Jiufeng Sun, Xiqing Li, Hanxiang Zeng, Zhi Xie, Changming Lu, Liyan Xi, Gert S de Hoog
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引用次数: 0
摘要
马尔尼菲青霉是东南亚免疫功能低下宿主中一种严重全身性疾病的病原。在本研究中,一种被称为环介导等温扩增(LAMP)的新方法被描述为快速和特异性检测物种,使用来自rRNA基因内部转录间隔区(ITS)的引物集。扩增产物可以用SYBR Green I在小瓶中肉眼检测,也可以用琼脂糖凝胶电泳检测。在65℃的水浴中反应1小时后,用LAMP法对纯培养物进行特异性扩增;未观察到与其他真菌包括其他双曲青霉的交叉反应。可检测到的DNA上限是两个副本。此外,利用马尼菲青霉病患者的石蜡包埋组织样本和竹鼠的组织样本进行特异性扩增。该方法为临床实验室的快速诊断提供了强有力的工具,并在生态学研究中具有应用潜力。
Development and evaluation of loop-mediated isothermal amplification (LAMP) for the rapid diagnosis of Penicillium marneffei in archived tissue samples.
Penicillium marneffei is the etiologic agent of a severe systemic disease in immunocompromised hosts in Southeast Asia. In the present study, a novel method, known as loop-mediated isothermal amplification (LAMP), is described for the rapid and specific detection of the species, using a primer set derived from the internal transcribed spacer (ITS) region of the rRNA gene. Amplification products can be detected macroscopically by visual inspection in vials using SYBR Green I as well as by electrophoresis on agarose gel. The LAMP assay resulted in specific amplification of P. marneffei ITS using pure cultures after a 1-h reaction at 65 degrees C in a water bath; no cross-reactivity with other fungi including other biverticillate penicillia was observed. The detectable DNA limit was two copies. In addition, specific amplification was achieved using paraffin wax-embedded tissue samples from patients with penicilliosis marneffei and tissue samples from bamboo rats. The method provides a powerful tool for rapid diagnostics in the clinical lab, and has potential for use in ecological studies.