蚊寨卡病毒核酸检测体系的建立与初步应用

发布时间：2018-01-20 11:23

本文关键词： 寨卡病毒核酸检测新型质粒标准分子白纹伊蚊　出处：《中国病原生物学杂志》2017年09期 　论文类型：期刊论文

【摘要】：目的建立敏感、特异的主要适用于蚊媒携带寨卡病毒(Zika virus,ZIKV)的核酸检测体系,构建新型质粒标准分子,并初步应用于广州地区白纹伊蚊野生种群ZIKV感染检测。方法从GenBank获取ZIKV全长序列,以MAGE6.0分析比对锚定保守区域作为靶标片段,结合Beacon Designer 7.5、Primer premier 6等设计引物及探针,采用逆转录PCR(RT-PCR)扩增ZIKV RNA,获得检测靶标片段RZ-1,运用Overlapping PCR在RZ-1中间插入50bp内含子(mRZ-1),克隆获得ZIKV新型质粒标准分子pBmRZ-1。构建并优化ZIKV巢式逆转录PCR(Nested RT-PCR)和荧光定量PCR(Real-time fluorescent quantitative PCR,qRT-PCR)检测体系,用pBmRZ-1及不同来源的ZIKV标本对检测体系进行敏感性和特异性测试,并用于广州白纹伊蚊野生种群ZIKV检测。结果成功构建ZIKV新型质粒标准分子pBmRZ-1及Nested RT-PCR和qRT-PCR检测体系。以pBmRZ-1测试最低检测极限(Limit of detection,LOD)Nested RT-PCR为5copies,qRT-PCR为1copy,qRT-PCR标准曲线拟合线性方程为Y=-3.387LOG(x)+41.43,相关系数R2=0.996,扩增效率为97.4%。该检测体系特异、敏感,能检出蚊虫和细胞感染的ZIKV,411份广州地区白纹伊蚊野生种群标本ZIKV检测阴性。结论成功构建了特异、敏感的ZIKV核酸检测体系,可用于蚊ZIKV检测,亦可用于细胞、血清标本的ZIKV检测。新型质粒标准分子pBmRZ-1能甄别标本中可能存在的来源于阳性参照的核酸污染。广州市白纹伊蚊野生种群未检出ZIKV。
[Abstract]:Objective to establish a sensitive and specific nucleic acid detection system for mosquito vector carrying Zika virus Zika virus ZIKV, and to construct a new plasmid standard molecule. It was applied to the detection of ZIKV infection in wild population of Aedes albopictus in Guangzhou. Methods the full-length ZIKV sequence was obtained from GenBank. MAGE6.0 analysis was used as the target fragment and Beacon Designer 7.5 was used as the target fragment. Primer premier 6 and other primers and probes were designed to amplify ZIKV RNAs by reverse transcriptase polymerase chain reaction (RT-PCR) to obtain the target RZ-1. Overlapping PCR was used to insert 50 BP intron mRZ-1 in the middle of RZ-1. Cloning of a novel plasmid pBmRZ-1.Construction and optimization of ZIKV nested reverse transcription PCR(Nested RT-PCR and fluorescence quantitative PCR1. Real-time fluorescent quantitative PCR. PBmRZ-1 and ZIKV samples from different sources were used to detect the sensitivity and specificity of the detection system. It was used to detect the wild population of Aedes albopictus in Guangzhou by ZIKV. Results the new plasmid standard molecules pBmRZ-1 and Nested of ZIKV were successfully constructed. RT-PCR and qRT-PCR detection systems-minimum detection limits for pBmRZ-1 testing. Limit of detection. The LOD)Nested RT-PCR was 5copiestio qRT-PCR (1copy). The linear equation of qRT-PCR standard curve fitting was YP3-3.387 LOGX) 41.43, and the correlation coefficient was R20.996. The amplification efficiency was 97.40.The detection system was specific and sensitive, and could detect the ZIKV of mosquito and cell infection. Conclusion A specific and sensitive ZIKV nucleic acid detection system was successfully constructed, which can be used to detect ZIKV and cells of Aedes albopictus in Guangzhou. ZIKV detection of serum samples. The new plasmid standard molecule pBmRZ-1 can identify the possible positive reference nucleic acid contamination in the samples. No ZIKV was detected in the wild population of Aedes albopictus in Guangzhou.
【作者单位】：南方医科大学公共卫生学院病原生物学系暨广东普通高校新发传染病防治重点实验室广东省热带病研究重点实验室;
【基金】：国家重点研发计划项目(No.2016YFC1200500) 广东省科技计划项目(No.2016A020251001) 广州市健康医疗协同创新重大专项(No.201508020263) 广州市对外科技合作重点项目(No.2012J5100026) 国家级大学生创新创业训练计划项目(No.201612121026)
【分类号】：R384.1
【正文快照】： ***amplification efficiency was 97.40%.ZIKV was successfully detected in infected mosquitos and c6/36cells using a nucleicacid detection system.Four hundred and eleven pools of Ae.albopictus in Guangzhou were tested but were all negative.Conclusion A spe

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