The development of a ligase mediated PCR with potential for the differentiation of serovars within Leptospira interrogans.
A ligase mediated polymerase chain reaction (LMPCR) was developed to amplify between the repetitive element, IS1533, of Leptospira and adjacent chromosomally located Bg/II restriction endonuclease enzyme sites. To do this, complimentary oligonucleotide linkers designed to anneal together with an overhanging Bg/II end were ligated to Bg/II digested DNA from 35 leptospiral reference strains and field isolates. This ligated DNA was used as template for PCR with oligonucleotide primers specific for the linker and for the repetitive element IS1533. The resultant amplicon profile hybridised a 102 bp region derived from the terminus of IS1533 thus confirming that amplicons generated by LMPCR contained part of IS1533. The number of fragments generated containing IS1533 was significantly fewer than that generated by RFLP but the LMPCR method has the potential to use far less template DNA and be quicker than standard RFLP. Obvious and reproducible interserovar differences were demonstrated by LMPCR whereas for 20 of 21 L. hardjo-bovis isolates tested no intraserovar differences were observed. Of those serovars known to possess IS1533 homologues and tested here by LMPCR, each produced a unique amplicon profile which hybridised the IS1533 terminus probe. The limited heterogeneity amongst hardjo-bovis isolates is discussed as is the potential contribution of this method to diagnosis, differentiation and the phylogenetics of the Leptospires.
Redstone
J S
JS
Bacteriology Department, Central Veterinary Laboratory, New Haw, Addlestone, Surrey, UK.
Woodward
M J
MJ
D000818:Animals; D001426:Bacterial Proteins; D001483:Base Sequence; D011088:DNA Ligases; D017931:DNA Primers; D015252:Deoxyribonucleases, Type II Site-Specific; D007921:Leptospira interrogans / Q000145:classification* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*
D001426:Bacterial Proteins; D017931:DNA Primers; C024404:BglII endonuclease; D015252:Deoxyribonucleases, Type II Site-Specific; D011088:DNA Ligases