通过实时聚合酶链反应检测和定量感染马匹和蜗牛中瑞氏埃立克体基因组DNA。

Detection and quantitation of Ehrlichia risticii genomic DNA in infected horses and snails by real-time PCR.

作者信息

Pusterla N, Leutenegger C M, Sigrist B, Chae J S, Lutz H, Madigan J E

机构信息

Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis 95616, USA.

出版信息

Vet Parasitol. 2000 Jun 10;90(1-2):129-35. doi: 10.1016/s0304-4017(00)00227-2.

DOI:10.1016/s0304-4017(00)00227-2

PMID:10828519

Abstract

A real-time quantitative PCR using the TaqMan fluorogenic detection system (TaqMan PCR) was established for identification of Ehrlichia risticii, the agent of Potomac horse fever (PHF). The TaqMan PCR identified an 85 base pair section of the 16S rRNA gene by use of a specific fluorogenic probe and two primers. This technique was specific for eight tested E. risticii strains. The TaqMan system identified 10 copies of a cloned section of the 16S rRNA gene of E. risticii. The sensitivity and specificity of the TaqMan PCR were similar to those of conventional nested PCR. The TaqMan PCR was evaluated on horses with infectious colitis and on freshwater stream snails collected from regions with a history of PHF. E. risticii could be detected in 22 of 153 (14.4%) horses with infectious colitis and in 25 of 234 (10.7%) snails in the TaqMan PCR. The same results were obtained in the conventional nested PCR. The Ehrlichia-load was in the range of 10,000-9,000,000 and 35,000-680, 000,000 Ehrlichia equivalents per microg leukocyte DNA and snail DNA, respectively.

摘要

建立了一种使用TaqMan荧光检测系统的实时定量PCR（TaqMan PCR）方法，用于鉴定波托马克马热（PHF）的病原体里氏埃立克体。TaqMan PCR使用特异性荧光探针和两条引物，鉴定16S rRNA基因的一个85碱基对的片段。该技术对8株受试里氏埃立克体菌株具有特异性。TaqMan系统能够鉴定出里氏埃立克体16S rRNA基因克隆片段的10个拷贝。TaqMan PCR的敏感性和特异性与传统巢式PCR相似。对患有感染性结肠炎的马匹以及从有PHF病史地区采集的淡水溪流蜗牛进行了TaqMan PCR评估。在TaqMan PCR中，153匹患有感染性结肠炎的马匹中有22匹（14.4%）检测到里氏埃立克体，234只蜗牛中有25只（10.7%）检测到该病原体。传统巢式PCR也得到了相同的结果。每微克白细胞DNA和蜗牛DNA中的埃立克体载量分别在10,000 - 9,000,000和35,000 - 680,000,000个埃立克体当量范围内。