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Development of a TaqMan probe-based insulated isothermal PCR (TiiPCR) for the detection of Acidovorax citrulli, the bacterial pathogen of watermelon fruit blotch

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Abstract

Watermelon (Citrullus lanatus) is an important crop of the Cucurbitaceae family in fruit production worldwide. During its production, bacterial fruit blotch (BFB) caused by Acidovorax citrulli (Acidovorax avenae subsp. citrulli) is an important limiting factor on the volume and value of crops. This pathogen is known as a seed-borne pathogen, and the infested seeds can be a primary source of inoculum. Hence, a rapid and sensitive method for detecting A. citrulli on seeds would be an important tool in the management of BFB. In this study, we sought to develop a method to detect A. citrulli bacterial cells based on a TaqMan probe-based insulated isothermal PCR (TiiPCR) assay. Firstly, the specific primers and probe were designed based on a specific DNA fragment from the genome of A. citrulli. Then, PCR amplification was performed with the plasmid DNA to adjust the components of the PCR reagents, such as the concentrations of primers, magnesium chloride, and Taq DNA polymerase. Results revealed that 10 copies of plasmid DNA were detectable within the modified reagents by TiiPCR. Moreover, 10 bacterial cells in each reaction tube were detectable at a 100 % detection rate in this condition with a fluorescent signal intensification over 1.8. Based on these results, we concluded that a specific, rapid, and sensitive method based on TiiPCR had been successfully developed to detect bacterial cells of A. citrulli.

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Acknowledgments

This work was supported by grants to Dr. Yi-Hsien Lin from the Ministry of Science and Technology, Taiwan (NSC 101-2313-B-020-024-MY3 and MOST- 104-2311-B-020-001). We also wish to thank GeneReach Biotechnology Corp. of Taichung, Taiwan, for providing of iiPCR device used in this study.

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Authors and Affiliations

  1. Department of Plant Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan

    Pei-Yi Wu, Lien-Chun Ho, Jun-Jie Chang & Yi-Hsien Lin

  2. Department of Plant Pathology, National Chung-Hsing University, Taichung, Taiwan

    Kuo-Ching Tzeng & Wen-Ling Deng

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  1. Pei-Yi Wu
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  2. Lien-Chun Ho
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  3. Jun-Jie Chang
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  4. Kuo-Ching Tzeng
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  5. Wen-Ling Deng
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  6. Yi-Hsien Lin
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Correspondence to Yi-Hsien Lin.

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Wu, PY., Ho, LC., Chang, JJ. et al. Development of a TaqMan probe-based insulated isothermal PCR (TiiPCR) for the detection of Acidovorax citrulli, the bacterial pathogen of watermelon fruit blotch. Eur J Plant Pathol 147, 869–875 (2017). https://doi.org/10.1007/s10658-016-1054-z

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