[Nucleotide sequence analysis of a species specific probe by an inserted fragment from recombinant plasmid pCX7 of L. interrogans sensu stricto serovar lai].
The etiological agents of leptospirosis are the pathogenic leptospires (L. interrogans sensu lato) which can be divided into 223 serovars organized into 23 serogroups. The serovar remains the basic taxon, but serotyping may now be accomplished and recognized by acceptable methods. Complementary molecular approaches are being used extensively to assess genetic relatedness amongst leptospires with restriction endonuclese analysis (REA), pulse field gel electrophoresis (PFGE) and DNA-DNA hybridization as well established tools. However, the method is cumbersome and unsuitable for routine application. To develop a sensitive and specific method for identification of pathogenic leptospires, a genomic library of L. interrogans sensu stricto serovar lai was constructed with the plasmid vector pUC9. A recombinant plasmid, designated pCX7 which has homologous fragment of pathogenic leptospires was screened from the bank. pCX7 could recognize pathogenic leptospiral DNA fragment 1.7 kb of strain 017 without cross hybridization to nonpathogenic leptospiral DNA. Inserted fragment of pCX7 DNA sequencing was performed by Dr. Yan Zhengxin (Max-Plank-Institut fur Biology, Tubingen, Germany). Insert fragment was cloned into pBluescript and sequenced by using ABI(Applied Bio. Systems, Model 373A). Nucleotide sequences were analyzed by Dr. Xiao Jianguo (Texas University Medical School and School of Public Health, Center for Infectious Diseases) using a suit of computer program (NIH). One open reading frame of 306 nucleotids were identified. There were identifiable initiation codons, terminators, pribnow box and sextama box within the sequenced regions. These results further confirmed that the little homology between L. interrogans sensu strito and L. borgpeterseni serovar javanica, L. inadai serovar ranarun and serovar manhao (L. genomospecies 2), L. biflexa serovar patoc, L. illini. pCX7 DNA probe could provide a base for identification and classification of leptospires.
Dai
B
B
Department of Pathophysiology, School of Basic Medical Sciences, Chengdu.
Xiao
J
J
Yan
Z
Z
Shen
C
C
Li
S
S
Fang
Z
Z
D004269:DNA, Bacterial / Q000235:genetics*; D015698:Genomic Library; D007921:Leptospira interrogans / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D016366:Open Reading Frames; D010957:Plasmids; D011994:Recombinant Proteins; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity
D004269:DNA, Bacterial; D011994:Recombinant Proteins