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<p><i>Diaporthe helianthi</i> is one of the main fungal pathogens responsible for causing phomopsis stem canker and significant yield losses of sunflowers. In this study, the calmodulin (<i>Cal</i>) gene of <i>D. helianthi</i> was selected to develop a rapid detection method involving recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a-based detection at 37&#xa0;°C. The developed detection system could complete the specific detection of <i>D. helianthi</i> in 45&#xa0;min, including 25&#xa0;min for RPA and 20&#xa0;min for CRISPR/Cas12 reaction. The detection system could be coupled with both lateral flow test strips and fluorescence signal reading modes. The detection limit for lateral flow assay was 1&#xa0;pg/μL genomic <i>D. helianthi</i> DNA (14&#xa0;copies/µL); The detection limit for fluorescence signal was 0.1&#xa0;pg/μL genomic DNA (1.4&#xa0;copies/µL), approximately 100&#xa0;times higher than that of the real-time PCR. Thus, the developed RPA/CRISPR-Cas12a system meets the need for portable detection of <i>D. helianthi</i> on-site at ports and in the field.</p>

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Rapid diagnosis of Diaporthe helianthi in sunflower using RPA/CRISPR-Cas12 and lateral flow assay

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年3月18日

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  • Ruirui Kuang,
  • Rong Lei,
  • Mingyue Li,
  • Xiwen Sun,
  • Weijun Duan,
  • Yifen Yang,
  • Junxia Shi,
  • Li Yang,
  • Zaifeng Fan,
  • Mengyuan lv,
  • Limei Li,
  • Pinshan Wu,
  • Xinyi Wang

摘要

Diaporthe helianthi is one of the main fungal pathogens responsible for causing phomopsis stem canker and significant yield losses of sunflowers. In this study, the calmodulin (Cal) gene of D. helianthi was selected to develop a rapid detection method involving recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a-based detection at 37 °C. The developed detection system could complete the specific detection of D. helianthi in 45 min, including 25 min for RPA and 20 min for CRISPR/Cas12 reaction. The detection system could be coupled with both lateral flow test strips and fluorescence signal reading modes. The detection limit for lateral flow assay was 1 pg/μL genomic D. helianthi DNA (14 copies/µL); The detection limit for fluorescence signal was 0.1 pg/μL genomic DNA (1.4 copies/µL), approximately 100 times higher than that of the real-time PCR. Thus, the developed RPA/CRISPR-Cas12a system meets the need for portable detection of D. helianthi on-site at ports and in the field.