Xiaoyun Chen, Kai Li, Yi Ji, Ziyue Zhang, Xin Qi, Lianming Lu, Xiaofu Wang, Cheng Peng, Min Wang, Junfeng Xu* and Liang Li*,
{"title":"Development of DNA Reference Materials of Citrus Huanglongbing Candidatus Liberibacter asiaticus","authors":"Xiaoyun Chen, Kai Li, Yi Ji, Ziyue Zhang, Xin Qi, Lianming Lu, Xiaofu Wang, Cheng Peng, Min Wang, Junfeng Xu* and Liang Li*, ","doi":"10.1021/acsagscitech.4c00001","DOIUrl":null,"url":null,"abstract":"<p ><i>Citrus</i> Huanglongbing (HLB) is a devastating disease within the <i>Citrus</i> industry. <i>Candidatus</i> Liberibacter asiaticus (<i>C</i>Las) 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 <i>C</i>Las detection. Here, we developed a series of DNA reference materials of <i>C</i>Las using 16S rDNA as the target gene and the SAND gene as the <i>Citrus</i> reference gene. The 16S rDNA gene fragment cloned by the NCBI sequence and <i>Citrus</i> DNA extracted by healthy <i>Citrus</i> 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) × 10<sup>3</sup> and (4.43 ± 0.39) × 10<sup>3</sup> 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) × 10<sup>2</sup> and (4.34 ± 0.37) × 10<sup>3</sup> cp/μL, respectively. In addition, this certified reference material will provide reliable quality control for detecting <i>C</i>Las.</p>","PeriodicalId":93846,"journal":{"name":"ACS agricultural science & technology","volume":"4 4","pages":"500–506"},"PeriodicalIF":2.3000,"publicationDate":"2024-03-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"ACS agricultural science & technology","FirstCategoryId":"1085","ListUrlMain":"https://pubs.acs.org/doi/10.1021/acsagscitech.4c00001","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"AGRICULTURE, MULTIDISCIPLINARY","Score":null,"Total":0}
引用次数: 0
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.