光敏生物素标记k-DNA打点杂交鉴定利什曼原虫种株

用光敏生物素标记利什曼原虫动基体DNA(k-DNA),以打点杂交鉴定利什曼原虫。结果表明,在前鞭毛体数为10~4～10~5时,此法能显示出利什曼原虫不同的种和株间有明显的区别,并且发现采自国内黑热病流行病学特征不同的地区的虫株间k-DNA 在同源程度上存在差异,但用传统分类法则无法加以鉴别。此法为快速、简便地鉴定利什曼原虫及诊断黑热病提供了新的途径。

IDENTIFICATION OF LEISHMANIA BY DOT BLOT HYBRIDIZATION WITH PHOTOBIOTIN LABELLED k-DNA

It has been shown that there are het-erogeneous sequences of k-DNA within dif-ferent species and strains of Leishmania byLopes and our previous report. Up to nowNick translation technique is most com-monly used to label k-DNA as a probe toidentify Leishmania, however, this techni-que is expensive and somewhat inconve-nient for field use because of the need ofDNA polymerase. In order to develop asimple, more rapid and cheaper systemfor the identification of Leishmania in fieldand clinic,we have used photobiotin label-led k-DNA as a probe and made the com-parison of the sensitivity between photobi-otin labelled probe and Biotin labelled probeby Nick translation. The results revealedthat detection of DNA by photobiotin wassensitive, as was shown by Biotin probe.The photobiotin was hybridized with 10~3-10~6promastigotes of different species and iso-lates of Leishmania which were dotted ontonitrocellular paper and denatured by alka-line, and the result showed the differencesbetween species and some isolates. It is ofinterest to point out that there are hetero-genicities between isolates of Leishmaniadonovani from patients in areas of hilland plain, which is in accordance with thedifferences of their epidemic evidence shownbefore. Suggestion has been made by ourresults that photobiotin probe for Dot blot isa practical and sensitive method for theidentification of Leishmania in field andclinic.