本文關(guān)鍵詞： 寨卡病毒 核酸檢測(cè) 新型質(zhì)粒標(biāo)準(zhǔn)分子 白紋伊蚊　出處：《中國(guó)病原生物學(xué)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(cè)體系,構(gòu)建新型質(zhì)粒標(biāo)準(zhǔn)分子,并初步應(yīng)用于廣州地區(qū)白紋伊蚊野生種群ZIKV感染檢測(cè)。方法從GenBank獲取ZIKV全長(zhǎng)序列,以MAGE6.0分析比對(duì)錨定保守區(qū)域作為靶標(biāo)片段,結(jié)合Beacon Designer 7.5、Primer premier 6等設(shè)計(jì)引物及探針,采用逆轉(zhuǎn)錄PCR(RT-PCR)擴(kuò)增ZIKV RNA,獲得檢測(cè)靶標(biāo)片段RZ-1,運(yùn)用Overlapping PCR在RZ-1中間插入50bp內(nèi)含子(mRZ-1),克隆獲得ZIKV新型質(zhì)粒標(biāo)準(zhǔn)分子pBmRZ-1。構(gòu)建并優(yōu)化ZIKV巢式逆轉(zhuǎn)錄PCR(Nested RT-PCR)和熒光定量PCR(Real-time fluorescent quantitative PCR,qRT-PCR)檢測(cè)體系,用pBmRZ-1及不同來源的ZIKV標(biāo)本對(duì)檢測(cè)體系進(jìn)行敏感性和特異性測(cè)試,并用于廣州白紋伊蚊野生種群ZIKV檢測(cè)。結(jié)果成功構(gòu)建ZIKV新型質(zhì)粒標(biāo)準(zhǔn)分子pBmRZ-1及Nested RT-PCR和qRT-PCR檢測(cè)體系。以pBmRZ-1測(cè)試最低檢測(cè)極限(Limit of detection,LOD)Nested RT-PCR為5copies,qRT-PCR為1copy,qRT-PCR標(biāo)準(zhǔn)曲線擬合線性方程為Y=-3.387LOG(x)+41.43,相關(guān)系數(shù)R2=0.996,擴(kuò)增效率為97.4%。該檢測(cè)體系特異、敏感,能檢出蚊蟲和細(xì)胞感染的ZIKV,411份廣州地區(qū)白紋伊蚊野生種群標(biāo)本ZIKV檢測(cè)陰性。結(jié)論成功構(gòu)建了特異、敏感的ZIKV核酸檢測(cè)體系,可用于蚊ZIKV檢測(cè),亦可用于細(xì)胞、血清標(biāo)本的ZIKV檢測(cè)。新型質(zhì)粒標(biāo)準(zhǔn)分子pBmRZ-1能甄別標(biāo)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(xué)公共衛(wèi)生學(xué)院病原生物學(xué)系暨廣東普通高校新發(fā)傳染病防治重點(diǎn)實(shí)驗(yàn)室廣東省熱帶病研究重點(diǎn)實(shí)驗(yàn)室;
【基金】：國(guó)家重點(diǎn)研發(fā)計(jì)劃項(xiàng)目(No.2016YFC1200500) 廣東省科技計(jì)劃項(xiàng)目(No.2016A020251001) 廣州市健康醫(yī)療協(xié)同創(chuàng)新重大專項(xiàng)(No.201508020263) 廣州市對(duì)外科技合作重點(diǎn)項(xiàng)目(No.2012J5100026) 國(guó)家級(jí)大學(xué)生創(chuàng)新創(chuàng)業(yè)訓(xùn)練計(jì)劃項(xiàng)目(No.201612121026)
【分類號(hào)】：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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