Standardization of PCR Detection of Leptospira.
2004
Aqa`ie Pur, Khosru | Safaviyyeh, Sediqeh | Vand Yusefi, Jalil | Moradi, S. | A`bd Ol-Lah Pur, GH.
Leptospira pomona and Leptospira icterohaemorrhagiae were used in developing these diagnostic methods. For evaluation of quality and rapidity, DNA extraction perfonned with three different methods e.g: boiling, phenol-chlorofonn-isoamyl alcohol and absorption on glass fibers. Two genomic noncoding target sequences on DNA were selected for amplification. The first pair of primers was G IIG2 and had a 285 bp product while the second pair was 590DIRII590REV2 with a 571bp product. PCR optimization was done and the sensitivity determined that was about 1 fg per tube equal to DNA of 1 Leptospira bacteriwn. The genome of none of near matching bacteria (based on BLAST search) had product with these two PCRs, which was another confinnation for specificity. RFLP with MboI for 285 and AiuI fo~ 571 bp products, respectively approved PCR accuracy. Electrophoresis were done on agarose gel and bands visualized \\\\-th ethidium bromide staining. Finally about 19 MAT positive sera were analysed with the both de\\&rdquoeloped PCR methods which the existance of Leptospira DNA in all of them was confinned.
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