Kositanont Uraiwan, Rugsasuk Songsak, Leelaporn Amornrut, Phulsuksombati Duangporn, Tantitanawat Sompong, Naigowit Pimjai
Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
Diagn Microbiol Infect Dis. 2007 Feb;57(2):117-22. doi: 10.1016/j.diagmicrobio.2006.07.014. Epub 2006 Oct 3.
A multiplex polymerase chain reaction (PCR) was developed for diagnosing leptospirosis and differentiating pathogenic and saprophytic leptospires. Specific primers were designed to amplify 23S rDNA from pathogenic Leptospira and saprophytic Leptospira spp. PCR products from 27 pathogenic and 5 (including 1 intermediate) saprophytic serovars were 615 and 316 base pairs (bp), respectively. After the restriction enzyme's digestion of PCR products, the fragments by SacI of pathogenic serovars and by PstI of saprophytic serovars were 339 and 276 bp and 202 and 114 bp, respectively. The PCR primers enabled amplification of DNA from L. meyeri serovar Ranarum as a pathogenic Leptospira spp. The PCR assay could detect 1 to 2 cells of leptospires and not amplify DNA from other 18 bacterial species. The sensitivity and specificity of this PCR in rat kidney, using isolation as gold standard, were 98.6% and 100%, respectively. The most appropriate sample preparation of blood for detecting DNA was buffy coat. Among the sample preparations from 7 laboratory-confirmed leptospirosis cases, leptospiral DNA was detected in all 7 buffy coat preparations, whereas leptospiral DNA was detected in only 3 plasma or serum samples. The PCR assay may be useful as a diagnostic tool for leptospirosis.
开发了一种多重聚合酶链反应（PCR）用于诊断钩端螺旋体病并区分致病性和腐生性钩端螺旋体。设计了特异性引物以扩增致病性钩端螺旋体和腐生性钩端螺旋体属的23S rDNA。来自27种致病性血清型和5种（包括1种中间型）腐生性血清型的PCR产物分别为615和316碱基对（bp）。PCR产物经限制性内切酶消化后，致病性血清型经SacI酶切的片段和腐生性血清型经PstI酶切的片段分别为339和276 bp以及202和114 bp。PCR引物能够扩增迈耶氏钩端螺旋体蛙血清型作为致病性钩端螺旋体属的DNA。该PCR检测方法可检测到1至2个钩端螺旋体细胞，且不会扩增其他18种细菌的DNA。以分离培养作为金标准，该PCR在大鼠肾脏中的灵敏度和特异性分别为98.6%和100%。用于检测DNA的血液最合适的样本制备方法是血沉棕黄层。在7例实验室确诊的钩端螺旋体病病例的样本制备中，所有7份血沉棕黄层样本中均检测到钩端螺旋体DNA，而仅在3份血浆或血清样本中检测到钩端螺旋体DNA。该PCR检测方法可能作为钩端螺旋体病的诊断工具。
