基于聚合酶链反应检测石蜡包埋组织中类鼻疽伯克霍尔德菌DNA的策略。
Strategies for PCR based detection of Burkholderia pseudomallei DNA in paraffin wax embedded tissues.
作者信息
Hagen R M, Gauthier Y P, Sprague L D, Vidal D R, Zysk G, Finke E-J, Neubauer H
机构信息
Institut für Mikrobiologie der Bundeswehr, Neuherbergstr. 11, D-80937 München, Germany.
出版信息
Mol Pathol. 2002 Dec;55(6):398-400. doi: 10.1136/mp.55.6.398.
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.
摘要
最近，欧洲已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。本报告描述了一种优化方案，用于快速、可靠地制备DNA，以用于两种不同的聚合酶链反应（PCR）检测，即：（1）针对核糖体蛋白亚基21（rpsU）基因属特异性序列的半巢式PCR检测；（2）针对编码丝状鞭毛蛋白（fliC）基因的巢式PCR检测。对各种伯克霍尔德菌属菌株、密切相关属的菌株以及实验感染小鼠的脾脏组织样本进行了研究。PCR与扩增子测序相结合具有高灵敏度和特异性。这些方法可在常规福尔马林固定和石蜡包埋样本中快速、灵敏且可靠地检测假鼻疽伯克霍尔德菌DNA，从而提供一种安全的诊断工具，并避免培养3类风险病原体。此外，该方法对于回顾性组织病理学研究可能有用。
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