本文關鍵詞： 寨卡病毒 核酸檢測 新型質粒標準分子 白紋伊蚊　出處：《中國病原生物學雜志》2017年09期 　論文類型：期刊論文

【摘要】：目的建立敏感、特異的主要適用于蚊媒攜帶寨卡病毒(Zika virus,ZIKV)的核酸檢測體系,構建新型質粒標準分子,并初步應用于廣州地區(qū)白紋伊蚊野生種群ZIKV感染檢測。方法從GenBank獲取ZIKV全長序列,以MAGE6.0分析比對錨定保守區(qū)域作為靶標片段,結合Beacon Designer 7.5、Primer premier 6等設計引物及探針,采用逆轉錄PCR(RT-PCR)擴增ZIKV RNA,獲得檢測靶標片段RZ-1,運用Overlapping PCR在RZ-1中間插入50bp內含子(mRZ-1),克隆獲得ZIKV新型質粒標準分子pBmRZ-1。構建并優(yōu)化ZIKV巢式逆轉錄PCR(Nested RT-PCR)和熒光定量PCR(Real-time fluorescent quantitative PCR,qRT-PCR)檢測體系,用pBmRZ-1及不同來源的ZIKV標本對檢測體系進行敏感性和特異性測試,并用于廣州白紋伊蚊野生種群ZIKV檢測。結果成功構建ZIKV新型質粒標準分子pBmRZ-1及Nested RT-PCR和qRT-PCR檢測體系。以pBmRZ-1測試最低檢測極限(Limit of detection,LOD)Nested RT-PCR為5copies,qRT-PCR為1copy,qRT-PCR標準曲線擬合線性方程為Y=-3.387LOG(x)+41.43,相關系數(shù)R2=0.996,擴增效率為97.4%。該檢測體系特異、敏感,能檢出蚊蟲和細胞感染的ZIKV,411份廣州地區(qū)白紋伊蚊野生種群標本ZIKV檢測陰性。結論成功構建了特異、敏感的ZIKV核酸檢測體系,可用于蚊ZIKV檢測,亦可用于細胞、血清標本的ZIKV檢測。新型質粒標準分子pBmRZ-1能甄別標本中可能存在的來源于陽性參照的核酸污染。廣州市白紋伊蚊野生種群未檢出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.
【作者單位】： 南方醫(yī)科大學公共衛(wèi)生學院病原生物學系暨廣東普通高校新發(fā)傳染病防治重點實驗室廣東省熱帶病研究重點實驗室;
【基金】：國家重點研發(fā)計劃項目(No.2016YFC1200500) 廣東省科技計劃項目(No.2016A020251001) 廣州市健康醫(yī)療協(xié)同創(chuàng)新重大專項(No.201508020263) 廣州市對外科技合作重點項目(No.2012J5100026) 國家級大學生創(chuàng)新創(chuàng)業(yè)訓練計劃項目(No.201612121026)
【分類號】：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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