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Volume 23 Issue 3
June 2008

    Jing LU, Li QIN, Guang-jie LIU, Si-ting ZHAO and Xiao-ping CHEN. Quantification of Simian Immunodeficiency Virus by SYBR Green RT-PCR Technique[J]. Virologica Sinica, 2008, 23(3): 189-195. doi: 10.1007/s12250-008-2896-0
    Citation: Jing LU, Li QIN, Guang-jie LIU, Si-ting ZHAO, Xiao-ping CHEN. Quantification of Simian Immunodeficiency Virus by SYBR Green RT-PCR Technique .VIROLOGICA SINICA, 2008, 23(3) : 189-195.  http://dx.doi.org/10.1007/s12250-008-2896-0
    shu

    SYBR Green 荧光定量PCR检测猴免疫缺陷病毒血浆病毒载量

    cstr: 32224.14.s12250-008-2896-0

    • 中国科学院广州生物医药与健康研究院

    • 在猴免疫缺陷病毒（SIV）感染模型研究中，血浆病毒RNA载量常用作评价病毒感染进程及疾病状态的重要指标。本文以SYBR green 为染料，建立了一步法实时荧光定量PCR用于精确定量血浆病毒载量。该方法成功用于检测SIVmac251和SIVmac239感染的CEM×174细胞培养上清中的病毒RNA载量，检测下限为每管反应10 copies。在建立的SIVmac251感染恒河猴模型中，该方法用于检测猴血浆病毒RNA载量，其检测下限为每毫升血浆215 copies，并且病毒载量的变化曲线也与其它的报道相符。该方法具有较高的灵敏度和精度，并且较branched-chain DNA（b-DNA）和基于TaqMan探针的荧光定量PCR方法更为简单、易行。

      Abstract

      Plasma viral RNA load is widely accepted as the most relevant parameter to assess the status and progression of Simian Immunodeficiency Virus (SIV) infections. To accurately measure RNA viral loads, a one-step fluorescent quantitative assay was established based on SYBR green Real-Time reverse transcription-polymerase chain reaction technique (RT-PCR). The assay with a lower detection limit of 10 copies per reaction was successfully applied to quantification of SIVmac251 and SIVmac239 virus stocks produced on CEM×174 cells. Moreover, the performance of the SYBR green real-time PCR was assessed in a SIVmac251 infected rhesus macaque. The result demonstrated that this technique can detect as less as 215 copies per milliliter of plasma and the dynamic pattern of viral load was similar with those based on other techniques from other reports. Our assay due to its convenience, sensitivity and accuracy could serve as a good alternative to branched-chain DNA (b-DNA) assay or real-time PCR assay based on TaqMan probes.

    Quantification of Simian Immunodeficiency Virus by SYBR Green RT-PCR Technique

    • Guangzhou Institute of Biomedicine and Health GIBH, Chinese Academy of Sciences, Guangzhou 510663, China

    • Corresponding author: Xiao-ping CHEN, chen_xiaoping@gibh.ac.cn
    • Received Date: 17 August 2007
      Accepted Date: 12 December 2007

      Fund Project: National 973 Program 2006CB504208Natural Science Foundation of Guangdong Province 07118293The Grant of Science and Technology Plans of Guangdong Province 2006B36005002

    • Plasma viral RNA load is widely accepted as the most relevant parameter to assess the status and progression of Simian Immunodeficiency Virus (SIV) infections. To accurately measure RNA viral loads, a one-step fluorescent quantitative assay was established based on SYBR green Real-Time reverse transcription-polymerase chain reaction technique (RT-PCR). The assay with a lower detection limit of 10 copies per reaction was successfully applied to quantification of SIVmac251 and SIVmac239 virus stocks produced on CEM×174 cells. Moreover, the performance of the SYBR green real-time PCR was assessed in a SIVmac251 infected rhesus macaque. The result demonstrated that this technique can detect as less as 215 copies per milliliter of plasma and the dynamic pattern of viral load was similar with those based on other techniques from other reports. Our assay due to its convenience, sensitivity and accuracy could serve as a good alternative to branched-chain DNA (b-DNA) assay or real-time PCR assay based on TaqMan probes.
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      Quantification of Simian Immunodeficiency Virus by SYBR Green RT-PCR Technique

        Corresponding author: Xiao-ping CHEN, chen_xiaoping@gibh.ac.cn
      • Guangzhou Institute of Biomedicine and Health GIBH, Chinese Academy of Sciences, Guangzhou 510663, China
      • Received Date: 17 August 2007
      • Accepted Date: 12 December 2007
      Fund Project:  National 973 Program 2006CB504208Natural Science Foundation of Guangdong Province 07118293The Grant of Science and Technology Plans of Guangdong Province 2006B36005002

      Abstract: Plasma viral RNA load is widely accepted as the most relevant parameter to assess the status and progression of Simian Immunodeficiency Virus (SIV) infections. To accurately measure RNA viral loads, a one-step fluorescent quantitative assay was established based on SYBR green Real-Time reverse transcription-polymerase chain reaction technique (RT-PCR). The assay with a lower detection limit of 10 copies per reaction was successfully applied to quantification of SIVmac251 and SIVmac239 virus stocks produced on CEM×174 cells. Moreover, the performance of the SYBR green real-time PCR was assessed in a SIVmac251 infected rhesus macaque. The result demonstrated that this technique can detect as less as 215 copies per milliliter of plasma and the dynamic pattern of viral load was similar with those based on other techniques from other reports. Our assay due to its convenience, sensitivity and accuracy could serve as a good alternative to branched-chain DNA (b-DNA) assay or real-time PCR assay based on TaqMan probes.

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