School of Breeding and Multiplication, School of Tropical Agriculture and Forestry, Hainan University, Sanya 572025, China.
Sanya Institute of China Agricultural University, Sanya 572025, China.
Int J Mol Sci. 2024 May 26;25(11):5773. doi: 10.3390/ijms25115773.
Southern stem canker (SSC) of soybean, attributable to the fungal pathogen , results in considerable losses of soybean in the field and has damaged production in several of the main soybean-producing countries worldwide. Early and precise identification of the causal pathogen is imperative for effective disease management. In this study, we performed an RPA-CRISPR/Cas12a, as well as LAMP, PCR and real-time PCR assays to verify and compare their sensitivity, specificity and simplicity and the practicality of the reactions. We screened crRNAs targeting a specific single-copy gene, and optimized the reagent concentrations, incubation temperatures and times for the conventional PCR, real-time PCR, LAMP, RPA and Cas12a cleavage stages for the detection of . In comparison with the PCR-based assays, two thermostatic detection technologies, LAMP and RPA-CRISPR/Cas12a, led to higher specificity and sensitivity. The sensitivity of the LAMP assay could reach 0.01 ng μL genomic DNA, and was 10 times more sensitive than real-time PCR (0.1 ng μL) and 100 times more sensitive than conventional PCR assay (1.0 ng μL); the reaction was completed within 1 h. The sensitivity of the RPA-CRISPR/Cas12a assay reached 0.1 ng μL genomic DNA, and was 10 times more sensitive than conventional PCR (1.0 ng μL), with a 30 min reaction time. Furthermore, the feasibility of the two thermostatic methods was validated using infected soybean leaf and seeding samples. The rapid, visual one-pot detection assay developed could be operated by non-expert personnel without specialized equipment. This study provides a valuable diagnostic platform for the on-site detection of SSC or for use in resource-limited areas.
南方茎溃疡病（SSC）由真菌病原体引起，导致田间大豆大量损失，并在全球几个主要大豆生产国造成了生产损失。早期和准确鉴定致病病原体对于有效疾病管理至关重要。在这项研究中，我们进行了 RPA-CRISPR/Cas12a 以及 LAMP、PCR 和实时 PCR 检测，以验证和比较它们的灵敏度、特异性和简便性以及反应的实用性。我们筛选了针对特定单拷贝基因的 crRNA，并优化了常规 PCR、实时 PCR、LAMP、RPA 和 Cas12a 切割阶段的试剂浓度、孵育温度和时间，用于检测。与基于 PCR 的检测方法相比，两种恒温检测技术，LAMP 和 RPA-CRISPR/Cas12a，具有更高的特异性和灵敏度。LAMP 检测的灵敏度可达 0.01 ng μL 基因组 DNA，比实时 PCR（0.1 ng μL）高 10 倍，比常规 PCR 检测（1.0 ng μL）高 100 倍；反应在 1 小时内完成。RPA-CRISPR/Cas12a 检测的灵敏度达到 0.1 ng μL 基因组 DNA，比常规 PCR（1.0 ng μL）高 10 倍，反应时间为 30 分钟。此外，使用感染的大豆叶片和种子样本验证了两种恒温方法的可行性。该研究提供了一种有价值的诊断平台，可用于现场检测 SSC 或在资源有限的地区使用。
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