A molecular diagnostic method for selected Ascosphaera species using PCR amplification of internal transcribed spacer regions of rDNA
A molecular diagnostic method for selected Ascosphaera species using PCR amplification of internal transcribed spacer regions of rDNA
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DOI：
10.1080/00218839.2005.11101150
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发表时间：
2005-01-01
影响因子：
1.9
通讯作者：
Jones, WA
中科院分区：
文献类型：
--
作者：
Murray, KD;Aronstein, KA;Jones, WA
囊腔菌属真菌与群居和独居的蜜蜂有关，在某些情况下是引起白垩病的病原体。作为形态学鉴定的补充，我们开发了一种简单的基于PCR的方法，选择Ascosphaera物种。我们利用rDNA内部转录间隔区的序列差异来设计物种特异性引物。分析涉及简单地对给定引物对的单个条带的存在或不存在进行评分。该方法可以区分已知与蜜蜂相关的四种Ascosphaera物种。它还区分Ascosphaera aggregata，苜蓿切叶蜂的白垩病病原体，从其他Ascosphaera与此蜂。我们希望该方法将是有用的，用于确定Ascosphaera文化的纯度，并可能是第一步，对蜜蜂和切叶蜂白垩病感染的早期检测方法的发展。我们还提出了一种新的，快速和可靠的方法来制备真菌DNA适合PCR扩增菌丝生长在液体或固体培养基上。
Ascosphaera spp. fungi are associated with social and solitary bees, in some cases as pathogens causing chalkbrood disease. As a supplement to morphological identification, we developed a simple PCR-based method for selected Ascosphaera species. We exploited sequence differences in the internal transcribed spacer regions of rDNA to design species-specific primers. Analysis involves simply scoring the presence or absence of a single band for a given pair of primers. The method can distinguish the four Ascosphaera species known to be associated with honey bees. It also distinguishes Ascosphaera aggregata, the chalkbrood pathogen of the alfalfa leafcutting bee, from other Ascosphaera species associated with this bee. We expect the method will be useful for determining purity of Ascosphaera cultures, and may be a first step toward development of an early detection method of chalkbrood infection in honey bees and leafcutting bees. We also present a new, quick and reliable method for preparing fungal DNA suitable for PCR amplification from mycelia grown in liquid or on solid media.