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谢 帆1, 2#, 龚 顺1, 2#, 王泽琼1, 肖玉雄1, 杜志强3, 仝 铸1, 何秀娟1, 邱文明1, 孙中海1, 潘志勇2, 肖 翠1*.柑橘黄龙病菌亚洲种分子检测引物评价及绝对定量PCR体系优化[J].植物保护,2024,50(3):234-246.
柑橘黄龙病菌亚洲种分子检测引物评价及绝对定量PCR体系优化
Molecular detection primer evaluation and absolute quantitative PCR system optimization of Candidatus Liberibacter asiaticus (CLas)
投稿时间:2023-07-31  修订日期:2023-10-11
DOI:10.16688/j.zwbh.2023396
中文关键词:  柑橘黄龙病  常规PCR  巢式PCR  绝对定量PCR  引物评价
英文关键词:Citrus Huanglongbing  conventional PCR  nested PCR  absolute quantitative PCR  primer evaluation
基金项目:国家重点研发计划(2021YFD1400802);湖北省农业科学院青年拔尖人才培养计划(Q2021015)
作者单位E-mail
谢 帆1, 2#, 龚 顺1, 2#, 王泽琼1, 肖玉雄1, 杜志强3, 仝 铸1, 何秀娟1, 邱文明1, 孙中海1, 潘志勇2, 肖 翠1* 1. 湖北省农业科学院果树茶叶研究所, 果树种质创新与利用湖北省重点实验室, 武汉 430064
2. 华中农业大学园艺林学学院, 果蔬园艺作物种质创新与利用全国重点实验室, 武汉 430070
3. 宜昌三峡百景宜农业科技发展有限公司, 宜昌 443600 
xiaocui@hbaas.com 
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中文摘要:
      柑橘黄龙病是对柑橘产业最具毁灭性的病害, 目前没有可用的有效药剂和抗病品种, 分子检测对黄龙病有效防控至关重要。本研究对国内外常用的常规PCR和巢式PCR检测引物进行评价, 针对多拷贝的nrdB和16S rDNA基因, 构建质粒标准品并筛选适用于绝对定量PCR的最佳质粒。结果表明, 使用Es Taq MasterMix对感染 Candidatus Liberibacter asiaticus (CLas)的柑橘样品进行常规PCR检测时, 在评价的16对引物中, OI1/OI2c、Las606/LSS和HLBF468/R877灵敏度最高, 推荐同时使用检测黄龙病菌含量低的样品;各组巢式PCR检测引物有其适用扩增体系, 部分引物用Es Taq MasterMix扩增时出现非特异性扩增, F1/B1→F3/B3则适用Es Taq MasterMix体系, 且最高可稳定特异检出105倍稀释感染CLas柑橘总DNA样品(2×10-3 ng/μL), 是灵敏度最高的引物组, OI1/OI2c→S3/S4在Es Taq MasterMix和Ex Taq DNA聚合酶体系中均可稳定特异检出104倍稀释感染CLas柑橘总DNA样品(2×10-2 ng/μL), 是适用扩增体系最广的引物组;构建的5个绝对定量PCR质粒标准品中, pnrdB83扩增效率最接近100%, 且在2次重复试验中波动最小, 稳定性最强, 并且作为标准品对黄龙病待测样品进行绝对定量时, 各样品在2次重复试验中的拷贝数差值最小, 是本研究筛选的最佳质粒。本研究的结果将为柑橘黄龙病菌的定性和定量分子检测提供参考。
英文摘要:
      Citrus Huanglongbing (HLB) is the most devastating disease for the citrus industry. Currently, there are no effective cure and disease-resistant varieties available. Molecular detection is crucial for the effective prevention and management of Huanglongbing. In this study, we evaluated the commonly and world-widely employed primers for conventional PCR and nested PCR detection, and constructed plasmid standards based on multiple-copy nrdB and 16S rDNA genes, and then screened the best plasmids suitable for absolute quantitative PCR. The results showed that among the 16 pairs of primers evaluated in this study, OI1/OI2c, Las606/LSS and HLBF468/R877 had the highest sensitivity when using Es Taq MasterMix for conventional PCR detection of Candidatus Liberibacter asiaticus (CLas) infecting citrus samples, which was recommended to detect samples with low CLas titer. Each nested PCR detection primer had their applicable amplification systems, and some primers showed strong non-specific amplification when amplified with Es Taq MasterMix, while F1/B1→F3/B3 were suitable for Es Taq MasterMix system, and could stably and specifically detect the 105-fold diluted citrus total DNA samples (2×10-3 ng/μL) infected with CLas, which was the primer set with the highest sensitivity. OI1/OI2c→S3/S4 could stably and specifically detect 104-fold diluted citrus total DNA samples (2×10-2 ng/μL) infected with CLas both in Es Taq MasterMix and Ex Taq DNA polymerase systems, which was the primer set with the widest applicable amplification systems. Among the five plasmid standards constructed for absolute quantitative PCR, pnrdB83 was the best plasmid whose amplification efficiency was closest to 100% and with the least fluctuation and strongest stability between two repeated experiments. Besides, when used pnrdB83 as a standard for absolute quantification of Huanglongbing samples, the copy number difference of each sample between the two repeated experiments is the smallest. The results will provide a reference for qualitative and quantitative molecular detection of CLas.
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