Bej A K, Mahbubani M H, Miller R, DiCesare J L, Haff L, Atlas R M
Department of Biology, University of Louisville, KY 40292.
Mol Cell Probes. 1990 Oct;4(5):353-65. doi: 10.1016/0890-8508(90)90026-v.
Detection of pathogens (Legionella species) and indicator bacteria (coliform bacteria) was achieved by multiplex (simultaneous) PCR amplification of diagnostic gene sequences and by hybridization to immobilized poly-dT-tailed capture probes using a dot- or slot-blot approach. Complex manipulations of primer concentrations and staggered additions of primers were required in order to achieve equal amplification of multiple genes. Multiplex PCR amplification of two different Legionella genes, one specific for L. pneumophila (mip) and the other for the genus Legionella (5S rRNA), was achieved by staggered amplification. Multiplex PCR amplification using differing amounts of primers specific for lacZ and lamB genes permitted the detection of coliform bacteria and those associated with human faecal contamination, including the indicator bacterial species E. coli and enteric pathogens Salmonella and Shigella. Hybridization of biotin-labelled amplified DNA, in which the biotin was incorporated during PCR amplification from biotinylated-dUTP, to immobilized 400-dT-tailed capture probes permitted specific and sensitive detection of target gene sequences. The sensitivity of colorimetric detection achieved by PCR amplification of target DNA was at a level equivalent to 1-2 bacterial cells, which is the same level of sensitivity obtained with radioactive detection. The simultaneous amplification of several genes and hybridization to immobilized capture probes with colorimetric detection is an effective, efficient and rapid detection method for various human bacterial pathogens.
通过对诊断基因序列进行多重（同时）PCR扩增，并采用斑点或狭缝印迹法与固定化的聚-dT尾捕获探针杂交，来检测病原体（军团菌属）和指示菌（大肠菌群）。为实现多个基因的同等扩增，需要对引物浓度进行复杂操作并交错添加引物。通过交错扩增实现了对两种不同军团菌基因的多重PCR扩增，一种是嗜肺军团菌特异性基因（mip），另一种是军团菌属特异性基因（5S rRNA）。使用不同量的针对lacZ和lamB基因的引物进行多重PCR扩增，可检测大肠菌群以及与人类粪便污染相关的细菌，包括指示菌大肠杆菌以及肠道病原体沙门氏菌和志贺氏菌。生物素标记的扩增DNA（其中生物素在PCR扩增过程中从生物素化的dUTP掺入）与固定化的400-dT尾捕获探针杂交，可实现对靶基因序列的特异性和灵敏检测。通过对靶DNA进行PCR扩增实现的比色检测灵敏度相当于1 - 2个细菌细胞，这与放射性检测获得的灵敏度水平相同。同时扩增多个基因并与固定化捕获探针杂交以及比色检测，是一种用于检测多种人类细菌病原体的有效、高效且快速的检测方法。
