纳米孔测序技术在实蝇类害虫检疫鉴定中的应用  

Preliminary investigation of quarantine and identification of Tephritidae based on nanopore sequencing technology

作　　者：姜帆 丁新华[3] 梁亮[4] 付开赟 郭文超 朱水芳[2] JIANG Fan;DING Xinhua;LIANG Liang;FU Kaiyun;GUO Wenchao;ZHU Shuifang(CAIQ Center for Biosafety,Sanya,Hainan 572025,China;Chinese Academy of Inspection and Quarantine,Beijing 100176,China;Key Laboratory of Integrated Pest Management on Crops in Northwestern Oasis,Institute of Plant Protection,Xinjiang Academy of Agricultural Sciences,Urumqi,Xinjiang 830091,China;Academy of Agricultural Planning and Engineering,Ministry of Agriculture and Rural Affairs,Beijing 100125,China)

机构地区：[1]三亚中国检科院生物安全中心,海南三亚572025 [2]中国检验检疫科学研究院,北京100176 [3]新疆农业科学院植物保护研究所,农业农村部西北荒漠绿洲作物有害生物综合治理重点实验室,新疆乌鲁木齐830091 [4]农业农村部规划设计研究院,北京100125

出　　处：《生物安全学报（中英文）》2024年第1期93-98,共6页Journal of biosafety

基　　金：三亚市科技创新专项项目(2022KJCX72);智力援疆创新拓展人才计划。

摘　　要：【目的】纳米孔测序技术是单分子实时测序技术的一种,正在被广泛应用于临床快速诊断及微生物检测等领域。本研究以实蝇这一类重要的检疫性有害生物为例,探究该技术在昆虫检疫鉴定中应用的可行性,为昆虫检疫鉴定提供新方法。【方法】分别采用一代测序技术和纳米孔测序技术,对14种经形态学鉴定的实蝇成虫进行DNA条形码测序,通过BOLD和NCBI数据库对测序结果进行比对分析,并比较2种测序技术所得序列结果准确性的差异。【结果】通过纳米孔测序,14个实蝇样品在44 min内获得181Mb bases,每个样品平均得到11280条reads,单个reads的准确度为92.10%~94.53%;经一致性序列校正,所有实蝇样品均可得到与一代测序结果完全一致的序列,序列分析结果与形态学鉴定结果完全一致。【结论】采用本研究的实验流程和数据分析方法,纳米孔测序技术可以应用于实蝇类害虫的检疫鉴定,测序结果准确、高效;本研究提供的实验方案同样适用于基于扩增子测序的物种鉴定,满足大规模样品的高通量精准鉴定需求。【Aim】Nanopore sequencing technology,a single-molecule real-time sequencing approach,plays a crucial role in rapid clinical diagnosis and microbiological detection.Using Tephritidae as a crucial example of quarantine pests,the applicability of nanopore sequencing technology was assessed for insect quarantine identification,offering a new assessment approach.【Method】We employed Sanger sequencing technology and nanopore sequencing to obtain the DNA barcodes of 14 Tephritidae species previously identified based on morphology.Each sequencing result was cross-referenced in the NCBI and BOLD databases to confirm species accuracy.We then compared the accuracy of sequences obtained through these two distinct sequencing technologies for each species.【Result】Nanopore sequencing generated 181-megabyte bases in just 44 min across 14 samples,with an average of 11280 reads per sample and individual read accuracy of 92.10%-94.53%.After sequence correction for consistency,the results of nanopore sequencing were consistent with those of Sanger sequencing,and the sequencing analysis findings were consistent with the outcome of morphological identification.【Conclusion】Our study demonstrates the full applicability of nanopore sequencing technology to quarantine the identification of Tephritidae using the experimental process and data analysis methods outlined here.The sequencing results proved to be both accurate and efficient.Additionally,the experimental protocol provided in this study is suitable for species identification based on amplicon sequencing,fulfilling the requirements for high-throughput and accurate identification of large-scale samples.

关 键 词：纳米孔测序 实蝇 昆虫 鉴定 扩增子 

分 类 号：S41-30[农业科学—植物保护]