Development of a new PCR primer system for selective amplification of Actinobacteria
Development of a new PCR primer system for selective amplification of Actinobacteria
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DOI：
10.1111/j.1574-6968.2010.02069.x
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发表时间：
2010-10-01
影响因子：
2.1
通讯作者：
Kaempfer, Peter
中科院分区：
文献类型：
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作者：
Schaefer, Jenny;Jaeckel, Udo;Kaempfer, Peter
放线菌在水损坏的建筑材料中的出现以及一些放线菌（例如多孢放线菌属，分枝杆菌属，诺卡氏菌属，等等），引导我们开发了一个检测系统来检测放线菌群落。设计了一个新的放线菌特异性引物系统Com 2xf/Ac 1186 r（基于16 SrRNA基因）。首先使用放线菌纲75个不同属的164个不同种的序列，通过计算机模拟研究了引物系统预期用途的充分性。为了测试复杂环境样品中的引物特异性，产生了四个16 S rRNA基因克隆文库（石膏材料、堆肥材料、堆肥植物和鸭舍生物气溶胶）。总体而言，87%的序列被分配到放线菌属。为了验证新设计的引物系统在水损害建筑材料中的适用性，筛选了18种不同水损害材料的16 S rRNA基因克隆文库，以确定它们是否属于放线菌。共88%的所有'放线菌阳性'检测质粒插入正确隶属。SSCP指纹图谱的结果清楚地显示了不同样品中放线菌特异性引物系统检测到的物种的差异。本研究中获得的总体结果表明，开发的引物系统适用于其预期用途。
The occurrence of Actinobacteria in water-damaged building materials as well as the clinical relevance of some Actinobacteria (e.g. Saccharopolyspora spp., Mycobacterium spp., Nocardia spp., etc.), led us to develop a detection system to examine the actinobacterial community. A new primer system, Com2xf/Ac1186r (16S rRNA gene based) specific for Actinobacteria was designed. The adequacy for the intended use of the primer system was first investigated in silico using sequences of 164 different species belonging to 75 different genera of the class Actinobacteria. To test the primer specificity in complex environmental samples, four 16S rRNA gene clone libraries were generated (plaster material, compost material, compost plant- and duck house bioaerosols). Overall, 87% of obtained sequences were assigned to actinobacterial genera. To verify the applicability of the new designed primer system in water-damaged building material, 16S rRNA gene clone libraries of 18 different water-damaged materials were screened for their affiliation to Actinobacteria. A total of 88% of all 'Actinobacteria-positive' detected plasmid inserts were affiliated correctly. Results of SSCP-fingerprinting clearly showed differences of the species detected by the Actinobacteria-specific primer system within the different samples. Overall results obtained in this study indicate the applicability of the developed primer system for its intended use.