Comparison of rapid, automated ribotyping and DNA macrorestriction analysis of Burkholderia pseudomallei
Comparison of rapid, automated ribotyping and DNA macrorestriction analysis of Burkholderia pseudomallei
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DOI：
10.1128/jcm.40.9.3198-3203.2002
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发表时间：
2002-09-01
影响因子：
9.4
通讯作者：
Sampson, J
中科院分区：
文献类型：
--
作者：
Inglis, TJJ;O'Reilly, L;Sampson, J
使用自动核糖体分型设备(RiboPrint)来确定一组假腮腺伯克霍尔德菌分离株的核糖型。在与限制性内切酶BanzIII和EcoRI的初步评估中，EcoRI的方法更具区分性。通过检测三个水平的细菌负荷来确定用EcoRI(EcoRI Ribotype)获得的核型的重复性。通过将机器优化的核糖体类型与Bionumerics软件分析的原始凝胶图像确定的类型进行比较，对制造商软件的性能进行了评估。将构建的假鼻疽杆菌EcoRI核型文库与脉冲场凝胶电泳法对XbaI酶切片段进行DNA宏限制性内切酶分析得到的核型进行比较。用EcoRI核糖体分型法对假鼻疽杆菌进行分型，可分型百分率为100%，识别指数为0.94。DNA大限制性内切酶分析提供的稍大的区分度(0.96)是以显著较长的6天处理时间为代价实现的，尽管该方法的成本仅为自动核糖体测序的一半。宏观限制分析的可分型性较低(97%)，除非添加硫脲步骤来中和Tris依赖的内切酶的作用。到目前为止分析的假鼻疽分离株的数字记录为未来的流行病学研究提供了有用的资源，并将有助于缩短应对时间，以应对进一步的类鼻疽疾病暴发或故意将假鼻疽作为生物危害释放。
An automated ribotyping device (RiboPrinter) was used to determine the ribotypes of a collection of Burkholderia pseudomallei isolates. In a preliminary evaluation with the restriction enzymes BanzIII and EcoRI, the protocol with EcoRI was more discriminating. The reproducibilities of the ribotypes obtained with EcoRI (EcoRI ribotypes) were determined by testing three levels of bacterial loads. The performance of the manufacturer's software was assessed by comparing the machine-optimized ribotypes with the type determined from the original gel image analyzed with Bionumerics software. The library of B. pseudomallei EcoRI ribotypes was then compared with the ribotypes obtained by DNA macrorestriction analysis of XbaI digests by pulsed-field gel electrophoresis. The typeability of B. pseudomallei by EcoRI ribotyping was 100%, and the discrimination index was 0.94. The slightly greater discrimination provided by DNA macrorestriction analysis (0.96) was achieved at the expense of a significantly longer processing time of 6 days, although the method was only half the cost of automated ribotyping. Typeability by macrorestriction analysis was lower (97%) unless a thiourea step was added to neutralize the action of Tris-dependent endonucleases. The digital record of B. pseudomallei isolates analyzed thus far provides a useful resource for future epidemiological studies and will help shorten the response time in the event of a further melioidosis outbreak or the deliberate release of B. pseudomallei as a biohazard.