率先利用尼特基因开发用于快速检测田间土壤中棉花根腐病致病菌--Macrophomina phaseolina (Tassi) Goid 的分子诊断测定法。

IF 3.5 4区 生物学 Q2 MICROBIOLOGY Journal of Basic Microbiology Pub Date : 2024-08-01 DOI:10.1002/jobm.202400325
Anil Kumar Saini, Mukesh Kumar, Karmal Singh, Mukul Kumar Bhambhu, Rohit Nain,  Garima,  Aakash, Shiwani Mandhania, Shubham Saini
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引用次数: 0

摘要

棉花根腐病是由棉花根腐霉菌(Macrophomina phaseolina)引起的,对棉花生产构成严重威胁,导致产量和质量大幅下降。及早准确地诊断土壤中的这种病原体对有效控制病害至关重要。本研究开创性地利用编码硝化酶的 nit 基因开发了一种分子诊断测定法,旨在快速检测田间土壤中的棉花褐斑病菌。该方法包括设计和验证针对硝基基因序列的引物,然后优化 PCR 条件以实现高效扩增。利用最先进的分子技术,该检测方法提供了一种新的方案,可准确识别土壤中是否存在高灵敏度和高特异性的相思豆菌。通过使用相柄霉和其他相关真菌的 DNA 进行 PCR 扩增,证实了所设计引物的特异性。灵敏度测试表明,PCR 法能可靠地检测出浓度低至 1 纳克的相思豆菌 DNA。此外,该诊断方法的性能还通过已知相思豆菌感染状况的田间土壤样本进行了严格评估,证明了其在实际应用中的可靠性和有效性。这项研究引入了一种用于检测相思豆菌的新型分子标记,并提供了一种快速、高效的方法,能以最少的时间和人力筛查大量土壤样本中的相思豆菌。
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Pioneering Nit Gene Exploitation to Develop Molecular Diagnostic Assay for Rapid Detection of Cotton Root Rot Incitant, Macrophomina phaseolina (Tassi) Goid, in Field Soil

Cotton root rot caused by Macrophomina phaseolina pose a significant threat to cotton production, leading to substantial yield and quality losses. Early and accurate diagnosis of this pathogen in soil is crucial for effective disease management. This study presents a pioneering investigation into the utilization of the nit gene encoding nitrilase for the development of a molecular diagnostic assay aimed at the rapid detection of M. phaseolina in field soils. The methodology involved the design and validation of primers targeting the Nit gene sequence, followed by the optimization of PCR conditions for efficient amplification. Leveraging state-of-the-art molecular techniques, the assay offers a novel protocol to accurately identify the presence of M. phaseolina in soil with high sensitivity and specificity. The specificity of the designed primers was confirmed through PCR amplification using DNA from M. phaseolina and other related fungi. Sensitivity tests demonstrated that the PCR assay reliably detected M. phaseolina DNA at concentrations as low as 1 ng. Furthermore, the performance of the diagnostic assay was rigorously evaluated using field soil samples with a known status of M. phaseolina infection, demonstrating its reliability and efficacy in real-world scenarios. This study introduces a novel molecular marker for the detection of M. phaseolina and offers a rapid and efficient means for screening M. phaseolina in large soil samples with minimal time and manpower.

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来源期刊
Journal of Basic Microbiology
Journal of Basic Microbiology 生物-微生物学
CiteScore
6.10
自引率
0.00%
发文量
134
审稿时长
1.8 months
期刊介绍: The Journal of Basic Microbiology (JBM) publishes primary research papers on both procaryotic and eucaryotic microorganisms, including bacteria, archaea, fungi, algae, protozoans, phages, viruses, viroids and prions. Papers published deal with: microbial interactions (pathogenic, mutualistic, environmental), ecology, physiology, genetics and cell biology/development, new methodologies, i.e., new imaging technologies (e.g. video-fluorescence microscopy, modern TEM applications) novel molecular biology methods (e.g. PCR-based gene targeting or cassettes for cloning of GFP constructs).
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