Identification of predominant human and animal anaerobic intestinal bacterial species by terminal restriction fragment patterns (TRFPs): a rapid, PCR-based method.
Identification of predominant human and animal intestinal tract anaerobes by conventional methods is cumbersome, time-consuming and less sensitive as compared to molecular methods. We have developed a molecular technique to identify most of the abundant intestinal microflora by polyermase chain reaction (PCR) amplification of a 16S rRNA gene fragment using a pair of universal PCR primers. The forward PCR primer was labelled with 6-carboxyfluorescein amino hexy (6-FAM) fluorescent dye to detect the terminal fragment of the PCR products after digestion with restriction enzymes. The PCR products were purified and digested with restriction enzymes and were analysed by capillary electrophoresis using an automated DNA sequencer. The data was analysed with GeneScan software 2.1. Eleven bacteria (Eubacterium biforme, E. limosum, Peptostreptococcus productus, Lactobacillus acidophilus, Bacteroides thetaiotaomicron, B. vulgatus, B. distasonis, Clostridium clostridiiforme, C. leptum, C. perfringens and Escherichia coli) that are predominant in human and animal intestinal tract were successfully identified by this rapid molecular technique. This protocol is rapid, accurate, sensitive and capable of identifying multiple organisms in a single sample.
Khan
A A
AA
Division of Microbiology, Food and Drug Administration, Jefferson, AR 72079, USA. akhan@nctr.fda.gov
Nawaz
M S
MS
Robertson
L
L
Khan
S A
SA
Cerniglia
C E
CE
D000818:Animals; D001421:Bacteria, Anaerobic / Q000145:classification* / Q000235:genetics; D017931:DNA Primers; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004275:DNA, Ribosomal / Q000032:analysis / Q000235:genetics; D004064:Digestive System / Q000382:microbiology*; D020459:Genes, rRNA; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D021521:Ribotyping / Q000379:methods*
D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D004262:DNA Restriction Enzymes