中東呼吸綜合征冠狀病毒半巢式PCR檢測(cè)方法的建立
發(fā)布時(shí)間：2019-05-05 12:37
【摘要】：目的建立特異、靈敏的半巢式PCR方法以期用于檢測(cè)中東呼吸綜合征冠狀病毒(MERS-CoV)。方法根據(jù)MERS-CoV N蛋白基因設(shè)計(jì)并合成特異性內(nèi)側(cè)、外側(cè)引物。通過(guò)條件優(yōu)化,建立MERS-CoV半巢式PCR檢測(cè)體系,擴(kuò)增產(chǎn)物測(cè)序后與目的序列進(jìn)行同源比對(duì)。以10倍系列稀釋重組質(zhì)粒為標(biāo)準(zhǔn)品,與普通PCR反應(yīng)進(jìn)行比較,檢測(cè)半巢式PCR方法的靈敏度。以其他6種呼吸道病原體及冠狀病毒基因?yàn)閷?duì)照,檢測(cè)半巢式PCR方法的特異性。結(jié)果使用建立的半巢式PCR方法擴(kuò)增的特異片段與GenBank中發(fā)表的MERS-CoV基因序列同源性為100%;半巢式PCR檢測(cè)方法的靈敏度為1.17×101拷貝,比普通一輪PCR擴(kuò)增的靈敏度(1.17×105拷貝)提高了104倍;用該方法檢測(cè)MERS-CoV基因?yàn)殛?yáng)性,其他6種呼吸道病原體及冠狀病毒基因檢測(cè)均呈陰性。結(jié)論建立的MERS-CoV半巢式PCR方法具有良好的準(zhǔn)確性、靈敏性和特異性,為MERS-CoV感染的診斷提供了一種新的、可靠的檢測(cè)手段。
[Abstract]:Objective to establish a specific and sensitive semi-nested PCR method for the detection of Middle East respiratory syndrome coronavirus (MERS-CoV). Methods specific medial and lateral primers were designed and synthesized according to MERS-CoV N protein gene. The MERS-CoV semi-nested PCR detection system was established by optimizing the conditions. The amplified products were sequenced and compared with the target sequence. The sensitivity of semi-nested PCR method was compared with that of ordinary PCR using 10-fold diluted recombinant plasmid as standard. Six other respiratory pathogens and coronavirus genes were used as controls to detect the specificity of the semi-nested PCR method. Results the specific fragment amplified by the semi-nested PCR method shared 100% homology with the published MERS-CoV gene sequence in GenBank. The sensitivity of semi-nested PCR was 1.17 脳 101copies, which was 104 times higher than that of conventional PCR amplification (1.17 脳 105copies). The other 6 respiratory pathogens and coronavirus genes were negative for the detection of MERS-CoV gene by this method. Conclusion the established MERS-CoV semi-nested PCR method has good accuracy, sensitivity and specificity, which provides a new and reliable method for the diagnosis of MERS-CoV infection.
【作者單位】： 軍事醫(yī)學(xué)科學(xué)院軍事獸醫(yī)研究所;吉林省人獸共患病預(yù)防與控制重點(diǎn)實(shí)驗(yàn)室;江蘇省動(dòng)物重要疫病與人獸共患病防控協(xié)同創(chuàng)新中心;中國(guó)農(nóng)業(yè)科學(xué)院生物技術(shù)研究所;
【基金】：國(guó)家科技支撐計(jì)劃項(xiàng)目(No.2013BAD12B04) 中國(guó)博士后科學(xué)基金項(xiàng)目(No.2013M541089)
【分類號(hào)】：R440;R511
，
本文編號(hào)：2469569
[Abstract]:Objective to establish a specific and sensitive semi-nested PCR method for the detection of Middle East respiratory syndrome coronavirus (MERS-CoV). Methods specific medial and lateral primers were designed and synthesized according to MERS-CoV N protein gene. The MERS-CoV semi-nested PCR detection system was established by optimizing the conditions. The amplified products were sequenced and compared with the target sequence. The sensitivity of semi-nested PCR method was compared with that of ordinary PCR using 10-fold diluted recombinant plasmid as standard. Six other respiratory pathogens and coronavirus genes were used as controls to detect the specificity of the semi-nested PCR method. Results the specific fragment amplified by the semi-nested PCR method shared 100% homology with the published MERS-CoV gene sequence in GenBank. The sensitivity of semi-nested PCR was 1.17 脳 101copies, which was 104 times higher than that of conventional PCR amplification (1.17 脳 105copies). The other 6 respiratory pathogens and coronavirus genes were negative for the detection of MERS-CoV gene by this method. Conclusion the established MERS-CoV semi-nested PCR method has good accuracy, sensitivity and specificity, which provides a new and reliable method for the diagnosis of MERS-CoV infection.
【作者單位】： 軍事醫(yī)學(xué)科學(xué)院軍事獸醫(yī)研究所;吉林省人獸共患病預(yù)防與控制重點(diǎn)實(shí)驗(yàn)室;江蘇省動(dòng)物重要疫病與人獸共患病防控協(xié)同創(chuàng)新中心;中國(guó)農(nóng)業(yè)科學(xué)院生物技術(shù)研究所;
【基金】：國(guó)家科技支撐計(jì)劃項(xiàng)目(No.2013BAD12B04) 中國(guó)博士后科學(xué)基金項(xiàng)目(No.2013M541089)
【分類號(hào)】：R440;R511
，
本文編號(hào)：2469569
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