Protocol for real-time PCR identification of anthrax spores from nasal swabs after broth enrichment
Protocol for real-time PCR identification of anthrax spores from nasal swabs after broth enrichment
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DOI：
10.1128/jcm.40.11.3956-3963.2002
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发表时间：
2002-11-01
影响因子：
9.4
通讯作者：
Pozzi, G
中科院分区：
文献类型：
--
作者：
Oggioni, MR;Meacci, F;Pozzi, G
设计了一种基于液体培养基中富集步骤的用于从鼻拭子诊断炭疽的大规模筛选方案。在可高压灭菌的小瓶和架子中进行生长孵育，以便对灭菌培养物进行实时PCR分析。用染色体标记rpoB和质粒标记lef的引物和探针建立双色PCR。设计特异性引物和探针，用于区分炭疽杆菌和B。蜡状芽孢杆菌，并用于区分Sterne疫苗株与田间分离株和最近炭疽生物恐怖袭击中使用的艾姆斯菌株。因此，本方案将实时PCR的高特异性和灵敏度与优异的生物安全性和处理大量样品所需的低操作时间相结合，这在涉及处理大量样品的控制程序期间是极其重要的。
A mass-screening protocol for the diagnosis of anthrax from nasal swabs based on an enrichment step in liquid medium was devised. Incubation for growth was performed in autoclavable vials and racks which allow real-time PCR analysis of sterilized cultures. A dual-color PCR was set up with primers and probes for the chromosomal marker rpoB and the plasmid marker lef. Specific primer and probe sets were designed for the differentiation of Bacillus anthracis from B. cereus and for the differentiation of the Sterne vaccine strain from field isolates and the Ames strain, which was used in the recent anthrax bioterrorist attack. The present protocol thus combines the high specificity and sensitivity of real-time PCR with excellent biosafety and the low hands-on time necessary for the processing of large numbers of samples, which is extremely important during control programs involving the processing of large numbers of samples.