Development of DNA Reference Materials of Citrus Huanglongbing Candidatus Liberibacter asiaticus

IF 2.3 Q1 AGRICULTURE, MULTIDISCIPLINARY ACS agricultural science & technology Pub Date : 2024-03-29 DOI:10.1021/acsagscitech.4c00001
Xiaoyun Chen, Kai Li, Yi Ji, Ziyue Zhang, Xin Qi, Lianming Lu, Xiaofu Wang, Cheng Peng, Min Wang, Junfeng Xu* and Liang Li*, 
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Abstract

Citrus Huanglongbing (HLB) is a devastating disease within the Citrus industry. Candidatus Liberibacter asiaticus (CLas) is one of the most prevalent HLB-associated strains that has not been cultured in vitro. To ensure the accuracy and comparability of the molecular diagnostic method for HLB detection, certified reference materials urgently need to be developed for CLas detection. Here, we developed a series of DNA reference materials of CLas using 16S rDNA as the target gene and the SAND gene as the Citrus reference gene. The 16S rDNA gene fragment cloned by the NCBI sequence and Citrus DNA extracted by healthy Citrus leaves are thoroughly mixed for preparation. Droplet digital PCR (ddPCR) was used as an accurate quantification method for 16S rDNA, and the SAND was established and optimized through this study. Nine laboratories collaborated in determining these two parameters, and the homogeneity and stability were adequate. The quantification results demonstrated that the copy number certified values and expanded uncertainty of 16S rDNA and SAND in the high-concentration reference material were (3.86 ± 0.34) × 103 and (4.43 ± 0.39) × 103 cp/μL, respectively. The copy number certified values and expanded uncertainty of 16S rDNA and SAND in the low-concentration reference material were (3.98 ± 0.36) × 102 and (4.34 ± 0.37) × 103 cp/μL, respectively. In addition, this certified reference material will provide reliable quality control for detecting CLas.

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开发柑橘黄龙病菌的 DNA 参考材料
柑橘黄龙病(HLB)是柑橘产业中的一种毁灭性病害。亚洲自由杆菌(CLas)是最普遍的 HLB 相关菌株之一,但尚未在体外培养。为确保 HLB 检测分子诊断方法的准确性和可比性,迫切需要开发出检测 CLas 的认证参考材料。在此,我们以 16S rDNA 为目标基因,以 SAND 基因为柑橘参考基因,开发了一系列 CLas DNA 参考材料。将通过 NCBI 序列克隆的 16S rDNA 基因片段与从柑橘健康叶片中提取的柑橘 DNA 充分混合后进行制备。本研究采用液滴数字 PCR(ddPCR)作为 16S rDNA 的精确定量方法,并建立和优化了 SAND 基因。九个实验室合作确定了这两个参数,其均一性和稳定性都很好。定量结果表明,高浓度参考物质中 16S rDNA 和 SAND 的拷贝数认证值和扩展不确定度分别为 (3.86 ± 0.34) × 103 cp/μL 和 (4.43 ± 0.39) × 103 cp/μL。低浓度参考物质中 16S rDNA 和 SAND 的拷贝数认证值和扩展不确定度分别为 (3.98 ± 0.36) × 102 和 (4.34 ± 0.37) × 103 cp/μL。此外,这种经认证的标准物质将为检测 CLas 提供可靠的质量控制。
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