Rapid, sensitive, microbial detection by gene amplification using restriction endonuclease target sequences.
The use of primers synthesized to eight class II restriction endonuclease target sequences, from Haemophilus parainfluenzae, Escherichia coli, Staphylococcus aureus, Salmonella infantis, Rhodobacter sphaeroides, Klebsiella pneumoniae, Bacillus amyloliquefaciens and Proteus vulgaris for single and multiplex PCR identification of the organisms is discussed. Results indicate that the method is sensitive and specific enough to detect single cells and attogram amounts of target DNA. It has also been demonstrated that the primers can be used in whole cell PCR for identification and whole cell PCR product recovery could be enhanced by the addition of gelatin or DMSO to PCR reaction mixtures. Other results have indicated that the method can be used for the definite identification of specific individuals present in mixed cultures or suspensions of organisms. The applicability of the method for detection of a specific strain within a group of closely related organisms has also been investigated and for that sequence/organism the results suggest that the proposed method is indeed very specific and discriminative. It is suggested that as more information becomes available regarding such sequences and their distribution, this approach could form the basis of a widescale, rapid, simple and cheap identification and/or typing system for bacteria.
Metherell
L A
LA
School of Chemical and Life Sciences, University of Greenwich, London, SE18 6PF, UK.
Hurst
C
C
Bruce
I J
IJ
D001419:Bacteria / Q000145:classification* / Q000201:enzymology* / Q000235:genetics; D015373:Bacterial Typing Techniques; D017931:DNA Primers / Q000235:genetics; D015246:Deoxyribonuclease EcoRI / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics*; D005784:Gene Amplification; D016133:Polymerase Chain Reaction / Q000379:methods*; D011511:Proteus / Q000235:genetics / Q000302:isolation & purification; D011532:Providencia / Q000235:genetics / Q000302:isolation & purification; D012475:Salmonella / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D013045:Species Specificity
D017931:DNA Primers; D015246:Deoxyribonuclease EcoRI; D015252:Deoxyribonucleases, Type II Site-Specific