Strategies for PCR based detection of Burkholderia pseudomallei DNA in paraffin wax embedded tissues.

Molecular pathology : MP Pub Date : 2002-12-01 DOI:10.1136/mp.55.6.398

R M Hagen, Y P Gauthier, L D Sprague, D R Vidal, G Zysk, E-J Finke, H Neubauer

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引用次数: 46

Abstract

Recently, several cases of melioidosis imported to Europe have been reported. The diagnosis of the acute or chronic infection remains challenging. This report describes an optimised protocol for fast and reliable DNA preparation for use in two different polymerase chain reaction (PCR) assays, namely: (1) a seminested PCR assay targeting a genus specific sequence of the ribosomal protein subunit 21 (rpsU) gene and (2) a nested PCR assay targeting the gene encoding the filament forming flagellin (fliC). Various strains of Burkholderia spp, strains of closely related genera, and spleen tissue samples of experimentally infected mice were investigated. The combination of PCR and sequencing of the amplicons resulted in high sensitivity and specificity. These procedures may allow rapid, sensitive, and reliable detection of B pseudomallei DNA in routinely formalin fixed and paraffin wax embedded samples, thus providing a safe diagnostic tool and avoiding the cultivation of a risk group 3 agent. In addition, this method could be useful for retrospective histopathological investigations.

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基于PCR检测石蜡包埋组织中假马氏伯克氏菌DNA的策略

最近，欧洲报告了几例输入性类鼻疽病例。急性或慢性感染的诊断仍然具有挑战性。本报告描述了一种用于两种不同聚合酶链反应(PCR)测定的快速可靠的DNA制备的优化方案，即:(1)针对核糖体蛋白亚基21 (rpsU)基因属特异性序列的半嵌套PCR测定和(2)针对编码细丝形成鞭毛蛋白(fliC)基因的嵌套PCR测定。研究了不同种类的伯克霍尔德氏菌及其密切相关属的菌株和实验感染小鼠的脾脏组织样本。PCR与扩增子测序相结合，具有较高的灵敏度和特异性。这些方法可以快速、灵敏、可靠地检测常规福尔马林固定和石蜡包埋样品中的假芽孢杆菌DNA，从而提供了一种安全的诊断工具，避免了风险组3剂的培养。此外，该方法可用于回顾性组织病理学调查。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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Molecular pathology : MP

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