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首页> 外文期刊>Investigative Genetics >Method: a single nucleotide polymorphism genotyping method for Wheat streak mosaic virus
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Method: a single nucleotide polymorphism genotyping method for Wheat streak mosaic virus

机译：方法：小麦条纹花叶病毒单核苷酸多态性基因分型方法

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Background The September 11, 2001 attacks on the World Trade Center and the Pentagon increased the concern about the potential for terrorist attacks on many vulnerable sectors of the US, including agriculture. The concentrated nature of crops, easily obtainable biological agents, and highly detrimental impacts make agroterrorism a potential threat. Although procedures for an effective criminal investigation and attribution following such an attack are available, important enhancements are still needed, one of which is the capability for fine discrimination among pathogen strains. The purpose of this study was to develop a molecular typing assay for use in a forensic investigation, using Wheat streak mosaic virus (WSMV) as a model plant virus. Method This genotyping technique utilizes single base primer extension to generate a genetic fingerprint. Fifteen single nucleotide polymorphisms (SNPs) within the coat protein and helper component-protease genes were selected as the genetic markers for this assay. Assay optimization and sensitivity testing was conducted using synthetic targets. WSMV strains and field isolates were collected from regions around the world and used to evaluate the assay for discrimination. The assay specificity was tested against a panel of near-neighbors consisting of genetic and environmental near-neighbors. Result Each WSMV strain or field isolate tested produced a unique SNP fingerprint, with the exception of three isolates collected within the same geographic location that produced indistinguishable fingerprints. The results were consistent among replicates, demonstrating the reproducibility of the assay. No SNP fingerprints were generated from organisms included in the near-neighbor panel, suggesting the assay is specific for WSMV. Using synthetic targets, a complete profile could be generated from as low as 7.15 fmoles of cDNA. Conclusion The molecular typing method presented is one tool that could be incorporated into the forensic science tool box after a thorough validation study. This method incorporates molecular biology techniques that are already well established in research and diagnostic laboratories, allowing for an easy introduction of this method into existing laboratories.
机译：背景资料2001年9月11日对世界贸易中心和五角大楼的袭击使人们更加关注对包括农业在内的美国许多脆弱地区进行恐怖袭击的可能性。农作物的集中特性，易于获得的生物制剂以及极有害的影响使农业恐怖主义成为潜在的威胁。尽管可以使用这种攻击后进行有效的刑事调查和归因的程序，但仍需要进行重要的改进，其中之一是能够对病原体进行精细区分。这项研究的目的是使用小麦条纹花叶病毒（WSMV）作为模型植物病毒，开发一种用于法医研究的分子分型测定法。方法该基因分型技术利用单碱基引物延伸来产生遗传指纹。选择外壳蛋白和辅助成分蛋白酶基因中的15个单核苷酸多态性（SNP）作为该测定的遗传标记。使用合成靶标进行了测定优化和灵敏度测试。从世界各地收集了WSMV菌株和野外分离株，并用于评估鉴别方法。针对由遗传和环境近邻组成的一组近邻测试了测定特异性。结果测试的每个WSMV菌株或野外分离株均产生唯一的SNP指纹，但在同一地理位置内收集的三个分离株却产生了难以区分的指纹。重复之间的结果是一致的，证明了测定的可重复性。没有从邻近面板中包含的生物生成SNP指纹，这表明该测定法对WSMV具有特异性。使用合成靶标，可以从低至7.15 fmole的cDNA生成完整的图谱。结论提出的分子分型方法是经过全面验证研究后可以整合到法医学工具箱中的一种工具。该方法结合了在研究和诊断实验室中已经建立的分子生物学技术，可以轻松地将该方法引入现有实验室。

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