Dinkel Anke, Njoroge Ernest M, Zimmermann Anja, Wälz Marcus, Zeyhle Eberhard, Elmahdi Ibrahim E, Mackenstedt Ute, Romig Thomas
Department of Parasitology, University of Hohenheim, Emil-Wolff-Strasse 34, 70599 Stuttgart, Germany.
Int J Parasitol. 2004 Apr;34(5):645-53. doi: 10.1016/j.ijpara.2003.12.013.
We describe the development of a specific and sensitive PCR/semi-nested PCR system for the rapid diagnosis of Echinococcus granulosus genotype G1, E. granulosus genotype G6/7, and Echinococcus ortleppi (G5). Diagnosis of G1 and the group G5/6/7 is performed by a simple PCR, while discrimination between E. ortleppi (G5) and G6/7 involves a subsequent semi-nested PCR step. The target sequence for amplification is part of the mitochondrial 12S rRNA gene. Specificity of the PCRs was 100% when evaluated with isolates of 16 species of cestodes, including Echinococcus multilocularis, Echinococcus equinus, E. ortleppi and three strains of E. granulosus (G1, G6 and G7). Sensitivity threshold was 0.25pg of DNA. This new approach was compared with published protocols of restriction fragment length polymorphism-PCR and sequencing of mitochondrial cytochrome c oxidase subunit 1 and NADH dehydrogenase 1 genes using Echinococcus isolates of human, sheep, goat, camel, cattle and pig origin from Kenya and Sudan. Additionally, two internal DNA probes were developed, one hybridising only with G1, the other with G5, G6 and G7 amplification products. Preliminary epidemiological results obtained with this PCR approach include the detection of a camel strain (G6) infection for the first time in a human patient from eastern Africa, and the first reports of E. ortleppi (G5) in livestock from Kenya and the Sudan.
我们描述了一种用于快速诊断细粒棘球绦虫G1基因型、细粒棘球绦虫G6/7基因型和奥氏棘球绦虫（G5）的特异性和灵敏性PCR/半巢式PCR系统的开发。通过简单的PCR对G1和G5/6/7组进行诊断，而区分奥氏棘球绦虫（G5）和G6/7则需要后续的半巢式PCR步骤。扩增的靶序列是线粒体12S rRNA基因的一部分。当用16种绦虫的分离株进行评估时，包括多房棘球绦虫、马棘球绦虫、奥氏棘球绦虫和三株细粒棘球绦虫（G1、G6和G7），PCR的特异性为100%。灵敏度阈值为0.25pg DNA。使用来自肯尼亚和苏丹的人、绵羊、山羊、骆驼、牛和猪源的棘球绦虫分离株，将这种新方法与已发表的限制性片段长度多态性-PCR以及线粒体细胞色素c氧化酶亚基1和NADH脱氢酶1基因测序方案进行了比较。此外，还开发了两种内部DNA探针，一种仅与G1杂交，另一种与G5、G6和G7扩增产物杂交。用这种PCR方法获得的初步流行病学结果包括首次在一名东非人类患者中检测到骆驼株（G6）感染，以及首次在肯尼亚和苏丹的家畜中报告奥氏棘球绦虫（G5）。
