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首页> 外文期刊>British Biotechnology Journal >Improved Sample Preparation for PCR-BasedAssays in the Detection of XanthomonadsCausing Bacterial Leaf Spot of Tomato
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Improved Sample Preparation for PCR-BasedAssays in the Detection of XanthomonadsCausing Bacterial Leaf Spot of Tomato

机译：改进的样品制备方法，用于基于PCR的检测细菌性黄斑病的番茄黄单胞菌检测

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Aims: To develop a sampling procedure for PCR-based screening of bacterial leaf spot (BLS)-causing xanthomonads without DNA extraction from infected tomato plants.Place and Duration of Study: University of Copenhagen, Denmark and Sokoine University of Agriculture, Morogoro, Tanzania between July 2008 and November 2010.Methodology: Flinders Technology Associates (FTA?) plant cards and Chromatography paper or Whatman? paper strips (WPS) were spotted with bacterial suspensions from 24-h-old cultures from reference strains of BLS-causing xanthomonads, or sap obtained by grinding or hand maceration of plant tissue, were used as templates in PCR reactions or isolation of live bacterial cells on Nutrient agar (NA) media. Samples were tested by PCR with Xan 7 genus/-specific Xanthomonas primers or in multiplex with 26S rRNA primers. Isolation of bacteria was done by streaking aliquots of 75 μl of a suspension from a disc (2-mm-punch by Harris Micro Punch?) in triplicate, removed from each of the FTA plant card and WPS onto NA media. Results: The FTA plant card spotted with pure cultures of reference strains of xanthomonads and sap from grinding or direct maceration of plant tissue resulted in more clear PCR bands (402 bp) and (594 bp of rRNA gene in multiplex) than the WPS samples. Sensitivity of detection by the FTA paper-based PCR was ≈ 5.0 x 102, while that of the WPS was > 1.0 x 103 CFU/ml. The WPS (but not the FTA) was proved to be useful for saving living bacteria cells for up to one week of storage at ambient temperatures.Conclusion: Both FTA plant card and WPS can be used for PCR detection of BLS-causing xanthomonads in tomato. However, the FTA plant card is recommended as it produced clearer PCR products than WPS. WPS is recommended for experiments requiring isolation of live bacterial cells on NA media.
机译：目的：开发一种采样方法，以基于PCR的方式筛选引起细菌叶斑（BLS）的黄原菌，而无需从感染的番茄植物中提取DNA。研究地点和期限：丹麦哥本哈根大学和坦桑尼亚莫罗哥罗的索科恩农业大学在2008年7月至2010年11月之间。方法：弗林德斯技术协会（FTA？）种植卡和色谱纸或Whatman？在纸条（WPS）上用来自BLS致病性黄单胞菌参考菌株的24小时培养物中的细菌悬液点样，或通过研磨或手工浸渍植物组织而获得的汁液，用作PCR反应或分离活细菌的模板营养琼脂（NA）培养基上的细胞。用Xan 7属/特异的Xanthomonas引物通过PCR或26S rRNA引物多重测试样品。分离细菌的方法是，从圆盘（Harris Micro Punch打孔机2毫米打孔）中一式三份地取75μl悬浮液的等分试样，从FTA植物卡和WPS的每张中取出，移至NA培养基上。结果：在FTA植物卡上标记了纯的黄单胞菌参考菌株培养物和来自植物组织研磨或直接浸渍的汁液，与WPS样品相比，产生了更清晰的PCR条带（402 bp）和594 bp（多重反应中的rRNA基因）。 FTA纸质PCR的检测灵敏度为≈5.0 x 102，而WPS的检测灵敏度为> 1.0 x 103 CFU / ml。事实证明，WPS（而非FTA）可用于保存活细菌细胞，使其在环境温度下保存长达一周。结论：FTA植物卡和WPS均可用于PCR检测番茄中引起BLS的黄单胞菌。 。但是，建议使用FTA植物卡，因为它产生的PCR产品比WPS更清晰。建议将WPS用于需要在NA培养基上分离活细菌细胞的实验中。

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