Globisporangium recalcitrans is an important phytopathogenic oomycete that causes soybean root rot. To develop a loop-mediated isothermal amplification (LAMP)-based system for rapid and specific detection of G. recalcitrans , four LAMP primers and two loop primers were designed with the Ribosomal DNA transcribed spacer 2 sequence (ITS2) as the target gene. The specificity and sensitivity of these primers were validated, and the system was also successfully applied to detect G. recalcitrans in soybean tissues after artificial inoculation and natural infection. The nucleic acid amplification reaction was performed under isothermal conditions at 63 °C for 60 min. Specificity was compared with those for 71 strains of G. recalcitrans , other Globisporangium spp. The minimum detection limit of the system was 100 fg·μL −1 for detecting fungal genomic DNA. This LAMP-based system provides a technique for specific detection of G. recalcitrans and rapid diagnosis of the disease it caused.
Trunk canker disease caused by Botryosphaeria dothidea with a prolonged latent infection phase poses a serious threat to Chinese hickory production. To further understand the epidemiological characteristics and develop reasonable management techniques, a quantitative loop-mediated isothermal amplification (q-LAMP) assay was developed to quantitatively monitor B. dothidea in hickory plants, water, and air samples. Specific primers were designed based on the different sites of the β-tubulin sequence between B. dothidea and other fungi commonly found on Chinese hickory. At the optimum reaction temperature of 65.9°C, this loop-mediated isothermal amplification (LAMP) assay can specifically distinguish B. dothidea from other tested fungi. The limit of detection of LAMP assays for B. dothidea was 0.001 ng/µl of pure genomic DNA and 10 spores per 1 ml of water. The q-LAMP assay enables rapid detection of B. dothidea within 60 min in hickory trunk, water in hickory forests, and spores captured on tapes. These results provide a powerful and convenient tool for monitoring B. dothidea , which could be applied widely in epidemiology, forecast, and management of tree canker disease.
Cao Li-Xia,Pan Xing-Run,Zhang Wan-Na,Lu Jing-Bo,Hu Zhi-Kang,Sheng Cheng-Wang,Peng Ying-Chuan
The fall armyworm (Spodoptera frugiperda), a lepidopteran pest of highly destructive, has developed resistance to organophosphate and carbamate insecticides through target-site mutations, including A201S and F290V in the ace-1 gene encoding acetylcholinesterase (AChE). Developing rapid and reliable methods to detect these mutations is crucial for monitoring resistance and guiding effective management strategies. Although S. frugiperda populations remain sensitive to the tested insecticides, the A201S and F290V mutations were identified in collected field samples. To facilitate detection, a sensitive and field-deployable loop-mediated isothermal amplification (LAMP) assay was developed, targeting these mutations within the ace-1 gene of S. frugiperda. The specificity and sensitivity of the LAMP assay was optimized for rapid detection, with reactions performed at 68 °C for 80 min. Results were visualized through color change and confirmed by gel electrophoresis. Validation using DNA from laboratory and field samples demonstrated complete agreement between the LAMP assay and DNA sequencing, affirming the accuracy and reliability of the method. The developed LAMP assay provides a simple, rapid, and effective tool for detecting the A201S and F290V mutations in the ace-1 gene of S. frugiperda. The assay’s simplicity, isothermal operation, and clear visual results make it a valuable tool for resistance monitoring and integrated pest management in agricultural settings.
危险品化学品经营许可证（带存储）