form) by alkaline phosphatase. We used the ECOL DNA primer set for amplification of ribosomal DNA of Escherichia coli to identify cells specifically at the single-cell level in a bacterial mixture. High-contrast images were obtained under an epifluorescence microscope with in situ PCR. By image analysis, E. coli cells in polluted river water also were detected.
我们应用HNPP（2-羟基-3-萘甲酸-2'-苯胺磷酸酯）进行直接原位PCR，以在单细胞水平常规检测特定细菌细胞。在载玻片上使用地高辛标记的dUTP进行PCR。用地高辛标记的PCR产物通过碱性磷酸酶标记的抗地高辛抗体和与固红TR结合的HNPP进行检测。碱性磷酸酶将其转化为HNP（去磷酸化形式）产生亮红色荧光信号。我们使用ECOL DNA引物组扩增大肠杆菌的核糖体DNA，以在细菌混合物中特异性地在单细胞水平鉴定细胞。通过原位PCR在落射荧光显微镜下获得高对比度图像。通过图像分析，还检测到了污染河水中的大肠杆菌细胞。
