Sensitive and rapid quantitative detection of anthrax spores isolated from soil samples by real-time PCR
Sensitive and rapid quantitative detection of anthrax spores isolated from soil samples by real-time PCR
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DOI：
10.1111/j.1348-0421.2003.tb03434.x
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发表时间：
2003-01-01
影响因子：
2.6
通讯作者：
Oh, HB
中科院分区：
文献类型：
--
作者：
Ryu, C;Lee, K;Oh, HB
环境样品中炭疽孢子的定量分析对于准确检测和风险评估至关重要，因为炭疽芽孢杆菌孢子已被证明是最有效的生物武器之一。采用TaqMan实时荧光定量PCR技术，设计特异性引物和探针，对致病性B进行鉴定。炭疽菌菌株的pag基因和cap基因分别位于两个质粒pXO 1和pXO 2上，以及sap基因编码的S层上。为了选择合适的环境样品中炭疽孢子的裂解方法，采用多重PCR技术对几种热处理和萌发方法进行了评价。其中，热处理悬浮有蔗糖和非离子洗涤剂的样品被认为是一种有效的孢子破碎方法，因为它检测到高达10（5）孢子/克土壤的多重PCR。B的系列稀释液。通过实时PCR检测到炭疽DNA和孢子分别达到0.1 ng/穆尔和10孢子/ml的水平，相关系数为0.99。用C-T值与土样系列稀释液进行比较，相关系数为0.99，可实现炭疽孢子的定量分析。此外，通过实时PCR，在3小时内检测到添加到土壤样品中的孢子高达10（4）个孢子/g土壤。因此，我们建立了一种快速、准确的环境炭疽孢子实时荧光定量PCR检测体系，避免了富集培养和DNA制备等费时费力的制备步骤。
Quantitative analysis of anthrax spores from environmental samples is essential for accurate detection and risk assessment since Bacillus anthracis spores have been shown to be one of the most effective biological weapons. Using TaqMan real-time PCR, specific primers and probes were designed for the identification of pathogenic B. anthracis strains from pag gene and cap gene on two plasmids, pXO1 and pXO2, as well as a sap gene encoded on the S-layer. To select the appropriate lysis method of anthrax spore from environmental samples, several heat treatments and germination methods were evaluated with multiplex-PCR. Among them, heat treatment of samples suspended with sucrose plus non-ionic detergent was considered an effective spore disruption method because it detected up to 10(5) spores/g soil by multiplex-PCR. Serial dilutions of B. anthracis DNA and spore were detected up to a level of 0.1 ng/mul and 10 spores/ml, respectively, at the correlation coefficient of 0.99 by real-time PCR. Quantitative analysis of anthrax spore could be obtained from the comparison between C-T value and serial dilutions of soil sample at the correlation coefficient of 0.99. Additionally, spores added to soil samples were detected up to 10(4) spores/g soil within 3 hr by real-time PCR. As a consequence, we established a rapid and accurate detection system for environmental anthrax spores using real-time PCR, avoiding time and labor-consuming preparation steps such as enrichment culturing and DNA preparation.