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首页> 美国卫生研究院文献>International Journal of Molecular Sciences >Detection of Mycosphaerella graminicola in Wheat Leaves by a Microsatellite Dinucleotide Specific-Primer
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Detection of Mycosphaerella graminicola in Wheat Leaves by a Microsatellite Dinucleotide Specific-Primer

机译：微卫星二核苷酸特异性引物检测小麦叶片中的白粉菌

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摘要

Early detection of infection is very important for efficient management of Mycosphaerella graminicola leaf blotch. To monitor and quantify the occurrence of this fungus during the growing season, a diagnostic method based on real-time PCR was developed. Standard and real-time PCR assays were developed using SYBR Green chemistry to quantify M. graminicola in vitro or in wheat samples. Microsatellite dinucleotide specific-primers were designed based on microsatellite repeats of sequences present in the genome of M. graminicola. Specificity was checked by analyzing DNA of 55 M. graminicola isolates obtained from different geographical origins. The method appears to be highly specific for detecting M. graminicola; no fluorescent signals were observed from 14 other closely related taxa. Primer (CT) 7 G amplified a specific amplicon of 570 bp from all M. graminicola isolates. The primers did not amplify DNA extracted from 14 other fungal species. The approximate melting temperature (Tm) of the (CT) 7 G primer was 84.2 °C. The detection limit of the real-time PCR assay with the primer sets (CT) 7 G is 10 fg/25 μL, as compared to 10 pg/25 μL using conventional PCR technology. From symptomless leaves, a PCR fragment could be generated two days after inoculation. Both conventional and real-time PCR could successfully detect the fungus from artificially inoculated wheat leaves. However, real-time PCR appeared much more sensitive than conventional PCR. The developed quantitative real-time PCR method proved to be rapid, sensitive, specific, cost-effective and reliable for the identification and quantification of M. graminicola in wheat.
机译：感染的早期检测对于有效处理格氏米氏菌叶斑是非常重要的。为了监测和量化这种真菌在生长期的发生，开发了一种基于实时PCR的诊断方法。使用SYBR Green化学试剂盒开发了标准和实时PCR分析方法，用于在体外或小麦样品中定量分析graminicola。微卫星二核苷酸特异引物是根据graminicola基因组中存在的序列的微卫星重复序列设计的。通过分析从不同地理来源获得的55 M. graminicola分离株的DNA来检查特异性。该方法似乎对检测格氏念珠菌具有高度特异性。从其他14个密切相关的类群中未观察到荧光信号。引物（CT）7 G从所有graminicola分离株中扩增了570 bp的特异性扩增子。引物未扩增从其他14种真菌中提取的DNA。 （CT）7 G底漆的近似熔化温度（Tm）为84.2°C。与使用常规PCR技术的10 pg / 25μL相比，使用引物对（CT）7 G进行实时PCR检测的检测限为10 fg / 25μL。接种两天后，可以从无症状的叶子中产生PCR片段。传统PCR和实时PCR均可成功地从人工接种的小麦叶片中检测出真菌。但是，实时PCR似乎比常规PCR更加灵敏。事实证明，所开发的定量实时PCR方法快速，灵敏，特异，经济高效且可靠，可用于鉴定和定量小麦中的小麦分枝杆菌。

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