摘要
目的基于全球统一DENV-1基因分型协同监测策略构建纳米孔测序溯源技术体系,并初步应用于患者血清样本测试。方法基于全球DENV整合序列资源数据库GISDD v1.2.1中DENV-1全基因组序列集,利用MEGA 7.0、primer-BLAST、MPprimer和GISDDrPrimer软件筛选、设计和评价特异性的靶向多重PCR引物对;提取血清样本RNA和逆转录;16对引物分配至2个引物池,多重PCR扩增靶标基因组序列;使用磁珠纯化,根据SQK-LSK109试剂盒操作流程构建文库,使用MinION测序;分析测序数据并组装基因组,以GISDD基因分型平台进行溯源分析。结果设计并鉴定了16对与GISDD所收录基因组序列具有高覆盖度的DENV-1引物,建立了DENV-1特异性多重PCR靶向纳米孔高通量测序技术体系。初步应用于13例广州登革热患者血清样本的全基因组检测,读长(reads)平均长度中值为1668.00 bp,测序平均质量中值为9.30,比对率中值为97.90%,比对上参考基因组NV46的平均长度中值为1677.90 bp,基因组平均深度中值为12886.40×。进一步基于GISDD基因分型和溯源分析发现,9个隶属DENV-1基因分支1E1、3个为1L1、1个为5C1。结论纳米孔测序DENV-1全基因组检测体系稳定可靠,可获得高质量DENV-1全基因组序列,为登革热疫情暴发中DENV-1基因组的快速侦检与溯源追踪提供了有效的技术方案,为建立登革热的全球协同监测控制体系奠定技术基础。
Objective Constructed a nanopore sequencing traceability technology system based on the globally unified DENV-1 genotyping collaborative monitoring strategy and applied it preliminarily to the serum sample testing of patients with dengue fever.Methods Utilizing the DENV-1 whole genome sequences set in the Global Integrated Sequence and Genotyping Database for DENV(GISDD v1.2.1),the specific targeted multiplex PCR primer pairs were screened,designed,and evaluated using MEGA 7.0,Prime-Blast,MPprimer,and GISDDrPrimer software.The RNA from the serum samples was extracted and subjected to reverse transcription.The 16 pairs of primers were designed into 2 primer pools for multiplex PCR amplification of the target genome sequences.The library was constructed using magnetic bead purification and the SQK-LSK109 kit according to the operational procedure,followed by MinION sequencing.The sequencing data were analyzed and DENV-1 genomes were assembled for traceability analysis using the GISDD genetic typing platform.Results Sixteen pairs of DENV-1 primers with high coverage to the DENV-1 genomes were designed and identified,establishing a DENV-1 specific multiplex PCR-based nanopore high-throughput sequencing system.It was preliminarily applied to the whole-genome detection of serum samples from 13 patients with dengue fever in Guangzhou.The median of average reads length was 1668.00 bp,the median of average reads quality was 9.30,the median alignment rate for the reference genome was 97.90%,and the median length aligned to the reference genome NV46 was 1677.90 bp,with a median genome depth of 12886.40×.The subsequent genotyping and tracing analyses using GISDD revealed that 9 genomes belonged to DENV-1 clade 1E1,3 to 1L1,and 1 to 5C1.Conclusions The nanopore sequencing-based DENV-1 whole-genome detection system contextualizing within GISDD described here is characterized by its stability and reliability,enabling the acquisition of high-quality DENV-1 genomes.This approach offers an efficient technical solution for the rapid identification and traceability of DENV genomes during dengue outbreaks,thereby establishing a solid foundation for developing a global collaborative surveillance and control system for dengue.
作者
胡敏玲
张晓晴
郭祥
刘晓华
Jone Jama Kpanda Ngobeh
李紫瑶
赵令斋
陈海洋
葛柳
曾淑
任文雯
张复春
周晓红
HU Minling;ZHANG Xiaoqing;GUO Xiang;LIU Xiaohua;Jone Jama Kpanda Ngobeh;LI Ziyao;ZHAO Lingzhai;CHEN Haiyang;GE Liu;ZENG Shu;REN Wenwen;ZHANG Fuchun;ZHOU Xiaohong(Institute of Tropical Medicine,Department of Pathogen Biology,School of Public Health,Southern Medical University,Guangdong Provincial Key Laboratory of Tropical Disease Research,Key Laboratory of Prevention and Control for Emerging Infectious Diseases of Guangdong Higher Institutes,Key Laboratory of Infectious Diseases Research in South China of Ministry of Education,Guangzhou,Guangdong 510515,China;Guangdong Huangpu Health Vocational Technical School,Guangzhou,Guangdong 510720,China;Guangzhou Eighth People's Hospital,Guangzhou Medical University,Guangzhou,Guangdong 510060,China)
出处
《中国热带医学》
CAS
北大核心
2024年第10期1166-1172,1216,共8页
China Tropical Medicine
基金
国家重点研发计划项目(No.2020YFC1200104)
国家自然科学基金项目(No.82072311)
广东省重点领域研发计划项目(No.2022B1111030002)。
关键词
登革病毒
纳米孔测序
全基因组
基因分型
Dengue virus(DENV)
nanopore sequencing
whole genome
genotyping
