Alternaria burnsii is the causal agent of cumin blight, a seed-borne disease of economic concern for all cumin growing areas. Current detection and identification methods for the pathogen are based on visual examination of morphological features, which are time-consuming and laborious. The present study describes conventional and real-time PCR assays for rapid and accurate detection of A. burnsii in cumin seeds. Based on sequence differences in Alternaria allergen a1 (Alt a1) gene, two primer pairs, Ab35/326 and AB177/403, were designed for conventional and real-time PCR assays, respectively. Both primer pairs amplified the expected target PCR fragment from A. burnsii genomic DNA. The sensitivity of conventional PCR with primer pairs Ab35/326 was 1 pg of genomic DNA and allowed the detection of pathogen in cumin seeds samples with 0.2% infestation rate. Real-time PCR assay was highly sensitive and allowed the quantification of 0.1 pg pathogen DNA. Also, this assay confirmed the presence of pathogen in cumin seeds up to 0.1% infestation level. The standard curve (r² = 0.99) showed a good correlation between fungal DNA quantities and Cq values. The specificity of primer pairs was confirmed by the absence of amplified product with DNA of related fungi species and healthy plant tissue. The assays developed in this study provide a rapid and sensitive tool for the detection and quantification of Alternaria burnsii in cumin seed.

中文翻译：

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常规和实时PCR检测试剂盒检测小茴香种子中链格孢菌的开发

交链孢霉是小茴香枯萎病的病原体，是所有小茴香生产地区经济上关注的种子传播疾病。当前的病原体检测和鉴定方法是基于目视检查形态特征，这既费时又费力。本研究中描述了一种用于快速和准确的检测的常规和实时PCR测定甲。 小茴香种子中的burnsii。根据链球菌变应原a1（Alt a1）基因的序列差异，分别设计了两个引物对Ab35 / 326和AB177 / 403，分别用于常规PCR和实时PCR分析。两个引物对均从A扩增了预期的目标PCR片段。 伯恩斯二世基因组DNA。使用引物对Ab35 / 326进行常规PCR的灵敏度为1 pg基因组DNA，可检测出孜然种子样品中的病原体，侵染率为0.2％。实时PCR检测具有很高的灵敏度，可以定量0.1 pg病原体DNA。而且，该测定法确认了孜然种子中病原体的存在，侵染水平高达0.1％。标准曲线（r ²  = 0.99）显示真菌DNA量与Cq值之间具有良好的相关性。引物对的特异性通过不存在具有相关真菌物种和健康植物组织的DNA的扩增产物来证实。在这项研究中开发的测定法提供了一种快速，灵敏的工具，用于检测和定量孜然种子中的链格孢菌。