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利用基因组特异性引物和Cas12a介导技术快速检测稻瘟菌小麦致病型
Houxiang Kang, Ye Peng, Kangyu Hua, Yufei Deng, Maria Bellizzi, Dipali Rani Gupta, Nur Uddin Mahmud, Alfredo S. Urashima, Sanjoy Kumar Paul, Gary Peterson, Yilin Zhou, Xueping Zhou, Md Tofazzal Islam, Guo-Liang Wang
工程（英文） ›› 2021, Vol. 7 ›› Issue (9) : 1326-1335.
PDF(2475 KB)
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利用基因组特异性引物和Cas12a介导技术快速检测稻瘟菌小麦致病型
Rapid Detection of Wheat Blast Pathogen Magnaporthe Oryzae Triticum Pathotype Using Genome-Specific Primers and Cas12a-mediated Technology
麦瘟病由稻瘟菌小麦致病型（MoT）引起，是一种存在于南美洲和孟加拉国的毁灭性病害。由于MoT通常不会在抽穗期之前使小麦显现染病症状，但在抽穗期其侵染会一直加剧，因此仅根据肉眼观察来使用杀菌剂是无效的。为了开发一种在苗期和营养期准确、灵敏检测MoT以控制病害蔓延的方法，我们对来自巴西的两个MoT分离株的基因组进行了测序，并确定了两个DNA片段：MoT-6098 和MoT-6099，它们存在于麦瘟病菌（MoT）基因组中，但不存在于感染稻瘟病菌（MoO）的水稻基因组中。利用聚合酶链反应（PCR），我们在来自南美洲和孟加拉国的53 株MoT和MoO分离株中证实了这两对标记引物的特异性。为了测试这两对标记引物的有效性，我们首先建立了一种环介导等温扩增（LAMP）方法。在等温条件下，这种方法在不使用常规PCR仪器的情况下也能检测MoT。随后，我们使用Cas12a 蛋白及导向RNA（gRNA）靶向MoT-6098 和MoT-6099 序列，两个靶标被识别后，均能激活Cas12a 非特异切割单链脱氧核糖核酸酶（ssDNAase）的活性，我们将依赖靶向Cas12a 激活ssDNase 活性，同时将重组酶聚合酶扩增（RPA）和核酸侧流免疫分析法（NALFIA）相结合，开发出一种准确、灵敏且经济高效地检测受感染小麦植株中MoT特异性DNA序列的方法。这种新技术可应用于田间麦瘟病和其他重要植物病害的快速检测。
Wheat blast, caused by the fungus Magnaporthe oryzae Triticum (MoT) pathotype, is a devastating disease persistent in South America and Bangladesh. Since MoT generally fails to cause visual symptoms in wheat until the heading stage when the infection would have advanced, disease control by fungicide application solely based on the detection of visual symptoms is ineffective. To develop an accurate and sensitive method to detect MoT at the seedling and vegetative stages for disease control, we sequenced the genomes of two MoT isolates from Brazil and identified two DNA fragments, MoT-6098 and MoT-6099, that are present in the MoT genome but not in the genome of the rice-infecting M. oryzae Oryzae (MoO) pathotype. Using polymerase chain reaction (PCR), we confirmed the specificity of the two markers in 53 MoT and MoO isolates from South America and Bangladesh. To test the efficiency of the two markers, we first established a loop-mediated isothermal amplification (LAMP) method to detect MoT at isothermal conditions, without the use of a PCR machine. Following this, we used the Cas12a protein and guide RNAs (gRNAs) to target the MoT-6098 and MoT-6099 sequences. The activated Cas12a showed indiscriminate single-stranded DNase (ssDNAase) activity. We then combined target-dependent Cas12a ssDNase activation with recombinase polymerase amplification (RPA) and nucleic acid lateral flow immunoassay (NALFIA) to develop a method that accurately, sensitively, and cost-effectively detects MoT-specific DNA sequences in infected wheat plants. This novel technique can be easily adapted for the rapid detection of wheat blast and other important plant diseases in the field.
麦瘟病 / 稻瘟菌小麦致病型 / Cas12a / 核酸侧流免疫分析 / 田间检测
Wheat blast / Magnaporthe oryzae / Triticum Cas12a / Nucleic acid rapid lateral flow immunoassay / Field detection
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