A molecular technique for identification of bacteria using small subunit ribosomal RNA sequences.
We have recently developed a novel molecular technique for identification of specific bacterial species within a complex mixture. The technique uses PCR to amplify small subunit ribosomal RNA (SSU rRNA) genes from a mixture of bacteria. One of the PCR primers is labeled with a fluorescent dye to allow detection of the amplified product. The PCR product is then digested with restriction enzymes and a capillary electrophoresis unit equipped with a laser-induced fluorescence detector is employed to analyze the restriction fragments. Only restriction fragments that contain the fluorescent-labeled primer are detected. Generally, the nucleotide sequence of the SSU rRNA genes is unique for each bacterial species. Consequently, the fluorescent-labeled restriction fragments from different bacterial species often have characteristic lengths. Thus, the different fluorescent peaks that appear in a capillary electropherogram correspond to labeled restriction fragments from different bacterial species. This protocol allows us to identify a number of different bacterial species in a complex mixture. Only a minute sample of bacterial DNA and a minimal amount of time (8-10 h) are required for this analysis. The protocol is sensitive, rapid and capable of identifying a broad spectrum of bacterial species.
Avaniss-Aghajani
E
E
University of California, Los Angeles.
Jones
K
K
Chapman
D
D
Brunk
C
C
D001419:Bacteria / Q000302:isolation & purification*; D001483:Base Sequence; D004586:Electrophoresis; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012335:RNA, Ribosomal / Q000235:genetics*
D012335:RNA, Ribosomal